Conference Program - ABRF 2011 - Association of Biomolecular ...

Meeting SponsorsThe Association of Biomolecular Resource Facilities (ABRF) and the ABRF 2011 Organizing Committee wouldlike to extend their gratitude for the generous support received from the following organizations:Meeting SponsorsNetworking Night at the RanchCyber Café Monday Munch & Mingle Meeting BagAward SponsorsABRF Annual Award for Outstanding Contributionsto Biomolecular TechnologiesABRF Poster AwardsMedia SponsorsABRF 2011 — Technologies to Enable Personalized Medicine • 5

Hotel FloorplanHotel Floorplan6 • ABRF 2011 — Technologies to Enable Personalized Medicine

Registration ServicesRegistration ServicesTicketed EventsFriday through TuesdayLevel 2, Ballroom Level Registration DeskOn–site registration will be open during the following hours:Friday, February 18 ........................................4:00 pm – 6:00 pmSaturday, February 19 ................................... 7:00 am – 6:00 pmSunday, February 20 ...................................... 7:00 am – 6:00 pmMonday, February 21 .................................... 7:00 am – 6:00 pmTuesday, February 22 .................................... 7:00 am – 6:00 pmMeeting RegistrationFull meeting registration includes: food functions served byABRF 2011, access into the Exhibit Hall, Receptions, ScientificSessions, Workshops, and Research Group Presentation Sessions.Note: All attendees are welcome to attend the Research GroupPresentation Sessions.Activities for which you have purchased tickets or guest ticketsrequire the actual ticket for entrance. Tickets are provided at thetime of check-in. Please be sure to check that you received ticketsfor all purchased items. Additional tickets can be purchased atthe On-site Registration Desk.Networking Night at the Ranch (Sponsored by )Guest Ticket ..................................................................................$50Exhibit Hall ONLY RegistrationExhibit Hall Only Registration ..................................................$35An Exhibit Hall Only Registration is available for those whowish to visit the Exhibition only. Exhibit Hall Only Registrationallows access into the exhibits, as well as all Research GroupPresentation Sessions.Registration ServicesOn-site FeesMember ....................................................................................... $510Non-Member ............................................................................ $635Student ........................................................................................ $195Membership + Meeting Registration ................................. $610ReceiptsRegistered participants will receive a receipt/confirmation viaemail. Participants who register on-site will receive a paperreceipt, if requested.Satellite Educational WorkshopsMember ....................................................................................... $325Non-Member ............................................................................ $425Please see page 28 for additional information about eachWorkshop listed below. Satellite Educational Workshops are notincluded in the regular meeting registration. You must purchasea separate registration to attend a specific Satellite EducationalWorkshop. Session Organizers will be checking badges for allSatellite Educational Workshops.• (SW1) Protein Purification for Mass Spectrometry• (SW2) An Introduction to Metabolomics• (SW3) Next Generation Sequencing Considerations ForCore Professionals• (SW4) Lean Management in Core FacilitiesBadgesBadges should be worn at all official functions of the meeting.Badge checkers will be stationed throughout the hotel. Onlythose with full meeting registrations will be allowed into sessions.If you forget or lose your badge, you may obtain a second badgeat registration with proof of registration.ABRF 2011 — Technologies to Enable Personalized Medicine • 7

Meals & RefreshmentsMeals& RefreshmentsMeeting registration includes all meals, receptions, andrefreshments provided by ABRF 2011, as well as one ticket tothe Networking Night at the Ranch (sponsored by ).Opening ReceptionSaturdayLevel 1, Exhibit Ballroom Foyer7:00 pm – 9:00 pmContinental BreakfastSunday through TuesdayLevel 1, Exhibit Ballroom Foyer7:00 am – 7:45 amMorning Refreshment BreakSunday through TuesdayLevel 2, Grand Oaks Ballroom, Exhibit Hall10:30 am – 11:00 amMunch & Mingle (Lunch)Sunday through TuesdayLevel 2, Grand Oaks Ballroom, Exhibit Hall12:00 pm – 1:30 pmAfternoon Refreshment BreakSunday through MondayLevel 2, Grand Oaks Ballroom, Exhibit Hall4:15 pm – 4:45 pmTuesdayLevel 2, Grand Oaks Ballroom N/S Foyer4:15 pm – 4:45 pmNetworking Night at the Ranch (sponsored by )SundayOffsite: Leon Springs Dance Hall7:30 pm – 10:30 pmShuttles to Leon Springs Dance Hall will be provided to all ABRF2011 Networking Night attendees. Shuttles will begin loadingpromptly at 7:00 pm to depart from the Exhibit Level Lobby.Shuttles to return to the JW Marriott San Antonio Hill CountryResort will also be provided. Please review the shuttle schedulefor further information.Closing ReceptionTuesdayJW Marriott San Antonio Hill Country Resort,Grand Ballroom Terrace7:00 pm – 9:00 pmClose out ABRF 2011 with the rest of your colleagues at the JWMarriott’s Grand Ballroom Terrace. Reminisce about the week’sevents with fantastic cocktails and hors d’oeuvres.8 • ABRF 2011 — Technologies to Enable Personalized Medicine

Presenter InformationPresenter InformationSpeaker Ready RoomSaturday through TuesdayLevel 2, Ballroom Level Registration DeskALL Presenters must check-in at the Speaker Ready Room at least2 hours prior to their scheduled presentation. Even if you havesubmitted your presentation in advance and have no changes,you must check and confirm that the presentation is correct.The hours of operation of the Speaker Ready Room are asfollows:Saturday, February 19 ................................... 7:00 am – 4:00 pmSunday, February 20 ...................................... 7:00 am – 4:00 pmMonday, February 21 .................................... 7:00 am – 4:00 pmTuesday, February 22 .................................... 7:00 am – 4:00 pmYou may also edit your presentation at this time. When youare finished reviewing your presentation and verify that it isready, the AV personnel will queue your presentation ontothe networked conference computers. The file will then betransferred to the computer network for presentation in thescheduled room.Meeting ManagementDonna Johnson, Strategic Account DirectorLauren Deaton, Meeting ManagerChris Cherkis, Exhibits & Sponsorship ManagerChezka Solon, Program & Registration ManagerABRF 2010c/o Courtesy Associates2025 M Street, NWSuite 800Washington, DC 20036Phone: +1 202-973-8670Fax: +1 202-331-0111Email: abrf@courtesyassoc.comPoster PresentationsSunday through TuesdayOver 140 posters will be on display all day Sunday throughTuesday. The Poster Presenters should be available for questionsat their display boards during the specific session times. PosterSession times are as follows:Poster Session I (Odd Numbered Posters)Sunday, February 20Level 2, Grand Oaks Ballroom, Exhibit Hall & Foyer10:30 am – 12:00 pmPoster Session II (Even Numbered Posters)Monday, February 21Level 2, Grand Oaks Ballroom, Exhibit Hall & Foyer10:30 am – 12:00 pmPoster Session III (All Posters)Tuesday, February 22Level 2, Grand Oaks Ballroom, Exhibit Hall & Foyer10:30 am – 12:00 pmOdd numbered posters will be presented during Poster SessionI. Even numbered posters will be presented during Poster SessionII. Please see page 60 for the full listing of poster presentations.Poster Session III on Tuesday, February 22 is a free period forattendees to review all posters.All posters should be set up at least one hour prior to theopening of the Exhibit Hall on Sunday, February 20. Posterpresenters have been given a special pass to allow them accessinto the Exhibit Hall during these hours. If you are missing thispass, please visit the Registration Desk to receive the access pass.All posters must be removed from the Exhibit Hall between thehours of 2:00 pm – 3:00 pm on Tuesday, February 22. Do notleave your presentation on the poster board. Posters remainingafter 3:00 pm on Tuesday will be discarded.10 • ABRF 2011 — Technologies to Enable Personalized Medicine

www.454.comSequence-Enrichment SolutionGS Junior andSequence CapturePerform targeted resequencing of any genomic region in your own labby combining two new Roche technologies: NimbleGen EZ ChoiceSequence Capture custom probes and the GS Junior Instrument.Use an easy, scalable workflow for sequence enrichment —Choose a simple workflow featuring gel-free sequencing librarypreparation, single-tube capture with stable DNA probes, and easyhybridization setup.Reduce sequencing costs — Multiplex sequencing samples withmolecular barcodes to maximize data output per run.To learn more, visit www.454.com Figure 1: The GS Junior Sequence Capture Protocol.1. The genomic DNA sample is fragmented and polished.2. Linkers are ligated to the fragments.3. The sample is hybridized to a library of DNA capture probes.4. Unbound fragments are washed away.5. The captured genomic DNA (bound to beads) is used directly as PCR template.6. The amplified, enriched sample is ready for high-throughput sequencing withthe GS Junior Instrument.For life science research only.Not for use in diagnostic procedures.454, 454 SEQUENCING, NIMBLEGEN, and GS JUNIOR are trademarks of Roche.© 2011 Roche Diagnostics. All rights reserved.Roche Diagnostics CorporationRoche Applied ScienceIndianapolis, Indiana

AwardsABRF Annual Award for Outstanding Contributions to Biomolecular TechnologiesPresentation & LectureSponsored by Agilent TechnologiesMonday, February 21Level 1, Exhibit Ballroom C3:15 pm – 4:15 pmAwardsThe ABRF Executive Board is pleased to announce that therecipient of the 2011 ABRF Annual Award for OutstandingContributions to Biomolecular Technologies is Sir Alec JohnJeffreys.Biological research is driven by technology.As new instruments and chemistries areconceived and implemented, new frontiersare created and new possibilities emerge.Technology, from the invention of thelight microscope to the deciphering ofthe Human Genome, opens doors intothe unknown that otherwise remain firmlysealed. The ABRF Award recognizes those pioneers responsiblefor developing these powerful new tools that serve as thefoundation of the modern biological research enterprise. TheABRF Award is sponsored by Agilent Technologies and ispresented at the annual ABRF meeting.About Sir Alec John JeffreysProf. Sir Alec Jeffreys studied biochemistry and genetics atMerton College, Oxford. He then began an EMBO PostdoctoralFellowship at the University of Amsterdam where, with Dr.Richard Flavell, he was one of the first to discover split genes. Hemoved in 1977 to the Department of Genetics at the Universityof Leicester where he currently holds the positions of Professorof Genetics and Royal Society Wolfson Research Professor.Sir Alec’s research at Leicester has focused on exploring humanDNA variation and the mutation processes that create thisdiversity. He was one of the first to discover inherited variationin human DNA, then went on to invent DNA fingerprinting,showing how it could be used to resolve issues of identityPast ABRF Award Winners1994 Frederick Sanger1995 Klaus Bieman1996 David Lipman1997 Lloyd Smith1998 Bruce Merrifield1999 Marvin Caruthers2000 Leroy Hood2001 Csaba Horváth2002 John Fenn2003 Franz Hillenkamp and Michael Karas2004 Edwin Southern2005 Stephen Fodor2006 Roger Tsien*2007 Don Hunt2008 Ruedi Abersold2009 Mathias Uhlen2010 Patrick O. Brown*also won the 2008 Nobel Prize in Chemistryand kinship. His current work concentrates on developingnew approaches to analyzing variation and mutation in humanchromosomes.Sir Alec’s work has received widespread recognition, includinghis election to the Royal Society in 1986 and a Knighthood forservices to genetics in 1994. Other awards include the Louis-Jeantet Prize for Medicine (2004), the Lasker Award (2005) andthe Heineken Prize (2006). He was also one of the four finalistsfor the Millennium Prize in 2008.12 • ABRF 2011 — Technologies to Enable Personalized Medicine

ABRF Outstanding Scientist/Technologist Travel AwardsRecipients of the ABRF Travel Awards are those ABRF members who have made outstanding contributions in their institutional corefacilities; have developed new biotechnologies with applications in core facilities; and/or have been active in ABRF activities.The 2011 awardees are:Pamela “Scottie” AdamsTrudeau InstituteSaranac Lake, NY, United StatesTaslimarif SaiyedCentre for Cellular and Molecular Platforms,NCBS-TIFRBangalore, IndiaElke Küster-SchöckProteomics and Genomics Coordinator,Microscopy AssociateCIAN (Cell Imaging and Analysis Network),McGill UniversityMontreal, QC, CanadaLaurence SandbergLoma Lind UniversityLoma Linda, CA, United StatesAwardsAnoja PereraStowers Institute for Medical ResearchKansas City, MO, United StatesToni WhistlerChronic Viral Diseases BranchCenters for Disease Control and PreventionAtlanta, GA, United StatesMichelle CiliaUnited States Department of Agriculture,Robert W. Holley Center for Agricultureand HealthIthaca, NY, United StatesABRF 2011 Travel Award Selection Committee:Michelle M. Detwiler (Chair), Roswell Park CancerInstituteDebbie Adam, University of British ColumbiaDr. Richard T. Pon, University of CalgaryDr. Satya P. Yadav, Cleveland Clinic FoundationResearch InstituteABRF 2011 — Technologies to Enable Personalized Medicine • 13

ABRF Robert A. Welch Outstanding ResearchGroup / Committee Member of the Year AwardThe ABRF Robert A. Welch OutstandingResearch Group/Committee Member ofthe Year Award recognizes the significantcontributions of members of the ABRFResearch Groups and Committees. The2011 award will be presented to JeffKowalak.The Journal of Biomolecular Techniques ( JBT)AwardThis annual award recognizes the best research article publishedeach year in the Journal of Biomolecular Techniques, the ABRFsociety publication. This year’s JBT award recipient is:The Comparison of Different Pre- and Post-Analysis Filters for Determination of Exon-LevelAlternative Splicing Events Using AffymetrixArraysAwardsABRF Lifetime Membership AwardThis award is given by the Executive Board to members withlongstanding contributions to the success of ABRF and whohave been keystones to its missions. The 2011 Award will bepresented to Lowell Ericsson.ABRF Poster AwardsSponsored by Waters CorporationWaters Corporation sponsors prizes for the four best researchposters presented at ABRF 2011. Eligible posters focus onthe latest scientific research results enabled by advanced lifesciences technologies, methods and software tools that facilitateapplications, and the latest technology developments in thebiotechnology field. This year ABRF Education Committeereviewed all poster abstracts submitted to the meeting andselected 11 semifinalists for award consideration.Toni Whistler, Cheng-Feng Chiang, Jin-Mann Lin,William Lonergan, and William C. ReevesChronic Viral Diseases Branch, Division of Viral andRickettsial Diseases, Centers for Disease Control andPrevention, Atlanta, GeorgiaPublished in the Journal of Biomolecular Techniques, 2010 April;21(1)FASEB MARC (Minority Access to ResearchCareers) Travel AwardsThe FASEB MARC Program sponsors ABRF 2011 Travel Awardsto help support the participation of full-time faculty/mentors,postdoctoral fellows, and students from minority institutionsand historically black colleges and universities.The 2011 Faculty/Mentors and Students Travel awardeesare:This year’s semifinalists are:• Mark Turner, Proteome Software, Inc. (Poster 137)• Christine Vogel, New York University (Poster 143)• Jenny Chuu, Life Technologies (Poster 149)• Jin Jen, Mayo Clinic (Poster 161)• Shripa Patel, Stanford University (Poster 169)• Ilene Magpiong, Mount Holyoke College (Poster 193)• Anand Narayanan, West Virginia University (Poster 197)• J. Will Thompson, Duke Institute for Genome Sciences andPolicy (Poster 199)• Stefan Duhr, University Munich, Schellingstrasse (Poster201)• Keith Fadgen, Waters Corporation (Poster 227)• Emily Freeman, Harvard School of Public Health (Poster231)The semifinalists are invited to give a short (5 minute) presentationof their research poster in front of a panel of judges on Sunday,February 20. The authors of the four top-scoring posters willpresent their work on Tuesday, February 22 during the WatersCorporation-sponsored Poster Award Session, 11:00 am – 12:00pm, Level 2, Grand Oaks Ballroom, Exhibit Hall, Demo Stage.Semifinalists are designated by ** next to their poster numberwithin the Poster Session Abstract Listing.• Dr. Natalia Vinas (Faculty), Jackson State University• Christina Bernard (Undergraduate Student), Jackson StateUniversity• Charnia Hall (Undergraduate Student), Jackson StateUniversityThe recipients of the 2011 Poster Presenter Travel Awardare:• Ilene Magpiong, Mount Holyoke College• Thurman Young, Langston University14 • ABRF 2011 — Technologies to Enable Personalized Medicine

The most accuratenext-gen sequencingtechnology available.Every Illumina sequencer is powered by TruSeq—the technology that delivers the most accuratehuman genome at any coverage. TruSeqproduces the highest yield of error-free reads.The most bases over Q30. The greatest numberof peer-reviewed publications—more than 1,400in the past four years.That’s Tru data quality.Get the proof. Go towww.illumina.com/TruSeqHiSeq 2000HiSeq 1000 HiScan SQGenome Analyzer IIx MiSeq Visit us at ABRF Booth #515Illumina Technology WorkshopSunday, February 20, 201112:00 p.m. – 1:30 p.m.Grand Oaks Ballroom: Rooms P-Q (Level 2)Complimentary lunch will be provided.

Program-at-a-GlanceProgram-at-a-GlanceTIME SATURDAY, FEBRUARY 19 SUNDAY, FEBRUARY 20 MONDAY, FEBRUARY 21 TUESDAY, FEBRUARY 227:00 am – 6:00 pmRegistration Open Registration OpenRegistration OpenRegistration Open7:00 am – 7:45 amContinential BreakfastContinential BreakfastContinential Breakfast7:45 am – 8:00 am8:00 am – 8:50 amSatellite EducationalWorkshops(8:00 am – 4:30 pm)(SW1) Protein Purificationfor Mass SpectrometryOpening Remarks Outstanding Scientist/Technologist Award Journal of Biomolecular Techniques AwardPlenary Session: Unlocking Biomarker Discovery:Unbiased Human Proteomics at High Scale,Sensitivity, and Accuracy — Larry Gold, Chairman,CEO of SomaLogicPlenary Session: En Route to the Era of GenomicMedicine — Eric Green, Director, National HumanGenome Research InstitutePlenary Session: Development by Selventa of aTherapeutic Diagnostic TM Stratification Biomarkerfor Drug Response in Ulcerative Colitis — David deGraaf, President & CEO, Selventa9:00 am – 10:30 am10:00 am – 6:30 pm10:30 am – 12:00 pm10:30 am – 11:30 am12:00 pm – 1:30 pm1:30 pm – 3:00 pm3:00 pm – 4:15 pm4:15 pm – 4:45 pm(SW2) An Introduction toMetabolomics(SW3) Next GenerationSequencingConsiderations for CoreProfessionals(SW4) Lean Managementin Core FacilitiesConcurrent Scientific SessionsS1 - Improving Human HealthS2 - Integration of Bioinformatics for Genomicsand Proteomics DataS3 - Single-cell Environmental GenomicsPoster Session I (Odd Numbered Posters)Coffee and Tea in Exhibit Hall (10:30 am – 11:00 am)Career Fair Presentation (11:30 am – 11:45 am)Meet the Speaker: Larry GoldLunch in Exhibit HallVendor Presentation in Meeting RoomsConcurrent Research Group PresentationsR1 - Joint Session: iPRG & sPRGR2 - Antibody Technology (ARG)R3 - Joint Session: DSRG & GVRGCoffee and Tea in Exhibit HallConcurrent Scientific SessionsS4 - EpigeneticsS5 - High Throughput Genome CentersS6 - Deep Mining of Complex Protein MixturesPoster Session II (Even Numbered Posters)Coffee and Tea in Exhibit HallMeet the Speaker: Eric GreenLunch in Exhibit Hall (sponsored by )Demo Stage Presentations in Exhibit HallVendor Presentation in Meeting RoomsConcurrent Research Group PresentationsR4 - Joint Session: PSRG & gPRGR5 - Joint Session: MARG & NARGW7 - Cellular 3D ImagingCoffee and Tea in Exhibit HallConcurrent Scientific SessionsS7 - NGS Technologies on the HorizonS9 - Proteomics StandardizationExhibit Hall Open Exhibit Hall Open Exhibit Hall Open (10:00 am – 2:00 pm)Poster Session IIIStatus of the NCRR (10:30 am – 11:00 am)Waters Poster Awards (11:00 am – 12:00 pm)Coffee and Tea in Exhibit HallMeet the Speaker: David de GraafLunch in Exhibit HallDemo Stage Presentations in Exhibit HallVendor Presentation in Meeting RoomsConcurrent Research Group PresentationsR6 - Joint Session: PERG & MIRGR7 - Light Microscopy (LMRG)R8 - Joint Session: PRG & MRGFriday, February 18th – Registration Open (4:00 – 6:00 pm)Concurrent WorkshopsW1 - The Diagnostics Core FacilityW2 - Database Searching in ProteomicsW3 - Protein TurnoverABRF Award Lecture: Sir Alec John Jeffreys(sponsored by Agilent Technologies)3:15 pm – 4:15 pmConcurrent WorkshopsW11 - Business of Running a Core FacilityW12 - Microarrays: Still AliveW15b - ETD Workshop Seminar (Part II)Coffee and Tea in Exhibit Hall4:45 pm – 6:00 pm6:00 pm –7:00 pmOpening Plenary Session:Raju KucherlapatiPaul C. Cabot Professor,Harvard Medical SchoolDepartment of Genetics(6:00 pm – 7:00 pm)Concurrent WorkshopsW4 - Protein Expression at Industrial ScaleW5 - Proteomics Tips & TricksW6 - Capillary ElectrophoresisABRF Affiliates and Chapters Open Mic Session(6:00 pm – 6:45 pm)Concurrent WorkshopsW8 - Production of Functional ProteinsW9 - Therapeutic AntibodiesW10 - Drug Targets from Network MiningCAC Hosted ABRF-Vendor Interface MeetingConcurrent WorkshopsW13 - Institutional Core ManagementW14 - NGS Software for Data ManagementW15b - ETD Workshop Seminar (Part II) (continued)ABRF Members Meeting7:00 pm – 11:00 pmOpening Reception7:00 pm – 9:00 pmNetworking Night at the Ranch(sponsored by )7:30 pm – 10:30 pmVendor Presentations in Meeting Rooms(7:30 pm – 9:00 pm)CAN-CC Meeting(7:00 pm – 8:00 pm)Closing Reception7:00 pm – 9:00 pmW15a - ETD Workshop Seminar (Part I)(8:00 pm – 11:00 pm)16 • ABRF 2011 — Technologies to Enable Personalized Medicine

Daily ProgramFriday, February 184:00 pm – 6:00 pm Registration Open — Level 2, Ballroom Level Registration DeskSaturday, February 197:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk7:00 am – 8:00 am Continental Breakfast — Level 2, Grand Oaks Ballroom P/Q8:00 am – 4:30 pm Satellite Educational WorkshopsSee page 28 for more detailed information.(SW1) Protein Purification for Mass SpectrometryLevel 2, Grand Oaks Ballroom R(SW2) An Introduction to MetabolomicsLevel 2, Grand Oaks Ballroom S(SW3) Next Generation Sequencing Considerations for Core ProfessionalsLevel 2, Grand Oaks Ballroom NDaily Program(SW4) Lean Management in Core FacilitiesLevel 2, Grand Oaks Ballroom O6:00 pm – 7:00 pm Opening Plenary Session — Level 1, Exhibit Ballroom CPersonalized Medicine: Opportunities and ChallengesRaju Kucherlapati, Paul C. Cabot Professor, Harvard Medical School Department of Genetics and Professor, Department ofMedicine, Brigham and Women’s Hospital7:00 pm – 9:00 pm Opening Reception — Level 1, Exhibit Ballroom FoyerSunday, February 207:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk7:00 am – 7:45 am Continental Breakfast — Level 1, Exhibit Ballroom Foyer7:45 am – 8:50 am Plenary Session — Level 1, Exhibit Ballroom CUnlocking Biomarker Discovery: Unbiased Human Proteomics at High Scale, Sensitivity, and AccuracyLarry Gold, Chairman, Founder, & CEO of SomaLogic, Inc.9:00 am – 10:30 am Concurrent Scientific Sessions(S1) Improving Human Health from the Ground UpLevel 2, Grand Oaks Ballroom R/SSession Organizer: Theodore W. Thannhauser, United States Department of Agriculture/Agricultural Research Service(S1-1) Vaccine and Therapeutic Protein Manufacture in PlantsTerence E. Ryan, iBio, Inc.(S1-2) Improving Human Nutrition from the Ground Up: Linking Agriculture to Human HealthRoss M. Welch, Department of Food Science, Cornell UniversityABRF 2011 — Technologies to Enable Personalized Medicine • 17

Sunday, February 20 (Continued)(S1-3) A Food Systems Approach to Address Poor Nutritional Health in Both Advanced and DevelopingEconomiesRobin D. Graham, Flinders University of South Australia(S2) Integration of Bioinformatics for Genomics and Proteomics DataLevel 2, Grand Oaks Ballroom P/QSession Organizer: Cathy Wu, Center for Bioinformatics & Computational Biology, University of Delaware(S2-1) Integrative Bioinformatics for Genomics and ProteomicsCathy Wu, Center for Bioinformatics & Computational Biology, University of Delaware(S2-2) Integrated Bioinformatics for MS-Based ProteomicsEric Deutsch, Institute for Systems Biology(S2-3) Skate Genome Project: Cyber-Enabled Bioinformatics CollaborationJames Vincent, University of Vermont(S3) Single-Cell Environmental GenomicsLevel 2, Grand Oaks Ballroom N/OSession Organizer: Rob Knight, University of ColoradoDaily Program(S3-1) Early Elements of a Pipeline for Single Bacterial Cell GenomicsDave Relman, Departments of Microbiology & Immunology, and of Medicine, Stanford University(S3-2) Assembling Complete Genomes from Complex MixturesFolker Meyer, Argonne National Laboratory, Mathematics and Computer Science Division(S3-3) Dining in with Trillions of Fascinating Friends: Exploring our Human Gut Microbiome in Healthand DiseaseJeff Gordon, Center for Genome Sciences and Systems Biology, Washington University School of Medicine10:00 am – 6:30 pm Exhibit Hall Open — Level 2, Grand Oaks Ballroom10:30 am – 11:00 am Morning Refreshment Break — Level 2, Grand Oaks Ballroom, Exhibit Hall10:30 am – 12:00 pm Poster Session I (Odd Numbered Posters) — Level 2, Grand Oaks Ballroom, Exhibit Hall10:30 am – 11:30 am Meet the Speaker — Level 2, Grand Oaks Ballroom, Exhibit Hall, Demo StageLarry Gold, Chairman, Founder, & CEO of SomaLogic, Inc.11:30 am – 11:45 am Career Fair Presentation — Level 2, Grand Oaks Ballroom, Exhibit Hall, Demo Stage12:00 pm – 1:30 pm Munch & Mingle — Level 2, Grand Oaks Ballroom, Exhibit Hall12:00 pm – 1:30 pm Vendor PresentationsSee page 120 for detailed information.1:30 pm – 3:00 pm Concurrent Research Group Presentations(R1) Joint Session: Proteome Informatics Research Group (iPRG) & Proteomics Standards Research Group(sPRG)Level 2, Grand Oaks Ballroom R/S(R1a) iPRG 2011: A Study on the Identification of Electron Transfer Dissociation (ETD) Mass SpectraSession Organizer & Presenter: Lennart Martens, Ghent University(R1b) ABRF-sPRG2011 Study: Development of a Comprehensive Standard for Analysis of Post-Translational ModificationsSession Organizer & Presenter: Alexander R. Ivanov, Harvard School of Public Health18 • ABRF 2011 — Technologies to Enable Personalized Medicine

(R2) Antibody Technology Research Group (ARG): Antibodies — Moving Closer to Personalized Therapeuticsand DiagnosticsLevel 2, Grand Oaks Ballroom P/QSession Organizers: Robert Carnahan, Vanderbilt University Medical Center, and Frances Weis-Garcia, Memorial Sloan-Kettering Cancer CenterPresenters: Brian Curtis, Blood Center of Wisconsin, John Harlan, Abbott Laboratories, and Robert Umek, Meso ScaleDiscovery(R3) Joint Session: Genomic Variation Research Group (GVRG) & DNA Sequencing Research Group (DSRG)Level 2, Grand Oaks Ballroom N/O(R3a): Evaluation of DNA Whole Genome Amplification Technologies for GenotypingSession Organizers & Presenters: Casey Dagnall and Amy Hutchinson, Core Genotyping Facility, SAIC-Frederick,Inc., NCI-Frederick(R3b): DNA Sequencing Research Group (DSRG)Session Organizer: Deborah Grove, Pennsylvania State University(R3b-1) Comparison of Custom Target Enrichment Methods; Agilent vs. NimblegenAnoja Perera, Stowers Institute for Medical Research Molecular Biology Facility(R3b-2) A Methodology Study for Metagenomics Using Next Generation SequencersSushmita Singh, BioMedical Genomics Center, University of Minnesota3:00 pm – 4:15 pm Concurrent Workshop Sessions(W1) The Diagnostics Core Facility: Harvesting the Promise of Personalized MedicineLevel 2, Grand Oaks Ballroom R/SSession Organizer: Katia Sol-Church, Nemours Biomedical Research, A.I. duPont Hospital for ChildrenDaily Program(W1-1) Win on Sunday, Sell on Monday: From the Exome Sequencing of One Boy to the Delivery ofClinical DiagnosticsMichael R. Tschannen, Human and Molecular Genetics Center, Department of Physiology, Medical College ofWisconsin(W1-2) Chromosomal Microarray Analysis in the ClinicLisa D. White, Microarray Core, Baylor College of Medicine(W1-3) Whole Genome Sequencing in the Clinical LaboratoryTina Hambuch, Illumina Clinical Services Laboratory, Illumina, Inc.(W2) Spectral and Sequence Database Searching in ProteomicsLevel 2, Grand Oaks Ballroom P/QSession Organizer: Lennart Martens, Ghent University(W2-1) ETD Performance and Complementarity to Other Fragmentation Methods for ProteomicAnalysisRobert Chalkley, University of California San Francisco(W2-2) Discovery, Identification and Localization of Post-Translational ModificationsNuno Bandeira, Center for Computational Mass Spectrometry, Department of Computer Science and EngineeringSkaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego(W2-3) Building and Using MS/MS Spectral Libraries for Peptide Identifications in ProteomicsHenry Lam, The Hong Kong University of Science and Technology(W3) Quantifying Protein Turnover by In Vivo Metabolic LabelingLevel 2, Grand Oaks Ballroom N/OSession Organizer: Kieran Geoghegan, Pfizer, Inc.ABRF 2011 — Technologies to Enable Personalized Medicine • 19

Sunday, February 20 (Continued)(W3-1) Stable Isotope Tracers Applied to Measuring Rates of Protein Synthesis and Breakdown inMuscle: Principles and ApplicationsRobert R. Wolfe, University of Arkansas Medical Sciences(W3-2) In Vivo Stable Isotope Labeling for Quantifying Amyloid-Beta Kinetics in Alzheimer’s Disease:Is it All in our Head?Kevin E. Yarasheski, Biomedical Mass Spectrometry Research Laboratory, Washington University School of Medicine4:15 pm – 4:45 pm Afternoon Refreshment Break — Level 2, Grand Oaks Ballroom, Exhibit Hall4:45 pm – 6:00 pm Concurrent Workshop Sessions(W4) Insights for Expression of Recombinant Proteins for Drug Target ValidationLevel 2, Grand Oaks Ballroom R/SSession Organizer: Francis Rajamohan, Pfizer, Inc.(W4-1) New Developments in BacMam VectorsFrederick M. Boyce, Massachusetts General Hospital, Department of Neurology(W4-2) Strategies for Optimized High-Throughput Cloning, Expression, and Purification ofRecombinant ProteinsRebecca Page, Brown University, Department of Molecular Biology, Cell Biology and Biochemistry, Center forGenetics, Genomics and ProteomicsDaily Program(W5) Proteomics Tips and Tricks: From Discovery to Protein-Protein InteractionsLevel 2, Grand Oaks Ballroom P/QSession Organizer: Michelle Cilia, United States Department of Agriculture/Agricultural Research Service, Robert W. HolleyCenter for Agriculture and Health(W5-1) Proteomics Tips and Tricks: From Discovery to Protein-Protein InteractionsMichelle Cilia, United States Department of Agriculture/Agricultural Research Service, Robert W. Holley Center forAgriculture and Health(W5-2) Protein Interactions and Topologies in CellsJames Bruce, University of Washington, Department of Genome Sciences(W5-3) The Virus-Host Interface: Exploring Dynamic Protein Interactions via Targeted ProteomicsIleana Cristea, Princeton University, Department of Molecular Biology(W5-4) Towards the Development of Proteomics Workflows for the Analysis of Samples Derived fromRefractory Plant TissuesTheodore W. Thannhauser, United States Department of Agriculture/Agricultural Research Service(W6) Current State and Future of Capillary Electrophoresis and Sanger SequencingLevel 2, Grand Oaks Ballroom N/OSession Organizer: Jan Kieleczawa, Pfizer, Inc.Presenters: Debbie Adam, NAPS Unit, Michael Smith Laboratories, University of British Columbia, Jan Kieleczawa, Pfizer,Inc., Peter Schweitzer, Cornell University, Eric Vennemeyer, LifeTechnologies, and Michael Zianni, Plant-Microbe GenomicsFacility, Ohio State University6:00 pm – 6:45 pm ABRF Affiliates and Chapters Open Mic Session — Level 2, Grand Oaks Ballroom N/O7:30 pm – 10:30 pm Networking Night — Offsite: Leon Springs Dance Hall*(Sponsored by )*Buses will begin boarding at 7:00 pm from the Level 1, Exhibit Hall Lobby. Buses will run continuously between 7:00 pmand 10:30 pm.20 • ABRF 2011 — Technologies to Enable Personalized Medicine

Monday, February 217:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk7:00 am – 7:45 am Continental Breakfast — Level 1, Exhibit Ballroom Foyer7:45 am – 8:50 am Plenary Session — Level 1, Exhibit Ballroom CEn Route to the Era of Genomic MedicineEric Green, Director, National Human Genome Research Institute9:00 am – 10:30 am Concurrent Scientific Sessions(S4) EpigeneticsLevel 2, Grand Oaks Ballroom R/SSession Organizer: Benjamin A. Garcia, Princeton University(S4-1) Next Generation Quantitative Proteomic Tools for Analyzing Histone ModificationsBenjamin A. Garcia, Princeton University(S4-2) Chromatin Dynamics in Melanoma: A Role for MacroH2AAvnish Kapoor, Department of Oncological Sciences, Mount Sinai School of Medicine(S4-3) Using Protein Domain Microarrays to Read the Histone CodeMark Bedford, Department of Carcinogenesis, Science Park, Research Division, The University of Texas MD AndersonCancer Center(S5) High-Throughput Genome CentersLevel 2, Grand Oaks Ballroom P/QSession Organizer: George Grills, Cornell UniversityDaily Program(S5-1) Overview of the Illumina Sequencing Platform at the Broad InstituteKristen Connolly, Process & Technology Development, Genome Sequencing Platform, The Broad Institute of MIT andHarvard(S5-2) Science and Technology at a High-Throughput Genome CenterLucinda Fulton, The Genome Center at Washington University School of Medicine(S5-3) High-Throughput Next Generation Sequencing Methods and ApplicationsDonna Muzny, Baylor College of Medicine, Human Genome Sequencing Center(S6) Strategies for Deep Mining of Complex Protein MixturesLevel 2, Grand Oaks Ballroom N/OSession Organizer: David Speicher, The Wistar Institute(S6-1) Coverage and Recovery of Upstream Protein Fractionation Methods in LC-MS/MS WorkflowsLeonard J. Foster, Centre for High-Throughput Biology, The University of British Columbia(S6-2) Improving the Comprehensiveness of Large-Scale Proteomics Experiments Using AdvancedComputational Tools and Accurate Multiple Hypothesis Testing StatisticsMichael J. MacCoss, Department of Genome Sciences, University of Washington(S6-3) In-Depth Analysis of Human and Mouse Plasma Using 3-D and 4-D Fractionation StrategiesDavid Speicher, The Wistar Institute10:00 am – 6:30 pm Exhibit Hall Open — Level 2, Grand Oaks Ballroom10:30 am – 11:00 am Morning Refreshment Break — Level 2, Grand Oaks Ballroom, Exhibit Hall10:30 am – 12:00 pm Poster Session II — Level 2, Grand Oaks Ballroom, Exhibit Hall10:30 am – 11:30 am Meet the Speaker — Level 2, Grand Oaks Ballroom, Exhibit Hall, Demo StageEric Green, Director, National Human Genome Research Institute12:00 pm – 1:30 pm Munch & Mingle — Level 2, Grand Oaks Ballroom, Exhibit Hall (Sponsored by )ABRF 2011 — Technologies to Enable Personalized Medicine • 21

Monday, February 21 (Continued)12:00 pm – 1:30 pm Vendor and Demo Stage PresentationsSee pages 120 and 126 for detailed information.1:30 pm – 3:00 pm Concurrent Research Group Presentations(R4) Joint Session: Protein Sequencing Research Group (PSRG) & Glycoprotein Research Group (gPRG)Level 2, Grand Oaks Ballroom R/S(R4a) PSRG 2011 Study: Sensitivity Assessment for Terminal Sequencing Techniques Using an UnknownProteinSession Organizer: Jim Walters, Sigma AldrichPresenters: Kwasi Mawuenyega, Washington University School of Medicine, Wendy Sandoval, Genentech, Inc., SteveSmith, University of Texas, and Jim Walters, Sigma Aldrich(R4b) gPRG: Toward Consensus on Glycan Analysis: Reliable Methods and ReproducibilitySession Organizer & Presenter: Joseph Zaia, Boston University(R5) Joint Session: Nucleic Acids Research Group (NARG) & Microarray Research Group (MARG)Level 2, Grand Oaks Ballroom P/QDaily Program(R5a) Determining miRNA Expression Levels in Degraded RNA Samples Using Real-Time RT-qPCR andMicroarray TechnologiesSession Organizer: Sridar Chittur, State University of New York at AlbanyPresenters: Sridar Chittur, State University of New York at Albany, and Scott Tighe, University of Vermont(R5b) Microarray Research Group Projects, 2010-11Session Organizer: Don A. Baldwin, Molecular Profiling Facility, University of PennsylvaniaPresenters: Nadereh Jafari, Northwestern University, and Natalia Reyero-Vinas, Jackson State University(W7) Cellular 3D Imaging*Level 2, Grand Oaks Ballroom N/OSession Organizer: Richard Cole, Wadsworth Center, New York State Department of Health(W7-1) 3D Cellular Imaging: Beyond the Simple Imaging ParadigmRichard Cole, Wadsworth Center, New York State Department of Health(W7-2) Basics of Colocalization AnalysesJudith Lacoste, Cell Imaging and Analysis Network, Department of Biology, McGill University*A Leica confocal microscope, provided by Leica Microsystems, will be featured in this session in Grand Oaks B.Leica Scanner — Dedicated to advancing biomolecular core facilitiesThe ABRF will host a Leica SP5 II confocal equipped with a tandem scanner. This Confocal covers a broad range ofrequirements in confocal imaging — with the full array of scan speeds at highest resolution. With its high-efficiency SPdetection (five channels simultaneously) and the optional AOBS (Acousto-Optical Beam Splitter), the system deliversbright, noise-free images with minimal photo damage at high speed. Additionally, there will be available the latestAutoquant’s advanced image deconvolution and 3D visualization software for life science researchers.Imaging sessions include:• Co-registration & deconvolution “hands-on”• Confocal microscope & deconvolution software on-site• Opportunity to image and analyze “your” sample (physical or digital file from your scope)• Performance testing: point spread functions, spectral calibration & beyond• Core management session/round tables• Networking for imaging core personnel3:15 pm – 4:15 pm ABRF Award Lecture: Sir Alec John Jeffreys — Level 1, Exhibit Ballroom C(Sponsored by Agilent Technologies.)22 • ABRF 2011 — Technologies to Enable Personalized Medicine

4:15 pm – 4:45 pm Afternoon Refreshment Break — Level 2, Grand Oaks Ballroom, Exhibit Hall4:45 pm – 6:00 pm Concurrent Workshop Sessions(W8) Successful Production of Functional ProteinsLevel 2, Grand Oaks Ballroom R/SSession Organizer: Jeff Culp, Pfizer, Inc.(W8-1) Successful Protein ProductionJeff Culp, Pfizer, Inc.(W8-2) Overcoming Problems in Protein Expression and PurificationBill Gillette, SAIC-Frederick, Inc., National Cancer Institute at Frederick(W8-3) When Proteins Misbehave, Try Adding a Little PressureRobert M. Petrovich, National Institute of Environmental Health Sciences Laboratory of Structural Biology(W9) Therapeutic Antibodies: Over-Hyped “Magic Bullet”, or Under-Explored Technology?Level 2, Grand Oaks Ballroom P/QSession Organizer: Dan L. Crimmins, Washington University School of MedicinePresenters: Dan L. Crimmins, Washington University School of Medicine, John Harlan, Abbott Laboratories, and FrancesWeis-Garcia, Memorial Sloan-Kettering Cancer Center(W10) Identification of Mechanism-Based Biomarkers and Drug Targets Using Pathway AnalysisLevel 2, Grand Oaks Ballroom N/OSession Organizer: Alexander Kel, geneXplain GmbH(W10-1) GeneXplain — Identification of Causal Biomarkers and Drug Targets in PersonalizedCancer PathwaysAlexander Kel, geneXplain GmbHDaily Program(W10-2) Pathway Analysis in Expression ProteomicsRoman Zubarev, Chemistry I Division, Department of Medical Biochemistry and Biophysics, Karolinska Institute(W10-3) Metabolic Biomarkers, Metabolic Networks, and Pathway AnalysisVladimir Tolstikov, University of California Davis Genome Center6:00 pm – 7:00 pm CAC Hosted ABRF-Vendor Interface Meeting — Level 1, Exhibit Ballroom C7:00 pm – 8:00 pm Core Administrators Network – Coordinating Committee (CAN-CC) Meeting — Level 1, Exhibit Ballroom C7:30 pm – 9:00 pm Vendor PresentationsSee page 120 for detailed information.8:00 pm – 11:00 pm (W15a) ETD Workshop Seminar Series PART I: Manual Interpretation of Electron Transfer Dissociation (ETD)Mass Spectra of Peptides* — Level 2, Grand Oaks Ballroom N/OPresenter: Donald F. Hunt, Departments of Chemistry and Pathology, University of VirginiaTuesday, February 22*Attendees must be registered for ABRF 2011 to attend the workshop and must also complete the ETD Workshop SeminarForm. Please visit the Registration Desk to sign up for the session.7:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk7:00 am – 7:45 am Continental Breakfast — Level 1, Exhibit Ballroom Foyer7:45 am – 8:50 am Plenary Session — Level 1, Exhibit Ballroom CDevelopment by Selventa of a Therapeutic Diagnosticin Ulcerative ColitisDavid de Graaf, President & CEO, SelventaTMStratification Biomarker for Drug ResponseABRF 2011 — Technologies to Enable Personalized Medicine • 23

Tuesday, February 22 (Continued)9:00 am – 10:30 am Concurrent Scientific Sessions(S7) Next Generation Sequencing Technologies on the HorizonLevel 2, Grand Oaks Ballroom R/SSession Organizer: Katia Sol-Church, Nemours Biomedical Research, A.I. duPont Hospital for Children(S7-1) Towards Optical DNA Sequencing Using Nanopore ArraysAmit Meller, Department of Biomedical Engineering & Department of Physics, Boston University(S7-2) Single Molecule Real-Time DNA Sequencing on the Surface of a Quantum-Dot NanocrystalPeter B. Vander Horn, Genetic Systems, Life Technologies(S7-3) Sequencing with Semiconductor ChipsGlenn Powell, Ion Torrent(S9) Proteomics StandardizationLevel 2, Grand Oaks Ballroom N/OSession Organizer: David B. Friedman, Proteomics Laboratory Mass Spectrometry Research Center, Vanderbilt University(S9-1) A Multi-Laboratory Study Assessing Robustness and Reproducibility of Plasma ReferenceSample for Benchmarking LC-MS Platform PerformanceJuan-Pablo Albar, ProteoRed, National Center for Biotechnology-CSICDaily Program(S9-2) Clinical Proteomic Technologies for Cancer: NIH-Funded Measurement ScienceChristopher Kinsinger, National Cancer Institute, Center for Strategic Scientific Initiatives, Office of Cancer ClinicalProteomics Research(S9-3) Why Reproducible Outcomes are Essential in Proteomic Research and WhyStandardization of Processes is Essential for Achieving ReproducibilityAndy Borthwick, Nonlinear Dynamics Limited10:00 am – 2:00 pm Exhibit Hall Open — Level 2, Grand Oaks Ballroom10:30 am – 11:00 am Morning Refreshment Break — Level 2, Grand Oaks Ballroom, Exhibit Hall10:30 am – 12:00 pm Poster Session III — Level 2, Grand Oaks Ballroom, Exhibit Hall10:30 am – 11:00 am Status of the NCRR — Level 2, Grand Oaks Ballroom P/QPresenter: Mark O. Lively, Wake Forest University School of Medicine11:00 am – 12:00 pm Poster Awards (Sponsored by Waters Corporation) — Level 2, Grand Oaks Ballroom, Exhibit Hall, Demo Stage10:30 am – 11:30 am Meet the Speaker — Level 2, Grand Oaks Ballroom, Exhibit HallDavid de Graaf, President & CEO, Selventa12:00 pm – 1:30 pm Munch & Mingle — Level 2, Grand Oaks Ballroom, Exhibit Hall12:00 pm – 1:30 pm Vendor and Demo Stage PresentationsSee pages 120 and 126 for detailed information.1:30 pm – 3:00 pm Concurrent Research Group Presentations(R6) Joint Session: Protein Expression Research Group (PERG) & Molecular Interactions Research Group (MIRG)Level 2, Grand Oaks Ballroom R/S(R6a) PERG Research Group Presentation: Refolding StudySession Organizer: Cynthia Kinsland, Cornell University(R6b) Conclusions from the MIRG 2010 Benchmark Study: Molecular Interactions in a ThreeComponent System and Presentation of 2011 Survey Results on Label-Free TechnologiesSession Organizer: Aaron Yamiuk, Bristol-Myers SquibbPresenters: Satya Yadav, Cleveland Clinic Foundation, and Aaron Yamiuk, Bristol-Myers Squibb24 • ABRF 2011 — Technologies to Enable Personalized Medicine

(R7) Light Microscopy Research Group (LMRG)Level 2, Grand Oaks Ballroom P/QSession Organizer: Robert Stack, Wadsworth Center, New York State Department of Health(R7-1) Point Spread Functions, Spectral Calibration, and BeyondRobert Stack, Wadsworth Center, New York State Department of Health(R7-2) Deconvolution: Core Concepts, Algorithms, and Advanced IssuesBrian Northan, MediaCybernetics(R8) Joint Session: Proteomics Research Group (PRG) & Metabolomics Research Group (MRG)Level 2, Grand Oaks Ballroom N/O(R8a) PRG-2011: Defining the Interaction Between Users and Suppliers of Proteomics ServicesSession Organizer: David Hawke, The University of Texas MD Anderson Cancer Center(R8b) Metabolomics Research Group 2011 StudySession Organizer: William R. Wikoff, University of California DavisPresenters: John M. Asara, Beth Israel Deaconess Medical Center, Vladimir Tolstikov and William R. Wikoff,University of California Davis3:00 pm – 4:15 pm Concurrent Workshop Sessions(W11) The Business of Running a Core FacilityLevel 2, Grand Oaks Ballroom R/SSession Organizer: Nicholas Ambulos, University of Maryland School of MedicinePresenters: Nicholas Ambulos, University of Maryland School of Medicine, and Steve Bobin, Dartmouth School of Medicine(W12) Microarrays: The Reports of My Death Have Been Greatly ExaggeratedLevel 2, Grand Oaks Ballroom P/QSession Organizer: Don Baldwin, University of Pennsylvania, Penn Molecular Profiling FacilityDaily Program(W12-1) Chip or Seq: Helping Clients ChooseDon Baldwin, University of Pennsylvania, Penn Molecular Profiling Facility, and Kevin Knudtson, University of Iowa(W12-2) Microarray Analysis of Fluorescence Activated Cell Sorter-Derived Cells: Creating Harmonybetween TechnologiesScott Tighe, University of Vermont(W12-3) Microarray Futures: Don’t Decommission Your Scanners Just YetSeth Crosby, Department of Genetics, Washington University School of Medicine(W15b) ETD Workshop Seminar Series PART II: Manual Interpretation of Electron Transfer Dissociation (ETD)Mass Spectra of Peptides*Level 2, Grand Oaks Ballroom N/OPresenter: Donald F. Hunt, Departments of Chemistry and Pathology, University of Virginia*Attendees must be registered for ABRF 2011 to attend the workshop and must also complete the ETD Workshop SeminarForm. Please visit the Registration Desk to sign up for the session.4:15 pm – 4:45 pm Afternoon Refreshment Break — Level 2, Grand Oaks Ballroom N/S Foyer4:45 pm – 6:00 pm Concurrent Workshop Sessions(W13) Institutional Core ManagementLevel 2, Grand Oaks Ballroom R/SSession Organizers: Susan Meyn, Vanderbilt University Medical Center, and Paula Turpen, University of Nebraska MedicalCenterPresenter: Pam Alexander, Morehouse School of Medicine, Julie Auger, University of California San Francisco, GregoryFarber, National Center for Research Resources, and Sheenah Mische, New York University Langone Medical CenterABRF 2011 — Technologies to Enable Personalized Medicine • 25

Tuesday, February 22 (Continued)(W14) Next Generation Sequencing Software for Data Management, Analysis, and VisualizationLevel 2, Grand Oaks Ballroom P/QSession Organizer: Kip Bodi, Tufts University School of Medicine(W14-1) Tools for Next Generation Sequencing Data AnalysisKip Bodi, Tufts University School of Medicine(W14-2) GenomeView: Visualizing the Next Generation of DataThomas Abeel, Broad Institute of MIT and Harvard, and VIB Department of Plant Systems Biology, Ghent University(W14-3) Galaxy Next Generation Sequencing Functionality from Sample Tracking to SNP CallingGreg Von Kuster, Pennsylvania State University(W15b) ETD Workshop Seminar Series PART II: Manual Interpretation of Electron Transfer Dissociation (ETD)Mass Spectra of Peptides (continued)Level 2, Grand Oaks Ballroom N/OPresenter: Donald F. Hunt, Departments of Chemistry and Pathology, University of Virginia*Attendees must be registered for ABRF 2011 to attend the workshop and must also complete the ETD Workshop SeminarForm. Please visit the Registration Desk to sign up for the session.Daily Program6:10 pm – 7:00 pm ABRF Member’s Meeting — Level 1, Exhibit Ballroom C7:00 pm – 9:00 pm Closing Reception — JW Marriott San Antonio Hill Country Resort, Grand Ballroom Terrace26 • ABRF 2011 — Technologies to Enable Personalized Medicine

The Future Is EmergingTake an integrated view of biology, with tools from Agilent. A new frontier is emerging. Advances in technologyhave given researchers the ability to study complex biological systems beyond individual components—and Agilentis committed to helping scientists harness this integrated approach. Agilent supports the range of omics platforms,with genomic, transcriptomic, proteomic, and metabolomic applications, enabling a multi-faceted view into biologicalprocesses. Informatics tools provide analysis for each of the omics, and offer integrated data sets across the biologicalsystem. Agilent is also collaborating with scientists to overcome the challenges of this emerging discipline, helpingfi nd answers to some of our most complex questions. Get the full picture at www.agilent.com/lifesciences/biology.© Agilent Technologies, Inc. 2010

Satellite Educational Workshop Sponsors(SW1) Protein Purification for Mass Spectrometry(SW2) An Introduction to Metabolomics(SW3) Next Generation Sequencing Considerations for Core ProfessionalsSatellite EducationalWorkshops(SW4) Lean Management in Core Facilities28 • ABRF 2011 — Technologies to Enable Personalized Medicine

Satellite Educational WorkshopsAll Satellite Educational Workshops will be held on Saturday, February 19 at the JW Marriott San Antonio Hill Country Resort.Attendees must register separately for all Satellite Educational Workshops. To register, please visit the Registration Desk on Level 2.For further information and for individual workshop schedules, please review the proceeding pages.SW1: Protein Purification for Mass Spectrometry8:00 am – 4:00 pm Level 2, Grand Oaks Ballroom ROrganizers & Presenters: Robert Carnahan, and W. Hayes McDonald, Vanderbilt University Medical CenterWorkshop Description: The workshop is intended as a practical guide for investigators wishing to purify proteins and proteincomplexes for mass spectrometry based analysis. It should be useful for individuals wishing to perform such purifications as well asfor mass spectrometrists who will be analyzing these types of samples. We will explore various considerations and possibilities indesigning and executing protein purifications intended for mass spectrometry and then focus on antibody-based affinity purifications.While there will not be time to take workshop participants through all of the hands-on aspects, our intent is to guide them throughdesigning and troubleshooting the various stages of the process. We will then utilize data generated from an actual hands-on course,and allow the participants to see and interpret some real data that they have “watched” being generated.Workshop Agenda7:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk7:00 am – 8:00 am Continental Breakfast — Level 2, Grand Oaks Ballroom P/Q8:00 am – 9:00 am Affinity Purification for Mass SpectrometryPresenter: Robert Carnahan, Vanderbilt University Medical Center9:00 am – 10:00 am Mass Spectrometry Analysis of Protein ComplexesPresenter: W. Hayes McDonald, Vanderbilt University Medical Center10:00 am – 10:30 am Morning Refreshment Break — Level 2, Grand Oaks Ballroom P/Q10:30 am – 12:00 pm Virtual “Hands-On” WorkshopPresenters: W. Hayes McDonald and Robert Carnahan, Vanderbilt University Medical CenterReagent generation and QCBiological OptimizationSample Preparation and ProcessingData Analysis12:00 pm – 1:00 pm Lunch — Level 2, Grand Oaks Ballroom P/Q1:00 pm – 2:30 pm Exploration of Experimental Design Considerations and their ImplicationsPresenters: W. Hayes McDonald and Robert Carnahan, Vanderbilt University Medical Center2:30 pm – 3:00 pm Afternoon Refreshment Break — Level 2, Grand Oaks Ballroom P/Q3:00 pm – 4:00 pm Alternative ApproachesPresenters: W. Hayes McDonald and Robert Carnahan, Vanderbilt University Medical CenterSatellite EducationalWorkshopsABRF 2011 — Technologies to Enable Personalized Medicine • 29

SW2: An Introduction to Metabolomics8:00 am – 4:30 pm Level 2, Grand Oaks Ballroom SOrganizer: William R. Wikoff, University of California DavisPresenters: William R. Wikoff, University of California Davis, and Pavel Aronov, Stanford UniversityWorkshop Description: Metabolomics can be defined as the systems-level investigation of small molecules and metabolites inbiological cells, tissues, and organisms. The course will provide the scientist with an overview of the field, with an emphasis on practical,mass spectrometry-based approaches, presented in a clear scientific framework. The entire process of metabolomics, from studydesign, sample preparation and extraction, chromatography, mass spectrometry, data processing and analysis will be presented.Topics will include: types of detectors and their relative merits for specific metabolomics applications, including quadrupole, triplequad, ion trap, TOF, QTOF, and FT-ICR. Ionization sources. Application of LCMS and GCMS, including a comparison of observablecompounds in these techniques. Brief coverage of specialized instruments: GC-QQQ, GC-TOF, chemical ionization. Methods forsample extraction. Targeted versus untargeted methods. Peak integration, data alignment, and software. Data analysis approaches willbe discussed, including uni- and multi-variate statistics, with an emphasis on selecting straightforward approaches appropriate for agiven problem. Basic statistics for metabolomics: types of t-tests, multiple testing correction, False Discovery Rate, basic multivariatemethods. Approaches to biomarker discovery; metabolomics in drug discovery. Review and discussion of selected publications fromthe literature (case studies). Some familiarity with mass spectrometry and analytical chemistry is preferred, but background will beprovided.Workshop AgendaSatellite EducationalWorkshops7:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk7:00 am – 8:00 am Continental Breakfast — Level 2, Grand Oaks Ballroom P/Q8:00 am – 8:30 am Introduction and Overview with Historical BackgroundPresenter: William Wikoff, University of California Davis8:30 am – 10:00 am Instrumentation for Metabolomics: Mass Spectrometry, Mass Analyzers, Resolution and Mass Accuracy.GC/MS: Separation, Mass Spec, and LibrariesPresenter: Pavel Aronov, Stanford University10:00 am – 10:30 am Morning Refreshment Break — Level 2, Grand Oaks Ballroom P/Q10:30 am – 12:00 pm Sample Preparation: Liquid Chromatography, Columns and MethodsPresenter: William Wikoff, University of California Davis12:00 pm – 1:00 pm Lunch — Level 2, Grand Oaks Ballroom P/Q1:00 pm – 2:00 pm Data Processing: MZmine, XCMS, Databases. Compound IdentificationPresenter: Pavel Aronov, Stanford University2:00 pm – 2:30 pm Statistics Part IPresenter: William Wikoff, University of California Davis2:30 pm – 3:00 pm Afternoon Refreshment Break — Level 2, Grand Oaks Ballroom P/Q3:00 pm – 3:30 pm Statistics Part IIPresenter: William Wikoff, University of California Davis3:30 pm – 4:30 pm Metabolomics Case Studies, Summary and DiscussionPresenter: William Wikoff, University of California Davis30 • ABRF 2011 — Technologies to Enable Personalized Medicine

SW3: Next Generation Sequencing Considerations For Core Professionals8:00 am – 4:30 pm Level 2, Grand Oaks Ballroom NOrganizer: Kevin L. Knudtson, University of IowaPresenters: Deborah Grove, Pennsylvania State University, Anoja G. Perera, Stowers Institute for Medical Research, and Peter Schweitzer,Cornell UniversityWorkshop Description: This workshop is intended for the laboratory professional looking to enhance the quality of their recentlyestablished Next Generation Sequencing (NGS) services. It is not intended for the seasoned NGS service provider. The organizationof the workshop will be similar to the workflow a core professional expert might take when working with an investigator on theirnext generation sequencing project. The early sessions will focus on platform choice, experimental design, and sample preparationconsiderations. The latter sessions will focus on the performance of specific applications.Workshop Agenda7:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk7:00 am – 8:00 am Continental Breakfast — Level 2, Grand Oaks Ballroom P/Q8:00 am – 8:30 am Overview and First ContactPresenter: Kevin Knudtson, University of Iowa8:30 am – 9:15 am Providing Next Gen Services Using the GS FLXPresenter: Kevin Knudtson, University of Iowa9:15 am – 10:00 am Providing Next Gen Services using the GAIIxPresenter: Anoja Perera, Stowers Institute for Medical Research10:00 am – 10:30 am Morning Refreshment Break — Level 2, Grand Oaks Ballroom P/Q10:30 am – 11:15 am Providing Next Gen Services using the HiSeq2000Presenter: Peter Schweitzer, Cornell University11:15 am – 12:00 pm Providing Next Gen Services using the SOLiDPresenter: Deborah Grove, Pennsylvania State University12:00 pm – 1:00 pm Lunch — Level 2, Grand Oaks Ballroom P/Q1:00 pm – 1:30 pm Agilent Presentation1:30 pm – 2:30 pm Specialized Protocols for Use with Next GenPresenters: Anoja Perera, Stowers Institute for Medical Research, and Peter Schweitzer, Cornell University2:30 pm – 3:00 pm Afternoon Refreshment Break — Level 2, Grand Oaks Ballroom P/Q3:00 pm – 3:45 pm Data Management /LIMSPresenters: Deborah Grove, Pennsylvania State University, Kevin Knudtson, University of Iowa, Anoja Perera, StowersInstitute for Medical Research, and Peter Schweitzer, Cornell University3:45 pm – 4:30 pm Panel DiscussionPanelists: Deborah Grove, Pennsylvania State University, Kevin Knudtson, University of Iowa, Anoja Perera, Stowers Institutefor Medical Research, and Peter Schweitzer, Cornell UniversitySatellite EducationalWorkshopsABRF 2011 — Technologies to Enable Personalized Medicine • 31

SW4: Lean Management in Core Facilities8:00 am – 4:30 pm Level 2, Grand Oaks Ballroom OOrganizer: Tim C. Hunter, Vermont Cancer CenterPresenters: Belynda Hicks, SAIC-Frederick, National Cancer Institute at Frederick, Nicholas Ambulos, University of Maryland School ofMedicine, Val Scott, The Jackson Laboratory, Rand Haley, Huron Consulting Group, Wayne Collins, Agilent Technologies, and Tim C.Hunter, University of VermontWorkshop Description: This workshop is intended for core directors, managers, and facility personnel that are interested in learningnew and creative approaches to core management during lean budget times. Sessions will focus on ways to reduce or maintain costswhile sustaining the activity necessary to continue meeting the demands of the scientific user base. Topics include: balancing yourcore budget, non-compensatory approaches to acknowledge high performing staff and foster professional development, negotiatingservice contracts, money saving tips, and decision making strategies for in-house or outsourcing analyses.Workshop AgendaSatellite EducationalWorkshops7:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk7:00 am – 8:00 am Continental Breakfast — Level 2, Grand Oaks Ballroom P/Q8:00 am – 8:05 am Introductions and Workshop Overview8:05 am – 9:00 am Cost Savings via Cost Avoidance: Quality Management for the Core LaboratoryPresenter: Belynda Hicks, SAIC-Frederick, National Cancer Institute at Frederick9:00 am – 10:00 am Novel Institutional Approaches to Managing Core Facilitiesduring Difficult Economic TimesPresenter: Rand Haley, Huron Consulting Group10:00 am – 10:30 am Morning Refreshment Break — Level 2, Grand Oaks Ballroom P/Q10:30 am – 11:30 am Building Synergistic Relationships with Institutional Leadership that Pays OffPresenter: Nicholas Ambulos, University of Maryland School of Medicine11:30 am – 12:00 pm Non-compensatory Approaches to Acknowledging High Performing Staffand Fostering Professional DevelopmentPresenter: Tim C. Hunter, University of Vermont12:00 pm – 1:00 pm Lunch — Level 2, Grand Oaks Ballroom P/Q1:00 pm – 2:00 pm How to Develop and Balance your Core BudgetPresenter: Val Scott, The Jackson Laboratory2:00 pm – 2:30 pm Can I Afford that Service Contract? Getting the Most for the LeastPresenter: Tim C. Hunter, University of Vermont2:30 pm – 3:00 pm Afternoon Refreshment Break — Level 2, Grand Oaks Ballroom P/Q3:00 pm – 3:30 pm Association of Laboratory Managers: Promoting Excellence in Laboratory ManagementPresenter: Wayne Collins, Agilent Technologies3:30 pm – 4:30 pm Interactive Roundtable Discussion on Lean Management in Core FacilitiesPanelists: Belynda Hicks, SAIC-Frederick, National Cancer Institute at Frederick, Nicholas Ambulos, University of MarylandSchool of Medicine, Val Scott, The Jackson Laboratory, Rand Haley, Huron Consulting Group, Wayne Collins, AgilentTechnologies, and Tim C. Hunter, University of Vermont32 • ABRF 2011 — Technologies to Enable Personalized Medicine

NotesNotesABRF 2011 — Technologies to Enable Personalized Medicine • 33

Plenary Session AbstractsPlenary SessionAbstracts(PS1) Personalized Medicine: Opportunitiesand ChallengesR. KucherlapatiHarvard Medical School Department of Genetics andProfessor, Department of Medicine, Brigham and Women’sHospital, Boston, MA, United StatesThe completion of the sequencing of the human genome has augureda new era in genetics. The genome program not only provided uswith the complete sequence but also lead to a revolution in manytechnologies that are critical to biology and medicine. The cost ofDNA sequencing has gone down significantly and is expected to godown further. This technology is enabling us to examine genetic andgenomic differences among and between individuals at a depth thathas not been possible before. The application of genetic and genomicknowledge to assess disease risk, to accurately diagnose disease and tohelp with determining the appropriate therapy is called personalizedmedicine. Our understanding about human genetic variation and howthis influences risk of many complex disorders is increasing at a rapidpace. Assessing individual risk may help prevent or postpone the onsetof human diseases. The genetics of human disease is complex and theuse of genetic information may help us better diagnose disease that inturn may alter management of the disease. In the case of cancer, somaticgenetic changes are helping stratify patients and helping choose themost appropriate drug or therapy to which the patients is most likelyto respond. All of these changes are bringing in a new era whereimplementation of the principles of personalized medicine wouldresult in better health for our population at an affordable cost.(PS2) Unlocking Biomarker Discovery: UnbiasedHuman Proteomics at High Scale, Sensitivity,and AccuracyL. GoldSomaLogic, Inc., Boulder, CO, United StatesUnbiased human proteomics should be used for biomarker discovery,given that one’s capacity to deduce (from the literature) sensiblebiomarkers for disease is severely constrained by our limitedknowledge of human biology. Empiricism will rule the field for yearsto come. We have worked for more than a decade on building aproteomics platform that allows such empiricism. The platform utilizesa special class of aptamers, called SOMAmers that have enhancedaffinities and specificities toward their target proteins. SOMAmerperformance is sufficient to allow our platform to operate without pairsof affinity reagents for each target protein — in other words, we do notuse “sandwiches.” Today we measure more than 1,000 human proteinsfrom human samples (plasma, serum, tissue extracts, or other matrices),using only a small volume of sample. The measurements have limitsof detection similar to those of good ELISA’s, and low CV’s (around5%). We anticipate additional content in the near future — as newcontent is added to the platform, performance appears to get better.To date we have analyzed more than 10,000 samples from individuals,and studied a number of diseases. In most cases we have found novel(and unexpected) biomarkers, which, when used together in a smallpanel, offer hopes for accurate diagnostic information for patients andphysicians. The first diagnostic products will enter the market soon.(PS3) En Route to the Era of Genomic MedicineE.D. GreenNational Human Genome Research Institute, Bethesda, MD,United StatesThe Human Genome Project s completion of the human genomesequence in 2003 was a landmark scientific achievement of historicsignificance. It also signified a critical transition for the field of genomics,as the new foundation of genomic knowledge started to be used inpowerful ways by researchers and clinicians to tackle increasinglycomplex problems in biomedicine. To exploit the opportunitiesprovided by the human genome sequence and to ensure the productivegrowth of genomics as one of the most vital biomedical disciplines ofthe 21st century, the National Human Genome Research Institute(NHGRI) is pursuing a broad vision for genomics research beyond theHuman Genome Project. This vision includes facilitating and supportingthe highest-priority research areas that interconnect genomics tobiology, to health, and to society. Current efforts in genomics researchare focused on using genomic data, technologies, and insights toacquire a deeper understanding of biology and to uncover the geneticbasis of human disease. Some of the most profound advances arebeing catalyzed by revolutionary new DNA sequencing technologies;these methods are already producing prodigious amounts of DNAsequence data, including from large numbers of individual patients.Such a capability, coupled with better associations between geneticdiseases and specific regions of the human genome, are acceleratingour understanding of the genetic basis for complex genetic disordersand for drug response. Together, these developments will usher in theera of genomic medicine.(PS4) Selventa Development of a TherapeuticDiagnostic TM Stratification Biomarker for DrugResponse in Ulcerative ColitisD. de GraafSelventa, Cambridge, MA, United StatesUsing a gene expression data set from Ulcerative Colitis patientstreated with Infliximab with objective response criteria as input dataand our proprietary Selventa analytics, we have developed a diseaseclassifier which identifies alternative molecular mechanisms driving thedisease beyond TNF-alpha, the target of Infliximab. This TherapeuticDiagnostic TM classifier identifies at least 3 more strong, independentdisease drivers, including IL-6, and provides the opportunity to stratifypatient a priori by molecular disease mechanism and associatedtreatment(s). In the case of IL6 we provide supporting evidence in arelated disease that indicates the complementary nature of the TNFalphaand IL-6 disease driving mechanisms, as a partial validation of theapproach. This approach does not rely on response data to developclassifiers and is easily generalized to deliver patient stratification forany specific disease population in Early Discovery with an optimizedproject portfolio as a result.34 • ABRF 2011 — Technologies to Enable Personalized Medicine

VISIT BOOTH 601 TO LEARN MORE!genetic research mattersdemand accuracyIntroducing the NEW 5500 Series SOLiD SequencersDiscover what comes from accuracy. The new, totally redesigned 5500 Series SOLiD Sequencers—now every lab can access a translational research platform with 99.99% accuracy.• Accurate detection of somatic mutations or rare genetic variants• Rapid and cost effective sample processing with flexible microfluidics FlowChip• Simple adoption into your lab with streamlined data analysis and a robust, easy to use platformThere’s a lot riding on the quality of your results.That’s why you need the 5500 Series SOLiD Sequencer in your lab.go to www.appliedbiosystems.com/solid5500DOWNLOAD THE FREE MOBILE APP AT HTTP://GETTAG.MOBI scan the barcodeto instantly access more information about the 5500 Series SOLiD SequencersLife Technologies offers a breadth of products DNA | RNA | protein | cell culture | instrumentsFOR RESEARCH USE ONLY. NOT INTENDED FOR ANY ANIMAL OR HUMAN THERAPEUTIC OR DIAGNOSTIC USE.© 2010 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners, unless otherwise noted.

Scientific Session AbstractsScientific SessionAbstracts(S1) Improving Human Health from the GroundUpT.W. ThannhauserUnited States Department of Agriculture/AgriculturalResearch Service, Ithaca, NY, United StatesThe focus of this scientific session will be the essential link betweenagriculture and human health that is grounded in biotechnology.Advances in medicine, in particular personalized medicine, willultimately demand equal and outstanding advances in agriculturalbiotechnologies to occur in parallel. Such advances promise tomake highly nutritious food accessible and affordable to most of theworld’s population. Speakers will describe the ways in which humansrely on agriculture, and increasingly, on agricultural technologies, tomeet their basic nutritional needs. Direct applications of agriculturalbiotechnologies for crop improvement, agricultural diversification andthe production of vaccines and other protein therapeutics will alsobe presented. Members of the ABRF can help to meet the growingdemand of agricultural research scientists for analytical advances andthroughput via the application of state-of-the-art biotechnologies toagricultural research.(S1-1) Vaccine and Therapeutic Protein Manufacturein PlantsT.E. RyaniBio, Inc., Newark, DE, United StatesThe need to contain costs, as well as the desire to expand patient accessto biotherapeutics has increased interest in non-traditional methodsof recombinant protein expression. The production of vaccines andbiotherapeutic proteins in whole plants holds the promise of dramaticallylowering the capital and operating costs for the manufacture of lifesavingdrugs, and can also be used in developing nations that lack asophisticated drug manufacturing infrastructure. A transient expressionsystem in whole plants using a combination Agrobacterium/viral genevector (iBio Launch TM ) has been developed to manufacture vaccinesand biotherapeutics in a variety of plant species (including Nicotianabenthamiana), and has been successfully scaled to pilot plant levelsof plant biomass (50kg). The speed of protein production in thissystem makes it highly effective for personalized vaccines or vaccinesthat counter pandemic threats. Influenza vaccines for H 5N 1and H 1N 1isolates generated in whole plants using this system have been shownto raise neutralizing antibodies in animal challenge models, and are nowin human clinical trials.(S1-2) Improving Human Nutrition from the GroundUp: Linking Agriculture to Human HealthR.M. WelchDepartment of Food Science, Cornell University, Ithaca, NY,United StatesMalnutrition is the leading cause of death globally. Both overt nutrientdeficiencies and diet-related chronic diseases account for over 20million deaths a year. The causes of malnutrition are complex but arerooted in dysfunctional food systems dependent on agricultural systemsthat have never had an explicit goal of improving human health. Thesedeaths are preventable. Linking agricultural systems to human healthcould provide sustainable solutions to malnutrition. Various agriculturaltools can be used to improve the health and felicity of people afflictedwith malnutrition. Biofortification is one tool that is currently beingemployed to address micronutrient malnutrition among resourcepoorfamilies in the developing world. Fertilizers provide another toolthat has been used successfully to address selenium, iodine and zincdeficiencies in several nations. There are numerous other “off the shelf”agricultural tools that could be used to improve the nutrient outputof farming systems and improve the health all people dependent onagricultural systems for their sustenance. These include: designingcropping systems to maximize nutrient output, using agronomicpractices to improve the nutritional and health promoting quality offood crops, re-diversifying cropping systems, and genetically modifyingcrops to be more nutritious and healthy. This can only be accomplishedif explicit links are made between the agriculture, nutrition and healthcommunities. Further, government policies should be reoriented toreflect the important roles that agriculture plays in the health of allpeople. We need to closely link agriculture to health if we want to findsustainable solutions to malnutrition globally.(S1-3) A Food Systems Approach to AddressPoor Nutritional Health in Both Advanced andDeveloping EconomiesR.D. GrahamFlinders University of South Australia, Adelaide, SouthAustraliaSince the retreat of the oceans, the land surface of planet Earth has forplant growth become widely deficient in nitrogen and phosphorus andto a smaller extent, potassium and sulphur. A century ago, agriculturewas focused on adding these nutrients to solve production problemsof otherwise good soils exhausted from many hundreds of yearsof cultivation and of new, more marginal lands being brought intoproduction. The use of mineral fertilisers such as Chilean saltpetre,superphosphate and potash, together with the other then-knownessential minerals, calcium and magnesium, brought production upto expectations, but to experienced eyes, anomalous results hinted atlimitations to production as yet unknown. The micronutrients, as theybecame to be known, contributed greatly to the 20th century cropproductivity. It is estimated that of the agricultural soils of the world,49% were low in zinc, 31% low in boron, 15% low in molybdenum, 14%low in manganese, 10% low in copper and just 3% deficient in iron. Thepost war explosion in the human population put great stress on our36 • ABRF 2011 — Technologies to Enable Personalized Medicine

food systems in the 1950s but the Green Revolution (1960-1980) morethan doubled world food production and mass starvation was avoided.Instead, there was in the 1980s and 90s a massive rise in micronutrientdeficiencies, especially in subsistence farming systems, which are stillunresolved today. It appears that the Green Revolution emphasison cereal production at the expense of pulses and other nutrientrichfoods is the cause, and new food systems capable of addressingall human nutritional needs are being developed, a multifactorialchallenge. Dealing with the complex nutritional requirements of newand effective food systems for the future is demanding new analyticalcapability to support plant breeders and agronomists in the field.(S2) Integration of Bioinformatics for Genomicsand Proteomics Data(S2-1) Integrative Bioinformatics for Genomics andProteomicsC.H. WuCenter for Bioinformatics and Computational Biology,University of Delaware, Newark, DE, United StatesSystems integration is becoming the driving force for 21st centurybiology. Researchers are systematically tackling gene functions andcomplex regulatory processes by studying organisms at different levelsof organization, from genomes and transcriptomes to proteomesand interactomes. To fully realize the value of such high-throughputdata requires advanced bioinformatics for integration, mining,comparative analysis, and functional interpretation. We are developinga bioinformatics research infrastructure that links data mining withtext mining and network analysis in the systems biology contextfor biological network discovery. The system features include: (i)integration of over 100 molecular and omics databases, along with gene/protein ID mapping from disparate data sources; (ii) data mining andtext mining capabilities for literature-based knowledge extraction; and(iii) interoperability with ontologies to capture functional propertiesof proteins and complexes. The system further connects with a dataanalysis pipeline for next-generation sequencing, linking genomics datato functional annotation. The integrative approach will reveal hiddeninterrelationships among the various components of the biologicalsystems, allowing researchers to ask complex biological questions andgain better understanding of biological and disease processes, therebyfacilitating target discovery.(S2-2) Integrated Bioinformatics for MS-BasedProteomicsE. DeutschInstitute for Systems Biology, Seattle, WA, United StatesA typical tandem mass spectrometry (MS/MS) proteomics workflowinvolves a series of steps including format conversion, spectrumidentification, peptide validation, protein inference, quantification,interpretation, and public repository deposition. This talk will providean overview of the proteomic bioinformatics resources developed atthe Institute for Systems Biology, covering the Trans-Proteomic Pipeline(TPP) and related tools, the PeptideAtlas public repository, and theemerging SRMAtlas resource. The TPP provides an easily-installablesuite of tools to enable users to perform nearly all steps in an MS/MSanalysis workflow. PeptideAtlas is a multi-species public compendiumof peptide and protein identifications assembled from a large numberof uniformly processed MS/MS experiments, along with tools touse the information in a variety of ways. SRMAtlas is a resource thatenables the design of selected reaction monitoring (SRM) experimentsbased on information from several different sources. In addition, theinterface of these resources with community standardization andcooperation efforts such as the Proteomics Standards Initiative and theProteomeXchange Consortium will be presented.(S2-3) Skate Genome Project: Cyber-EnabledBioinformatics CollaborationJ. VincentUniversity of Vermont, Burlington, VT, United StatesThe Skate Genome Project, a pilot project of the North EastCyberinfrastructure Consortium, aims to produce a draft genomesequence of Leucoraja erinacea, the Little Skate. The pilot projectwas designed to also develop expertise in large scale collaborationsacross the NECC region. An overview of the bioinformatics andinfrastructure challenges faced during the first year of the project willbe presented. Results to date and lessons learned from the perspectiveof a bioinformatics core will be highlighted.(S3) Single-Cell Environmental Genomics(S3-1) Early Elements of a Pipeline for SingleBacterial Cell GenomicsD.A. RelmanDepartments of Microbiology & Immunology, and ofMedicine, Stanford University, Stanford, CA, United StatesA critical aspect of microbial community analysis is to understandthe contributions of individual cells to overall community structureand function. Together with Steve Quake and Paul Blainey from theDepartment of Bioengineering, we have built a microfluidics device andplatform that permits sequence-based identification of single bacterialcells using fluorescence in situ hybridization, single cell sorting usinglaser tweezers, and subsequent on-chip whole genome amplification(WGA). Current trapping performance enables the sorting of up to 60cells per hour, and complete processing of 30 - 46 cells, 0 – 16 negativecontrols, and two positive controls per day on a routine basis. Eachdevice enables 48 simultaneous single-cell WGA reactions, which canbe monitored in real-time with SYBR green fluorescence. In conjunctionwith other developments described in this Session, we envision a nearfuturecapability for microbial community genomic analysis at a level ofresolution not previously imagined.(S3-2) Assembling Complete Genomes fromComplex MixturesF. MeyerArgonne National Laboratory, Mathematics and ComputerScience Division, Argonne, IL, United StatesCharacterizing genomes of un-culturable microbes (most specieson earth) requires new approaches for genome assembly fromenvironmental or biomedical samples that often contain manyhundreds or thousands of species. Here we show we can reconstructthe complete genome (Candidatus Sulfuricurvum sp) via short-readmetagenomics and novel approaches for assembly based on simpleScientific SessionAbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 37

Scientific SessionAbstractsstatistical principles. While previous examples of complete genomesequences from metagenomes stem from samples of very limitedcomplexity (>10 OTUs), this sequence was obtained from a complexmix of over 300 OTUs. The reason traditional genome assemblyapproaches fail are varying abundance levels and strain variation andwe show a simple approach to overcome those hurdles. This novelapproach will allow the assembly of genomes and via metabolic modelsderived from the genomic sequences, hopefully the cultivation of keyspecies from diverse environments/enrichment cultures.(S3-3) Dining in with Trillions of Fascinating Friends:Exploring Our Human Gut Microbiome in Healthand DiseaseJ.I. GordonCenter for Genome Sciences and Systems Biology,Washington University School of Medicine, St. Louis, MO,United StatesOur genetic landscape is a summation of the genes embedded inour human genome and in the genomes of our microbial symbionts(the microbiome). Similarly, our metabolic features (metabotypes)are an amalgamation of human and microbial traits. Therefore,understanding of the range of human genetic and metabolic diversitymeans that we must characterize our microbiomes, which containat least several hundred-fold more genes than our human genome,as well as the factors that influence the properties of our microbialcommunities (microbiota). The results should provide an additionalperspective about contemporary human biology as we assesshow our changing lifestyles, cultural norms, socioeconomic status,and biosphere are influencing our microbial ecology and healthstatus. I will discuss the results of our group’s ongoing metagenomicstudies of the interrelationships between diet and the structure anddynamic operations of the human gut microbiome. We believe thatunderstanding these interrelationships is important for advancing ourappreciation of the nutritional value of food ingredients, for creatingnew nutritional guidelines for humans at various stages of their lifespan,and for developing new ways to deliberately manipulate the propertiesof the gut microbiota to prevent or treat various diseases. We havedeveloped a translational medicine pipeline that involves metagenomicanalyses of the gut microbial communities of adult mono- and dizygotictwins living in the USA who are lean, or concordant or discordant forobesity, and twins aged 0-3 years living in developing countries whodevelop normally, or who become malnourished and are treated witha ready-to-use therapeutic food (RUTF). Intact fecal communities fromthese individuals, or ‘personal’ culture collections that capture themajority of bacterial diversity in their microbiota, are then transplantedinto germ-free mice, which are fed the diets of the human donors, orsystematically manipulated derivatives of these diets. The impact ofdiet and microbiota on these humanized mice, including the degreeto which the human donor’s physiologic/metabolic phenotypes can betransmitted to gnotobiotic animals via microbiota transplants, are thenstudied using a variety of methods.(S4) Epigenetics(S4-1) Next Generation Quantitative ProteomicTools for Analyzing Histone ModificationsB.A. GarciaPrinceton University, Princeton, NJ, United StatesHistones are small proteins that package DNA into chromosomes,and a large number of studies have showed that several singlepost-translational modification sites on the histones are associatedwith both gene activation and silencing. Nevertheless, what typeof effect distinct combinations of simultaneously occuring histonemodifications (Histone Codes or patterns) have upon cellular eventsis poorly understood. The main reason for this lack of knowledge isthat robust high-throughput methods for quantitative characterizationor even qualitative identification of combinatorial Histone Codesby any standard biological, immunological or physical techniquedo not exist. We plan to specifically address this deficiency bydeveloping novel mass spectrometry based proteomic methods andaccompanying bioinformatics to quantitatively characterize molecularlevel descriptions of combinatorial Histone Codes, and apply thesemethods to study how these dynamic Histone Codes influence geneexpression under different biological conditions. Here we presentinitial proteomics data that describes: (i) high-throughput comparisonof histone modifications from multiple cellular states (ii) developingmass spectrometry methods for quantitative tracking of combinatorialHistone Codes (iii) monitoring in vivo Histone Code dynamics, and(iv) investigating the role of Histone Code interpreting proteins inrecognizing distinct Histone Codes. Ultimately, we will work towardsthe goal of taking any defined part of the genome and accuratelyquantifying the Histone Codes, detecting all the non-histone proteinsthat reside on these distinct pieces of chromatin, and then mappingthis proteomic data back to specific genomic locations, therefore takinga proteomic snapshot of what that chromosome landscape looks likeduring any nuclear event. These studies in combination with biologicalexperiments will help provide a systems biology outlook on geneexpression that will lay down the basic scientific foundation to advanceseveral applications, such as stem cell reprogramming and cancerprogression.38 • ABRF 2011 — Technologies to Enable Personalized Medicine

(S4-2) Chromatin Dynamics in Melanoma: A Role forMacroH2AA. Kapoor 1,2 , M. Goldberg 1,2 , L. Cumberland 1,2 *,K. Ratnakumar 1,2 *, M. Segura 4,6 , P. Emanuel 2,3 ,S. Menendez 4,6 , C. Vardabasso 1,2 , G. LeRoy 7 , C. Vidal 2,3 †,D. Polsky 4,5,6 , I. Osman 5,6 , B. Garcia 7 , E. Hernando 4,6 ,E. Bernstein 1,21Department of Oncological Sciences, Mount Sinai Schoolof Medicine, New York, NY, United States; 2 Departmentof Dermatology, Mount Sinai School of Medicine, NewYork, NY, United States; 3 Department of Pathology, MountSinai School of Medicine, New York, NY, United States;4Department of Pathology, New York University LangoneMedical Center, New York, NY, United States; 5 Departmentof Dermatology, New York University Langone MedicalCenter, New York, NY, United States; 6 InterdisciplinaryMelanoma Cooperative Group, New York UniversityLangone Medical Center, New York, NY, United States;7Department of Molecular Biology, Princeton University,Schultz Laboratory, Princeton, NJ, United States; †PresentAddress: Department of Dermatology, Saint Louis UniversitySchool of Medicine, St. Louis, MO, United States; *Theseauthors contributed equally to this work.Cancer is a disease consisting of both genetic and epigenetic changes.Although increasing evidence demonstrates that tumour progressionentails chromatin-mediated changes such as DNA methylation, therole of histone variants in cancer initiation and progression currentlyremains unclear. Histone variants replace conventional histones withinthe nucleosome and confer unique biological functions to chromatin.Using well characterized, paired series of murine and human melanomacells lines, we probed the epigenetic profile of melanoma. Analysisof histones from both series using multiplexed quantitative massspectrometryrevealed changes in several histone posttranslationalmodifications and histone variants. The loss of mH2A isoforms,histone variants generally associated with condensed chromatin andfine-tuning of developmental gene expression programs is positivelycorrelated with increasing malignant phenotype of melanoma cellsin culture and human tissue samples. Knockdown of mH2A isoformsin melanoma cells of low malignancy results in significantly increasedproliferation and migration in vitro and growth and metastasis invivo. Restored expression of mH2A isoforms rescues these malignantphenotypes in vitro and in vivo. We demonstrate that the tumourpromotingfunction of mH2A loss is mediated, at least in part, throughdirect transcriptional upregulation of CDK8. Suppression of CDK8, acolorectal cancer oncogene inhibits proliferation of melanoma cells,and knockdown of CDK8 in cells depleted of mH2A suppresses theproliferative advantage induced by mH2A loss. Moreover, a significantinverse correlation between mH2A and CDK8 expression levels existsin melanoma patient samples. Taken together, our results demonstratethat mH2A is a critical component of chromatin that suppresses thedevelopment of malignant melanoma, a highly intractable cutaneousneoplasm.(S4-3) Using Protein Domain Microarrays to Read theHistone CodeM.T. BedfordMD Anderson Cancer Center, Department of MolecularCarcinogenesis, Smithville, TX, United StatesFor cells to survive, differentiate, and grow, information has to betransferred from the cell surface to the nucleus. This process is referredto as signal transduction. A hallmark of cancer is the deregulation ofsignal transduction pathways. Signaling events in eukaryotic cells involvethe assembly and disassembly of large protein-protein complexes.These diverse associations are mediated through interactions ofa limited number of modular signaling units or protein-domains.Protein interactions involving domains are often regulated by posttranslationalmodification (PTM – like phosphorylation, methylationand acetylation) of the smaller protein motif within the ligand. Wehave developed a chip-size protein microarray that harbors a displayof over 300 modular protein-interacting domains including SH2, SH3,PDZ, FHA, 14-3-3, WW, Chromo, Tudor, PHD and MBT domains. Inthe emerging proteomic era, it is becoming easier to identify proteinsusing tryptic digestion followed by mass spectrometric approaches.These same methods also detect sites of posttranslational modificationon proteins. Many of these posttranslational modifications likelygenerate docking sites for protein modules. We have developedprotein-domain microarray technology to help identify proteins thatcan interact with motifs that are either methylated or phosphorylated.This high-throughput approach facilitates the rapid identificationof protein-protein interactions in vitro. Further in vivo studies areneeded to confirm that these interactions do indeed occur in biologicalsystems. Protein domains are cloned into a GST expression vector, andrecombinant protein is produced in bacteria. These fusion proteinsare then arrayed onto nitrocellulose coated glass slides using a robot.These slides are probed with biotinylated peptides that are preconjugatedto streptavidin-Cy3. The peptides used in this experimentare synthesized as 15 mers, and both the modified and unmodifiedforms of the peptides are tested on the array. In this manner, we canidentify novel methyl- and phospho-dependent interactions. We havebuilt three types of arrays: (1) A phospho-tyrosine reader harbors 70SH2 domains and 5 PTB domains (total = 75 domains). (2) A phosphothreonine/serinereader that harbors 7 14-3-3 domains, 5 FHAdomains, 15 BRCT domains and a WW domain (total = 28 domains).(3) An epigenetic reading array that harbors methyl and acetyl readers.This array is composed of 50 tudor domains, 22 bromo domains, 36PHD domains, 17 MBT domains, 11 WD40 domains, 9 SANT domains,28 chromo domains, 15 PWWP domains, 5 BRK domains, 5 CWdomains, and 9 Ank repeats (total = 207 domains). More and moreposttranslational modifications are being discovered on proteins. Theroles of many of these methylation and phosphorylation events oftenremain obscure. This approach provides an easy way for a researcherto identify potential binding partners for their favorite proteins.These arrays thus offer researchers tools to get at “mechanism”. Onceinvestigators know that they are working with a clearly functional PTM,they can proceed with confidence to generate modification specificantibodies and interrogate the signaling pathway that is engaged bythe identified PTM-driven protein-protein interaction.Scientific SessionAbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 39

Scientific SessionAbstracts(S5) High-Throughput Genome Centers(S5-1) Overview of the Illumina Sequencing Platformat the Broad InstituteK. ConnollyProcess & Technology Development, Genome SequencingPlatform, The Broad Institute, Cambridge, MA, United StatesThe constant increase in quality and quantity of Next-Generationsequencing data necessitates a parallel growth in sample preparationand a scalable tracking system. The Broad Institute’s Illumina SequencingPlatform handles a variety of applications and comprises Illumina’s latesthardware, software and kit releases, and a high-throughput samplepreparation process. With our automated sample preparation and QCprocesses, we have been able to meet our increased capacity goals ofup to 3,840 libraries per week, and have reduced our rework rate to5% through attaining target cluster densities with high reproducibility.We continue to work closely with Illumina to develop the sequencingtechnology, using data to drive process improvements and exploringmethods to improve GC bias. Maximizing platform-wide efficiency ispossible through the implementation and continuous development oftools for process quality control and centralized communication, suchas our real-time run monitoring dashboard and JIRA tracking system.We were able to convert rapidly from GAIIxs to HiSeq2000s byestablishing and using an enterprise Knowledge Management system,with which we can efficiently accumulate and disseminate a changingknowledge base. All of these improvements are applicable to both theGA and HiSeq platforms.(S5-2) Science and Technology at a HighThroughput Genome CenterL. Fulton, R. Wilson, The Genome Center ProductionGroupThe Genome Center at Washington University School ofMedicine, St. Louis, MO, United StatesThe Genome Center (GC) at Washington University School of Medicinehas developed a state of the art genomics facility. Our scientists workon a variety of cutting edge projects with researchers from aroundthe world. These collaborative research projects lead to cutting edgeadvances in the field of genomics. The structural organization at theGC reflects these efforts and is centered around six major scientificareas: Transcriptome Sequencing, Genome Assembly, Whole GenomeSequencing, Human Microbiome, Human Genetics, and TargetedResequencing. These specific scientific areas are supported by onecentral data production pipeline. Attributes of this pipeline includedetailed sample screening protocols, sample barcoding capabilitiesthat allow for a broad range of sample cohorts, multiplatform dataproduction, and the ability to select from more than one method ofsequencing strategies. All of this is supported by one centralized LIMSgroup dedicated to maintaining and developing the data productioncapabilities. The technology development group investigates newtechniques and instrumentation prior to any changes in the main dataproduction pipeline. Only robust protocols and instrumentation areallowed into the data production pipeline. This strategy allows TheGenome Center to run a base data production pipeline while constantlyinfusing high quality advances. Sequence data for each project is sentinto an advanced analysis pipeline built to conduct a multitude ofassessments. When needed, validation (a second sequence event) canbe used to confirm variants detected by the analysis software.(S5-3) High-Throughput Next GenerationSequencing Methods and ApplicationsD. Muzny 1 , M. Wang 1 , I. Newsham 1 , Y.Q. Wu 1 , H. Dinh 1 ,C. Kovar 1 , J. Santibanez 1 , A. Sabo 1 , J. Reid 1 , M. Bainbridge 1 ,E. Boerwinkle 2 , T. Albert 3 , R. Gibbs 11Baylor College of Medicine, Human Genome SequencingCenter, Houston, TX, United States; 2 University of TexasHealth Science Center at Houston, School of Public Health,Houston, TX, United States; 3 Roche NimbleGen, Inc.,Madison, WI, United StatesSecond Generation high-throughput sequencing technologies haverevolutionized the genome sequencing applications and will ultimatelyhave great impact on personalized medicine. The increase in capacityof both the AB/Life Technologies SOLiD 4.0 and Illumina HiSeqinstrumentation and the ability of the platforms to multiplex sampleshas led to process innovations impacting many ongoing projectsat the HGSC. Applications have ranged from regional and wholeexome capture sequencing to the use of whole genome shotgun fordeep coverage and determining structural rearrangements. Internaladvancements have complemented the higher capacity instrumentationthrough the implementation of library automation, low DNA inputsamples, capture hybridization multiplexing and application of readmapping tools such as BFAST and BWA. Development of sample intakeprocedures, LIMS tracking and defined reporting metrics has enabledNexGen sequencing pipelines that can effectively deliver targetedand whole genome shotgun data for thousands of samples. Thesetechnical advancements to the pipeline have allowed us to achievea rate of ~1500 libraries/captures per month. To date the center hascompleted over 5000 exome and regional capture libraries for TheCancer Genome Atlas (TCGA), NIMH Autism, Cohorts for Heart andAging Research in Genomic Epidemiology (CHARGE-S) and 1000Genomes Project. Development of these applications and methods willbe discussed along with key data metrics, process management andpipeline organization.(S6) Strategies for Deep Mining of ComplexProtein Mixtures(S6-1) Coverage and Recovery of Upstream ProteinFractionation Methods in LC-MS/MS WorkflowsL.J. FosterCentre for High-Throughput Biology, The University of BritishColumbia, Vancouver, BC, CanadaThe proteome of any cell or even any subcellular fraction remainstoo complex for complete analysis by one dimension of liquidchromatography-tandem mass spectrometry (LC-MS/MS). Hence, toachieve greater depth of coverage for a proteome of interest, mostgroups routinely subfractionate the sample prior to LC-MS/MS sothat the material entering LC-MS/MS is less complex than the originalsample. Protein and/or peptide fractionation methods that biochemistshave used for decades, such as strong cation exchange chromatography(SCX), isoelectric focusing (IEF) and SDS-PAGE, are the most commonprefractionation methods used currently. There has, as yet, been nocomprehensive, controlled evaluation of the relative merits of thevarious methods, although some binary comparisons have been made.We will discuss the most popular methods for fractionating samples atboth the protein and peptide level, demonstrating quantitatively which40 • ABRF 2011 — Technologies to Enable Personalized Medicine

are the best methods for optimal recovery and proteome coverage.A novel approach for fractionating samples at the level of proteincomplexes will also be discussed.(S6-2) Improving the Comprehensiveness of Large-Scale Proteomics Experiments Using AdvancedComputational Tools and Accurate MultipleHypothesis Testing StatisticsM.J. MacCoss, J. Egertson, B. Frewen, L. Käll, W. NobleDepartment of Genome Sciences, University of Washington,Seattle, WA, United StatesMass spectrometry based technology for the analysis of complexprotein mixtures has improved at an amazing rate. With each newinstrument release, mass spectrometers have become more sensitiveand have faster MS/MS data acquisition speeds. Furthermore,instruments are continuously improving the dynamic range, massaccuracy, and resolution of the resulting mass spectrometry data. Allof these developments have increased the number of peptides thatcan be identified and quantified without extending the overall analysistime. While the technological hardware advances that are required toincrease the number of peptide identifications by 50% with a constantanalysis time is monumental, we have been able to demonstrate thatincrease in performance without increasing the analysis time at all. Toaccomplish this, we have made use of improved database searchingalgorithms, spectrum library searching, use of chromatographicretention time, powerful machine learning tools, accurate multiplehypothesis testing statistics, and many more. Strategies will bediscussed on how to increase the comprehensiveness of any datasetusing improved data analysis strategies.(S6-3) In-Depth Analysis of Human and MousePlasma Using 3-D And 4-D Fractionation StrategiesD.W. SpeicherThe Wistar Institute, Philadelphia, PA, United StatesIn-depth profiling of plasma proteomes can potentially identify noveldisease biomarkers. But few biomarkers identified by proteomicapproaches have advanced to early-stage clinical testing because theyoften are not sufficiently disease specific. Major challenges in plasmaproteome analysis include the very wide dynamic range of proteinconcentrations, the high protein complexity, and the substantialheterogeneity of most protein concentrations in the normal humanpopulation. Because most disease-specific biomarkers are present inblood at very low concentrations, extensive fractionation is requiredprior to LC-MS/MS analysis. In general, more fractionation will result ingreater depth of analysis, but there is a point of diminishing return foreach fractionation method and throughput decreases as the numberof LC-MS/MS runs per proteome increases. A common feature of mostcurrent plasma profiling methods is to first immunodeplete as manyhigh abundance plasma proteins as possible, followed by extensiveprotein fractionation of the depleted plasma prior to trypsin digestionand LC-MS/MS. In addition, reliable quantitative comparisons areneeded for most types of studies. While all quantitative methods havestrengths and weaknesses, label-free quantitative comparison of LC-MSsignals is increasing in popularity and seems adequately reproduciblefor most studies. Our laboratory commonly uses two alternative plasmaproteome analysis strategies. One powerful approach utilizes a 3-Dprotein/peptide profiling method consisting of depleting 20 abundantproteins followed by 1-D SDS PAGE, fractionation of the gel lane into20 to 60 fractions and LC-MS/MS analysis. Proteins can be quantitativelycompared using label-free analysis of ion current patterns from the MSfull scans. An even greater depth of analysis can be achieved using a4-D protein/peptide profiling strategy utilizing microscale solutionisoelectrofocusing of proteins prior the SDS gel in the 3-D scheme,although throughput is substantially reduced.(S7) Next Generation Sequencing Technologieson the Horizon(S7-1) Towards Optical DNA Sequencing UsingNanopore ArraysA. MellerDepartment of Biomedical Engineering, Boston University,Boston, MA, United StatesNext generation DNA sequencing methods that utilize nanometersizepores have been subject of numerous studies in past years. Oneof the compelling features of the nanopore technique lies in its abilityto electrophoretically focus long DNA strands towards the pore areaand thread the molecules inside the pore in a highly efficient manner.Thus extremely small copy numbers of the target DNA are requiredfor analyses†, circumventing the need for costly and time-consumingtarget amplification. One of the major bottlenecks for the realizationof a viable nanopore-based DNA sequencing has been the ability tosimultaneously read the electrical signals from hundreds to thousandsof nanopores densely fabricated on sub-millimeter size silicon chip. Toaddress this issue we develop an extremely high throughput singlemoleculeDNA sequencing technique, which employs optical, widefieldreadout from DNA molecules electrically mobilized throughthe nanopores. Our method consists of two steps: First, target DNAmolecules are converted according to pre-determined code, which isrecognized by molecular beacons with four types of fluorophores (eachuniquely corresponding to one of the four DNA bases). Solid-statenanopores are then used to sequentially strip off the beacons, leadingto a series of photon bursts that can be detected with a custom mademicroscope. Notably the method circumvents the use of enzymes in thereadout stage, and is thus not affected by their limited processivity andlifetime. Here we demonstrate the feasibility of this method using a twocolor model system, and show for the first time, individual nucleotiderecognition from multiple nanopores simultaneously‡, allowingstraightforward parallelization of our system to nanopore arrays. †.Wanunu, M., W. Morrison, Y. Rabin, A. Y. Grosberg, and A. Meller. 2010.Electrostatic Focusing of Unlabeled DNA into Nanoscale Pores using aSalt Gradient. Nature Nanotechnology 5:160-165. ‡. McNally, B., A.Singer, Z. Yu, Y. Sun, Z. Weng, and A. Meller. 2010. Optical Recognitionof Converted DNA Nucleotides for Single-Molecule DNA SequencingUsing Nanopore Arrays. Nano Letters 10:2237-2244.Scientific SessionAbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 41

Scientific SessionAbstracts(S7-2) Single Molecule Real-Time DNA Sequencingon the Surface of a Quantum-Dot NanocrystalP.B. Vander HornGenetic Systems, Life Technologies, Carlsbad, CA, UnitedStatesA single molecule, long read-length, real-time sequencingby-synthesistechnology has been developed by buildinga sequencer directly on the surface of a ~ 10 nm quantumdotnanocrystal. Fluorescence resonance energy-transfertechnology (FRET) is utilized for DNA sequence detection, inwhich signals from the quantum-dot labeled DNA polymeraseplus 4 DNA-base-specific acceptor dyes are simultaneouslydetected. Precisely engineered sequencing-grade Qdot TMnanocrystals are smaller than current commercially availablematerials (to increase FRET signals), and have an exctinctioncoefficient ~100X greater than organic-dyes, allowing for verylow levels of excitation power to be used while sequencing,Acting as the FRET donor, the Qdot TM -polymerase generates acorrelated “photon-dip” for every inserted based (termed the“quantum-correlation-signal”), allowing for more accurate basecalling.Because the sequencer is not physically bound to anysolid substrate, it can be exchanged (like a reagent) during midsequenceruns, effectively replacing damaged non-functioningpolymerases mid-reaction. Each exchange cycle lengthens theeffective read-length of the sequencer. In this manner, theread-length can be continuously extended without “gaps”.Expanding upon this flexibility, after sequencing a particularlength of DNA, the newly synthesized strand can be selectivelyremoved. The original genomic DNA strand is then re-primed,Qdot TM -polymerase sequencers are rebound, and the identicalgenomic DNA strand can be sequenced again, greatly increasingthe net accuracy and not requiring circularization of genomictemplates. In combining these features, the desired accuracyand read-length can be “tuned” by adjusting the number ofreagent exchange cycles. Because each sequencing reaction canbe completed in minutes, multiple exchange experiments canbe performed per sequencing hour. These Qdot TM -polymerasesequencers can also bind to ultra-long DNA segments (>10kb)at multiple positions along the length of the DNA andsequence while moving “horizontally” (parallel to TIRF field),enabling the possibility of “ordered-reads” for long-phasedhaplotype sequencing. Examples of real-time sequencing ofhomopolymeric, patterned, and complex templates will beshown.(S9) Proteomics StandardizationD.B. FriedmanProteomics Laboratory Mass Spectrometry Research Center,Vanderbilt University, Nashville, TN, United StatesThe needs and issues related to establishing inter-laboratorystandardization in quantitative proteomics will be highlighted bypresentations from three international initiatives. By establishing theneed for standardization, each group will also highlight the tremendousamount of instrument and experimental variation that is also measuredwhen trying to determine real biological changes. The Spanish-basedProteoRed consortium addresses multiple proteomics platforms,ranging from MS-based to gel-based. The NIH CPTAC network isfocused on MS-based studies, and the UK-based FixingProteomicsinitiative covers mostly gel-based methods. Results from thesestudies will demonstrate complement and overlap in technology andapproaches used between these groups, all motivated by the universalgoal of standardizing these complex technologies across laboratories.(S9-1) A Multi-Laboratory Study AssessingRobustness and Reproducibility of PlasmaReference Sample for Benchmarking LC-MS PlatformPerformanceJ.P. Albar 5 , A. Campos 1,4 , E. Oliveira 1,4 ,S. Martínez-Bartolomé 3,4 , F. Canals 2,41Barcelona Science Park, Barcelona, Spain; 2 Vall d’HebronUniversity Hospital Research Institute, Barcelona, Spain;3Centro Nacional de Biotecnologia-CSIC, Madrid, Spain;4ProteoRed Consortium, Spanish National Institute ofProteomics, Madrid, Spain; 5 ProteoRed, National Center forBiotechnology-CSIC, Madrid, SpainAn increasingly common request for proteomics core facilities isdetermining qualitative and quantitative differences among clinicalsamples such as plasma, CSF, or urine. One of the missions of theSpanish Network of Proteomics Facilities (ProteoRed-ISCIII) is to assistits proteomics core facilities in evaluating their capabilities to performqualitative and quantitative proteomics analysis. This year, in an attemptto represent a realistic experiment scenario that might be requested toa proteomics core facility, we provided a moderately complex plasmastandard reference sample to be used for routine QC monitoring oflaboratory instrumentation. The ProteoRed Plasma Reference (PPR)sample is a subset of highly abundant well-characterized human plasmaproteins with a number of isoforms, in addition to 4 spiked-in proteins,altogether distributed over 5 orders of magnitude in concentration.The PPR sample was recently stress tested in the latest ProteoRedMulticenter Experiment (PME6) that counted with the participationof 17 proteomics facilities using a wide range of LC-MS platforms. Werequested the sample be analyzed in a single LC-MS run in experimentaltriplicate (3 different digestions). Evaluation of the results submitted bythe study participants revealed moderate discrepancies at the peptideidentification level, and poor overlap at the protein identificationlevel. In an attempt to identify the source of such irreproducibility,raw data of 8 laboratories (24 LC-MS runs) were reanalyzed centrallyusing a standardized data analysis pipeline, which included proteininference using ProteinProphet software. We found that the majorityof protein identification discrepancies across submitted reports ofthese 8 laboratories were due to inconsistencies on how data analysisand computational tools group and/or infer proteins. Immunoglobulin42 • ABRF 2011 — Technologies to Enable Personalized Medicine

variable chain identifications were particularly conflicting throughoutidentification lists, even in the centralized analysis. Using a series ofLC-MS performance metrics, we benchmarked the performance of 8LC-MS instruments (Orbitraps) and identified system components thatvary the most across laboratories.(S9-2) Clinical Proteomic Technologies for Cancer:NIH-Funded Measurement ScienceC.R. Kinsinger, E. Boja, T. Hiltke, M. Mesri, A. Rahbar,R. Rivers, H. RodriguezNational Cancer Institute, Center for Strategic ScientificInitiatives, Office of Cancer Clinical Proteomic Research,Bethesda, MD, United StatesIn 2006, the National Cancer Institute (NCI) launched the ClinicalProteomic Technologies for Cancer initiative (CPTC). The overallmission of this initiative was to foster the building of an integratedfoundation of proteomic technologies, data, analysis systems, andreagents and reference materials to systematically advance theapplication of protein science to accelerate discovery and clinicalresearch in cancer. Specifically, the CPTC was charged to addressissues of variability and irreproducibility in proteomic measurements.During the past five years, CPTC investigators have focused onassessing proteomic platforms involving mass spectrometry. Interlaboratorystudies have addressed variability in both unbiased andtargeted mass spectrometric methods. These studies have producedreference materials and data, performance metrics, standard operatingprocedures, and guidance for the community on the current abilityof mass spectrometry for proteomics. Outputs from the technologyassessment aspects of CPTC have leveraged additional developments.First, CPTC inter-laboratory studies provided a basis for engaging theFDA on the metrological requirements for approval in vitro diagnosticmultivariateindex assays. Second, the NCI developed a follow-onfunding opportunity that applies the technology pipeline developedin the first phase of CPTC.(S9-3) Why Reproducible Outcomes are Essentialin Proteomic Research and Why Standardisation ofProcesses is Essential for Achieving ReproducibilityA. Borthwick, W. DracupNonlinear Dynamics, Newcastle upon Tyne, United KingdomIn both 2D electrophoresis and LC-MS proteomic analysis we are dealingwith highly complex samples, and there are many complex processesinvolved which in turn can be affected by a host of parameters and issuessuch as reagent batches, column performance, even the temperature ofthe lab. This complexity means that it can be very difficult to generatethe same results from the same samples in different labs and even inthe same lab at different times. This in turn makes it very difficult forlabs to build upon published results, a fundamental principle of thescientific method. Quality Control (QC), based on the use of standardsto monitor levels of technical variation in industrial processes isfundamental in the output of a reproducible product. We argue thatbecause proteomic analysis is significantly more challenging than mostindustrial processes, employing standards and the standardisation ofprocesses in proteomics experiments is key to arriving at reproducibleoutcomes. Focusing mainly on 2D electrophoresis and to some extenton LC-MS we examine the importance of standardisation and howsuch standards may be applied to Proteomic research in order tofacilitate reproducible discoveries. Using studies carried out with singleand multi-users from within and between different laboratories, wedescribe our experiences of achieving standardisation using Standardsamples to provide feedback on the reproducibility of each stage andas well as the complete proteomic workflow.Scientific SessionAbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 43

Workshop Session AbstractsWorkshop SessionAbstracts(W1) The Diagnostics Core Facility: Harvestingthe Promise of Personalized Medicine(W1-1) Win on Sunday, Sell on Monday: From theExome Sequencing of One Boy to the Delivery ofClinical DiagnosticsM.R. TschannenHuman and Molecular Genetics Center, Department ofPhysiology, Medical College of Wisconsin, Milwaukee, WI,United StatesFor several years, there have been discussions about using bothSanger and whole genome sequencing in clinical practice. In late2009, the Medical College of Wisconsin initiated the infrastructureto streamline the delivery of current and emerging DNA technologiesinto state-of-the-art molecular diagnostics. The online publication ofour initial case in Genetics of Medicine in late 2010 further intensifiedour efforts in this endeavor. However, being relatively new to thefield of NextGen sequencing, we began with the addition of Sangerdiagnostic sequencing to our already successful research core, whichat that point had been in operation for almost ten years. This was agreat undertaking, as typically, independent research laboratoriesperforming cutting-edge science lack the financial resources andbreadth of experience to launch their custom product or applicationto the diagnostic industry. An independent research laboratory is ableto resolve these shortages by partnering with a core laboratory staffedwith diagnostic expertise. Due to our lack of diagnostic experience, wequickly aligned the research core to a consortium of individuals withclinical experience to allow us to benefit from established diagnosticfacilities on campus. Difficulties faced at the onset of diagnostic startupwere many, including large issues such as accreditation program (CAPvs. CLIA), SOP generation and validation, competency and proficiencytesting, and reimbursement, as well as smaller problems like semiannualpipette calibration, temperature monitoring, and inventorycontrol. The purpose of this talk is to give insight into efficient ways toresolve these problems, both large and small, and transform a decadeor more of research expertise into a viable diagnostic laboratory.(W1-2) Chromosomal Microarray Analysis in theClinicL.D. WhiteMicroarray Core, Baylor College of Medicine, Houston, TX,United StatesCurrent Array Comparative Genomic Hybridization (aCGH) orChromosomal Microarray Analysis (CMA) performed at BaylorCollege of Medicine utilizes the latest microarray technology to detectunbalanced chromosome abnormalities associated with over 210 clinicaldisorders with exon by exon coverage of over 1,700 genes. We haveperformed approximately 35,000 postnatal CMA tests. Additionally,inclusive of our reported experience with 300 prenatal cases (PMID19012303), we now have CMA results on approximately 800 clinicalprenatal samples. This talk will cover the utility CMA for chromosomalabnormality detection in the clinical lab as well as development anddeployment of clinical tests from the core lab perspective.(W1-3) Whole Genome Sequencing in the ClinicalLaboratoryT. Hambuch, M. Laurent, B. Sickler, A. Liao, P. Cotter,S. Jain, Y. Lyan, J. Bernd, J.O. Daniel, P. Poggio, M. Ross,D. BentleyIllumina Clinical Services Laboratory, San Diego, CA, UnitedStatesThe advent of routine whole genome sequencing creates an opportunityto provide an accurate, comprehensive and cost-effective catalogue ofgermline variation for an individual. The process of sequencing anddelivering genomes for individual use must be driven by clinical andeducational opportunities balanced by addressing ethical concerns.Using guidelines issued from professional and accrediting agenciesas well as an independent ethics board, we developed and launchedindividual genome sequencing (IGS) as a physician-led service. Aphysician orders the sequence and obtains informed consent from theindividual; the sample is sequenced within a CLIA certified laboratoryand after a series of quality checks the sequence is returned to thephysician for communication back to the individual. The sequencingplatform and process were validated for accuracy and precision, andaccredited following review by a College of American Pathologist(CAP) inspection team. Each individual genome is sequenced at >30fold coverage using paired-end reads of 100 base pairs. Resultingsequence information is provided for >93% of the NCBI36 genome, theremainder being mostly recently duplicated repeats where ambiguousread alignment is not permitted in our ELAND analysis. On average, wedetect over 3 million SNPs most of which are previously documentedin dbSNP129. The overall accuracy of our base calling is measured as>99.99% and the accuracy for SNP calling is >99.7% based on multiplemethodlogical assessments. The aim of the Illumina Clinical ServicesLaboratory is to make Individual Genome Sequencing accurate,accessible and clinically relevant for physicians and patients through afully accredited process. Here we have established baseline processes,tools, and policies to maximize the benefit to patients and minimizepotential misuse. Additionally, our ongoing efforts to develop clinicallyrelevant interpretation tools for physicians are described in a separateabstract (see M. Ross). Individual Genome Sequencing has the capacityto replace current genetic testing with a near-complete description ofthe sequence of an individual. Considering this potential, it is essentialto engage policy makers and ethicists so that appropriate policiesare developed around information access and use of whole genomeinformation.44 • ABRF 2011 — Technologies to Enable Personalized Medicine

(W2) Spectral and Sequence DatabaseSearching in ProteomicsL. MartensGhent University, Ghent, BelgiumThis session will present an overview of more advanced methods tomatch acquired MS/MS spectra to peptides, including strategies todetect (unexpected) protein modifications, and the use of spectrallibraries for peptide identification by spectrum-to-spectrum matching.Introduced by some of the world’s foremost experts in these proteomicsinformatics challenges, this session is meant to provide a pragmaticintroduction to the topics for interested researchers, thus making thesemethods directly adoptable in the lab.(W2-1) ETD Performance and Complementarityto Other Fragmentation Methods for ProteomicAnalysisR. ChalkleyUniversity of California San Francisco, San Francisco, CA,United StatesRadical-driven fragmentation approaches present an alternative to thewell-established collisional cleavage approaches that have dominatedproteomic research up until now. With the recent availability ofelectron transfer dissociation (ETD) in commercial quadrupole iontrap and hybrid instruments, this technology is now accessible to manyresearchers. It has been described as a complementary approachto CID, and decision tree approaches have been employed where achoice between CID or ETD is made depending on the precursor m/zand charge. In this presentation I will discuss the performance of ETD forpeptide identification, drawing heavily on data acquired as part of the2011 iPRG study ‘Identification of Electron Transfer Dissocation (ETD)Mass Spectra’. Results will be compared to analyses of the same sampleusing CID and HCD, decision tree approaches and the differencein measuring data in the ion trap versus in the orbitrap detector. Acomparison of search engine performance will be presented andmethods for improving database search engine analysis of ETD datawill also be discussed.(W2-2) Discovery, Identification and Localization ofPost-Translational ModificationsN. BandeiraCenter for Computational Mass Spectrometry, Departmentof Computer Science and Engineering Skaggs School ofPharmacy and Pharmaceutical Sciences University ofCalifornia, San Diego, CA, United StatesMass spectrometry based analysis of post-translational modificationscommonly report thousands of modified-peptide identificationsaccompanied by both precisely and ambiguously localizedmodification sites. Since these identifications often motivate extensivefollow up studies, the confident identification of the peptide andaccurate localization of the modification site(s) remains one of themajor challenges in computational proteomics. As revealed by the2010 iPRG study on identification of phosphopeptides and localizationof phosphorylation sites, participants only attempted to call themodification sites for less than 2 out of every 3 identified spectraand actually disagreed on over 20% of all cases where at least twoparticipants called a modification site. In this talk we will cover currentand novel methods for identification of post-translationally modifiedpeptides and automated determination of site localization confidencescores and false discovery rates.(W2-3) Building and Using MS/MS Spectral Librariesfor Peptide Identifications in ProteomicsH. LamThe Hong Kong University of Science and Technology, ClearWater Bay, Hong Kong, ChinaSpectral library searching is an emerging approach in peptideidentifications from tandem mass spectra, a critical step in proteomicdata analysis. In this approach, a spectral library is first meticulouslycompiled from a large collection of previously observed and identifiedpeptide MS/MS spectra. An unknown spectrum is then identified bycomparing it to all the candidates in the spectral library for the mostsimilar match. Thanks to the reduction of search space to only thepreviously discovered peptides, and the use of real, experimentallyobserved reference spectra for more precise spectral matching, thisapproach is considerably faster and more sensitive than the popularalternative of sequence searching. This talk will explain the basicprinciples of spectral library building and searching, describe itsadvantages and limitations, and provide a starting point for researchersinterested in adopting this new approach in their data analysis. It willalso discuss the future outlook on the evolution and utility of spectrallibraries in the field of proteomics.(W3) Quantifying Protein Turnover by In VivoMetabolic Labeling(W3-1) Stable Isotope Tracers Applied to MeasuringRates of Protein Synthesis and Breakdown in Muscle:Principles and ApplicationsR.R. WolfeUniversity of Arkansas for Medical Sciences, Little Rock, AR,United StatesMuscle is in a constant state of turnover, meaning that it is continuouslysynthesized and broken down. The balance between the rates ofsynthesis and breakdown determines if an individual is gaining orloosing muscle mass. It is therefore of interest in a variety of physiologicalcircumstances to quantify the rates of muscle protein synthesis andbreakdown. Tracer methodology using both radioactive and stableisotopes has been used in a wide variety of kinetic studies, includingmeasurement of synthetic and breakdown rates of various compounds.Stable isotopes are particularly suited for the study of muscle proteinmetabolism, as multiple amino acid tracers can be used simultaneously,and multiple labels can be used with any individual amino acid,including labeling the nitrogen with 15N. Two basic approaches canbe used to measure muscle protein synthesis. The direct incorporationof a labeled amino acid is the most conventional. This techniqueinvolves infusion or injection of tracer and measurement of subsequentincorporation into muscle protein over time. The measurement of theprecursor enrichment, which is usually taken to be the free intracellularpool of the tracer amino acid, is necessary to calculate the actual rateof synthesis. Alternatively, the rate of muscle protein synthesis can bederived from the rate of uptake of an amino acid from blood. In thisWorkshop SessionAbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 45

Workshop SessionAbstractscase incorporation of the amino acid into protein must be its onlymetabolic fate within the muscle. The basic principle of measuringmuscle protein breakdown is to determine the rate at which intracellularamino acid tracer is diluted by the appearance of unlabeled amino acidthat is not coming from the plasma. There are different approachesto accomplishing this measurement, and the choice of the optimaldepends of the method used to measure protein synthesis. Ideally,methods to measure synthesis and breakdown are compatible (i.e., thesame units) to enable calculation of the balance between synthesis andbreakdown to determine if there is a net gain or loss in muscle protein.(W3-2) In Vivo Stable Isotope Labeling forQuantifying Amyloid-beta Kinetics in Alzheimer’sDisease: Is it All in our Head?K.E. YarasheskiBiomedical Mass Spectrometry Research Laboratory,Washington University School of Medicine, St. Louis, MO,United StatesMass spectrometry has revolutionized the manner in which we identify,characterize, and quantify proteins. In combination with in vivo stableisotope labeling strategies, mass spectrometry-based analyses canprovide valuable information about human amino acid and proteinkinetics, protein production (synthesis), and clearance (proteolysis)rates. Dysregulated or imbalanced protein synthesis and degradationrates is the basis for many clinical disorders. These protein kineticrates can be quantified in vivo and serve to identify potential targetsfor novel drug therapies. Our group uses an intravenous infusion of13C6-Leu, cerebral spinal fluid (CSF) sampling, affinity isolation ofrelevant proteins, and tandem mass spectrometry to quantify 13C6-Leu incorporation and removal rates from CSF amyloid-beta andapolipoproteins in Alzheimer’s disease patients. This approach hasidentified slower amyloid-beta clearance rates as a primary lesion thatmay explain an accumulation of amyloid plaques in Alzheimer’s disease.(W4) Insights for Expression of RecombinantProteins for Drug Target Validation(W4-2) Strategies for Optimized High-ThroughputCloning, Expression and Purification of RecombinantProteins in E. coliR. PageBrown University, Department of Molecular Biology, CellBiology and Biochemistry, Center for Genetics, Genomicsand Proteomics, Providence, RI, United StatesThe arrival of structural genomics, in addition to large-scale effortsinitiated in pharmaceutical companies, have resulted in the developmentof numerous new methods and strategies to minimize the time requiredto optimize the cloning, expression and purification of novel proteindrug targets. Here, I will present both best practices and unusual (‘lastditch’) methods that are used to successfully express and purify bothprokaryotic and eukaryotic proteins in E. coli. Recent developments inprotein expression, including co-expression with protein partners andbacterial chaperones will be presented. In addition, a comparison ofthe best solubility enhancing tags, and methods for subsequent tagremoval, will be presented. Finally, I will also describe two case studieswhich required highly tailored expression protocols for the productionof a bacterial toxin and eukaryotic phosphatase. In summary, the focusof this lecture will be to provide practical information that researchersactively involved in protein purification can readily implement intotheir own workflows. This work was supported in part by a medicalresearch grant from the American Cancer Society (RSG-08-067-01-LIB)and an NSF-CAREER award (MCB 0952550).(W5) Proteomics Tips and Tricks: FromDiscovery to Protein-Protein Interactions(W5-1) Proteomics Tips and Tricks: From Discoveryto Protein-Protein InteractionsM. CiliaUnited States Department of Agriculture/AgriculturalResearch Service, Robert W. Holley Center for Agricultureand Health, Cornell University, Ithaca, NY, United StatesProteins are the functional constituents of cells. The diverse physicalproperties of proteins enable them to be multifunctional and also posechallenges to analyzing their abundance, subcellular localization, andpotential interacting partners using proteomics. Some samples areespecially recalcitrant and pose additional challenges in proteomicanalysis due to the unanticipated modification of proteins. Thesechallenges extend from initial protein extraction to analysis usingmass spectrometry. Broadly, the focus of workshop will be to discussthe benefits and limitations of using proteomics to study proteinidentification, protein quantification, protein structure, and proteinproteininteractions. Examples will be drawn from different biologicaldisciplines, including cell biology, host-pathogen biology, anddevelopmental biology to demonstrate how scientists are solving theproblems posed by their discovery methods and biological systems.The workshop will include presentations from each panelist followedby an open discussion and question and answer session.(W5-2) Protein Interactions and Topologies in CellsJ. BruceUniversity of Washington, Department of Genome Sciences,Seattle, WA, United StatesLife on earth has evolved to utilize proteins as functional molecules dueto the wide diversity of structures and physical properties this class ofmolecules can exhibit. However, the divergent properties that criticallysupport life also pose fundamental challenges in all efforts to measurethe proteome. As a result, most proteomics experiments only samplea small subset of expressed molecules and typically appear biasedtowards cytoplasmic proteins. Furthermore, detection of proteinproteininteractions is even more challenging. For example, most largescalemethods such as the yeast two-hybrid, tandem affinity purification,and co-IP methods are predicated on the production/maintenanceof native protein structures and co-localization of native interactingpartners. These two requirements can result in failure to identify manybone fide interactions and in false discovery because many interactions46 • ABRF 2011 — Technologies to Enable Personalized Medicine

may not survive cell lysis steps and non-relevant interactions can formin cell lysates during sample preparation. Significant progress onmany of these issues has been achieved in recent years, including theuse of cryogenic cell lysis techniques. Alternatively, covalent linkageof interacting proteins within cells as accomplished with chemicalcross-linking has long held potential for protein interaction studies.If successful, chemical cross-linking approaches mitigate the needto maintain native interactions and structures during subsequentproteome sample preparation and may offer unique insight on proteininteractions and structures present within cells. However, the analysis ofcross-linked peptides presents a different set of challenges related todynamic range and detection specificity. This presentation will highlightadvancements in technology, informatics and sample preparation stepswe have pursued to enable cross-linked peptide measurements from invivo cross-linking experiments and will illustrate the unique informationthat can be derived from this approach.(W5-3) The Virus-Host Interface: Exploring DynamicProtein Interactions via Targeted ProteomicsI. CristeaPrinceton University, Department of Molecular Biology,Princeton, NJ, United StatesDynamic protein interactions carry out the majority of the processeswithin a cell, including cellular responses to environmental stimuliand pathogens. Isolation and characterization of protein complexescan provide invaluable insights into their biological functions. Thedevelopment of approaches that can access stable and transientinteractions is invaluable for numerous fields of study, including thatof temporal and spatial virus-host protein interactions. Viruses haveco-evolved with their hosts, developing remarkable mechanisms forsubverting cellular processes for their own benefit. The study of virushostinteractions has therefore emerged as a driving force in infectiousdisease research. Despite these efforts, the protein interactomeremains in large part uncharted, and our knowledge of mechanismscontrolling the outcome of an infection is limited. Modern proteomicstechniques are currently emerging as powerful tools, bringing a newperspective to the field of virology. This presentation will describe theintegration of targeted proteomics with genetic, molecular biology,and bioinformatics techniques for studying dynamic virus-host proteinassociations. Strategies for isolating protein complexes, quantifyinginfection-triggered changes in interactions, and assessing interactionspecificity will be presented. In studies of human cytomegalovirus(HCMV) infection, we discovered parallel processes occurring at distinctcellular sites during the assembly of infectious virions. Additionally, wehave observed that certain viral proteins recruit chromatin-remodelingenzymes, such as histone deacetylases, indicating a possible mean ofcontrolling virus or host gene expression. In addition to revealing theirfunctional roles during infection, our studies provided insights intothe regulation of these enzymes outside the context of infection. Acombinatorial proteomics approach, incorporating CID, HCD and ETDpeptide fragmentation using a nLC LTQ Orbitrap Velos-ETD, identified17 in vivo phosphorylation sites on HDAC5. Functional phosphomutantscreening and live cell imaging allowed the characterization of novel siteswithin functional domains, and identified a previously unrecognizedregulatory point of its nuclear import.(W5-4) Towards the Development of ProteomicsWorkflows for the Analysis of Samples Derived fromRefractory Plant TissuesT.W. ThannhauserUnited States Department of Agriculture/AgriculturalResearch Service, Ithaca, NY, United StatesCarrying out proteomic analyses in plant tissues involves dealing witha number of specialized challenges that can make protein extractionand quantification significantly more difficult than in other organisms.In addition to having relatively low protein concentrations, plant tissuesare often rich in proteases, protease inhibitors and other materials thatimpede protein analysis. These compounds include lipids, tannins,polysaccharides, and a large variety of secondary metabolites. Theextent of the problems encountered is dependent on tissue typestudied. Two major research thrusts in our lab involve detaileddevelopmental and time-course studies on staple crops and fruits. Anunderstanding of the proteins involved in the development of fruits,seeds, tubers and other plant organs will enhance our ability to controlthe agronomically-important traits of these crops including stabilityin storage, disease resistance, and vitamin and mineral content. Tworecent studies that have posed unique challenges for proteome analysisinclude 1) comparing protein expression in red ripe tomato fruit to thatin mature green tomato fruit and 2) studying the effects of storage onpotato tubers. This talk will focus on the problems encountered in theserecalcitrant tissue types and our efforts to provide plant scientists witha broader array of extraction and chemical modification protocols thanis represented by those that constitute the existing paradigm. Effortsto move away from narrowly defined prescriptive methods and movetowards results-based methods will also be discussed.(W6) Current State and Future of CapillaryElectrophoresis and Sanger SequencingJ. Kieleczawa 1 , D. Adam 2 , P. Schweitzer 3 , E. Vennemeyer 4 ,M. Zianni 51Pfizer, Inc., Cambridge, MA, United States; 2 NAPS Unit,Michael Smith Laboratories, University of British Columbia,Vancouver, BC, Canada; 3 DNA Sequencing and GenotypingLab, Cornell University, Ithaca, NY, United States;4LifeTechnologies, Carlsbad, CA, United States; 5 Plant-Microbe Genomics Facility, Ohio State University, Columbus,OH, United StatesCapillary Electrophoresis remains a widely used, often irreplaceable,sequencing technology continuing to experience increasing demands.We believe that the speed, simplicity and high quality of reads for lowvolumesequencing needs will maintain the viability of this technologyfor many years to come. Although the main thrust of developmentin the sequencing space is now in NGS area, there are still stepswhich can be improved or adjusted as other methods mature. In thissession we propose to cover: The effect of long-term storage of DNAunder different conditions (temp/buffers) on sequencing, spectralcharacteristics, integrity and transformation efficiency; A Descriptionof the Apollo system; and the Future of Capillary Electrophoresis andSanger sequencing. In addition to the featured talks, there will be aquestion & answer component in which participants are encouraged tobring up any issues relevant to this topic.Workshop SessionAbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 47

Workshop SessionAbstracts(W7) Cellular 3D Imaging(W7-1) 3D Cellular Imaging: Beyond the SimpleImaging ParadigmR. ColeWadsworth Center, New York State Department of Health,Albany, NY, United StatesThe modern microscope has advanced so far beyond what Leeuwenhoekinvented, it’s doubtful he would even recognize it as a microscope.Modern microscopes, or more acutely, imaging workstations, arean integrated compilation of optoelectronic components. It is nowpossible to produce much more than simple images using thesesystems. Quantitation of various metrics are now possible with theseadvanced instruments. With the advent of fluorescently-taggedproteins/organelles, it is possible to actually quantify sub-cellularamounts of these transgenically produced or exogenously labeledmacromolecules/complexes. Spectral un-mixing of overlapping signalsfrom within a single image (fluorescent and absorbent) has blurreddistinction between imaging and spectroscopy. It is now possible toget chemical data from techniques such as unmixing in addition tomore conventional modes such as polarized light microscopy. Theability to determine if two macromolecules are precisely co-localized(< 100 Å apart) is now routinely accomplished in both living and fixedpreparations with Fluorescence Resonance Energy Transfer (FRET). Thelatest technological advances has been achieving resolution beyondAbbe’s predicted limits. This has been accomplished both throughhardware and software improvements, such as Stimulated EmissionDepletion (STED), and Structured Illumination Microscopy (SIM) toname a couple.(W7-2) Basics of Colocalization AnalysesJ. LacosteCell Imaging and Analysis Network, Department of Biology,McGill University, Montreal, QU, CanadaFor many fluorescence microscopy-based projects, colocalizationanalyses are often the main objectives of the study, and detecting“yellow” is considered a proof of colocalization. However, the “yellow”statement is essentially a qualitative one which opens the door tomore quantitative approaches. This presentation will provide aquick overview of colocalization analysis methods. First the requiredpreliminary steps and words of caution will be discussed. Secondly,intensity correlation coefficient -based methods (both the traditionaland more modern ones) will be presented, and object-based analysismethods will next be introduced. Lastly, it is important to rememberthat colocalization studies are dependent on quantitative microscopyand as such requires careful considerations.(W8) Successful Production of FunctionalProteins(W8-1) Successful Protein ProductionJ. CulpPfizer, Inc., Groton, CT, United StatesSuccessful production of functional proteins is more than animmunoreactive band on a Western blot. Availability of multipleexpression vectors make accessible a variety of expression systems andparallel expression approaches can speed results and increase chanceof success. The next hurdle is isolation of the protein target in sufficientamounts and with sufficient purity to support subsequent experimentalwork. Occasionally, protein refolding is the only method available toachieve the desired protein. Finally, sufficient characterization ofthe purified protein is required to verify that the protein is in thebiologically relevant state. Often, the protein is heterogeneous withpost-translational modifications and may be aggregated or in multipleoligomeric complexes.(W8-2) Overcoming Problems in Protein Expressionand PurificationB. GilletteSAIC-Frederick, Inc., National Cancer Institute at Frederick,Frederick, MD, United StatesWe use a parallel, micro-scale protein purification approach to screenpurification outcomes for multiple expression constructs from multipleexpression systems. Method development and optimization for leadcandidates are then performed on the same platform. Scale-up fromthe micro-scale format (10-160 microliter column volumes available)to bench-top scale (5-100 milliliter column volume) has provenpredictive in terms of qualitative and quantitative results. The increasein throughput allows us to follow a “purify first” approach that, whilestill providing data on expression levels and solubility, also producesvaluable purification information within the same time-frame as atraditional solubility assessment approach.(W8-3) When Proteins Misbehave, Try Adding aLittle PressureR.M. PetrovichNational Institute of Environmental Health SciencesLaboratory of Structural Biology, Research Triangle Park,NC, United StatesThe most economical way to produce the mg quantities of purifiedprotein required by many studies still requires expression in E. coli.Unfortunately, between 50% and 70% the time, a given protein willnot express properly in bacteria. Of those that are correctly folded, apercentage of them will either lose activity during purification or coldstorage once purified. To overcome these obstacles we have begunusing high hydrostatic pressure refolding (Barofold) which allows us torefold protein at high concentration (1 mg/ml or higher) without addingand removing chaotropic agents.48 • ABRF 2011 — Technologies to Enable Personalized Medicine

(W9) Therapeutic Antibodies: Over-Hyped“Magic Bullet”, or Under-Explored Technology?D.L. Crimmins 1 , J.E. Harlan 2 , F. Weis-Garcia 31Washington University School of Medicine, Saint Louis, MO,United States; 2 Abbott Laboratories, Abbott Park, IL, UnitedStates, 3 Memorial Sloan-Kettering Cancer Center, New YorkCity, NY, United StatesBringing the vision that monoclonal antibodies (mAbs) could bemedicinal “magic bullets” into practice has been a long and challengingjourney which began in 1975 when Kohler and Milstein made it possibleto generate these homogenous, highly specific homing devices. In thelast 35 years, many hurdles have slowed the manifestation of this dream,including: target location; host species, specificity, and subclass of themAb; immunogenicity of murine mAbs; mAb pharmacokinetics (i.e.hepatic and renal clearance); and mAb form (i.e. natural, conjugatedto a toxin or radionuclide, humanized murine mAbs, scFvs). The workinvolved in overcoming these factors explains why few mAb’s have beenmade it to the clinic in more than 3 decades later. Nonetheless, over20 mAbs have been FDA-approved for human use since 1994. Sincethe total global therapeutic monoclonal antibody market increasedfrom $40 billion in 2009 to $45 billion in 2010, pursuit of these “magicbullets” is unlikely to wane anytime soon. This workshop is a roundtableformat what where we will collectively discuss what we have learned onthis long road of bringing mAbs to the clinic and try and look forwardto where we are going.(W10) Identification of Mechanism-BasedBiomarkers and Drug Targets Using PathwayAnalysis(W10-1) GeneXplain — Identification of CausalBiomarkers and Drug Targets in Personalized CancerPathwaysA. Kel 1 , F. Kolpakov 2 , V. Poroikov 3 , G. Selivanova 41geneXplain GmbH, Wolfenbuettel, Germany; 2 Instituteof Systems Biology Ltd., Novosibirsk, Russia; 3 Institute ofBiomedical Chemistry of Russian Academy of MedicalSciences, Moscow, Russia; 4 Microbiology and Tumor BiologyCenter (MTC), Karolinska Institutet, Stockholm, SwedenWe have developed a geneXplain TM platform (www.genexplain.com)for causal interpretation of data coming from microarray, proteomics,miRNA and ChIP-chip/seq experiments. GeneXplain TM applies aunique upstream analysis approach based on implementation ofmachine learning and graph topological analysis algorithms in orderto identify causality keynodes in the network of gene regulation andsignal transduction and combines it with full genome sequence analysisand chemoinformatics methods for drug discovery. The power of thisapproach is that we are trying to identify causal biomarkers — thosewhich are more than just correlating with disease or treatment outcomebut which are parts of the disease mechanism, which may differ inpatient cohorts. Such personalized networks are analyzed in order tofind key nodes — most important nodes triggering the disease. Thesekeynodes and genes directly influenced by them are considered aspromising biomarkers which can discriminate patients into cohortsfrom the disease mechanism point of view. In the current study, weanalyzed a large scale gene expression and ChIP-seq data from astudy of a breast cancer samples treated with antineoplastic agentsincluding the novel drug compounds — RITA and Nutlin, targeting p53and Mdm2. We analyzed promoters of downregulated pro-survivalgenes and identified combinations of transcription factors involvedin their regulation. Topological modeling of the signal transductionnetwork upstream of these transcription factors revealed key-nodes— potent master-regulators of the cell survival program that preventefficient apoptosis of cancer cells. We considered these key-nodeproteins (e.g. PI3K subunits) as causal biomarkers as well as prospectivetargets for novel anticancer drug combinations. We applied acheminformatics computer tool PASS to these targets and identifiedtwo novel prospective antineoplastic chemical compounds which wereexperimentally validated in a cellular assay confirming their synergisticpotential in highly selective triggering of apoptosis of cancer cells.(W10-2) Pathway Analysis in Expression ProteomicsR.A. ZubarevChemistry I Division, Department of Medical Biochemistryand Biophysics, Karolinska Institutet, Stockholm, SwedenProteomics studies have revealed unexpected plasticity and dynamicnature of the human proteome. The paradigm that the time evolutionof a biological system can be described by abundance variation ofrelatively few “regulated” proteins has been shuttered, being replacedby the growing understanding that the whole proteome is regulated,and no protein remains unaffected when the system undergoestransition from one state to another. This finding underlines theimportance of systems biology analysis of expression proteomics data.Systems biology shifts the analytical focus from thousands of proteins tohundreds of signaling pathways, thus reducing the number of entities tobe analyzed. Application of these methods required the developmentof novel systems biology tools, such as the pathway search engine (PSE[1-3]). These tools can only be effective when they are quantitative, i.e.predict not only the activated pathway, but also the relative degree of itsactivation. Introducing the quantitative aspect in systems biology is oneof the greatest challenges this field is facing today, since the final goalof pathway analysis, which is the creation of a quantitative predictingmodel of the biological process under investigation. 1. Zubarev, R. A.;Nielsen, M. L.; Savitski, M. M.; Kel-Margoulis, O.; Wingender, E.; Kel,A. Identification of dominant signaling pathways from proteomicsexpression data, J. Proteomics, 2008, 1, 89-96. 2. Stahl, S.; Fung, Y.M.E.;Adams, C. M.; Lengqvist, J.; Mörk, B.; Stenerlöw, B.; Lewensohn, R.;Lehtiö, J.; Zubarev, R. A.; Viktorsson, K. Proteomics and Pathway AnalysisIdentifies JNK-signaling as Critical for High-LET Radiation-inducedApoptosis in Non-Small Lung Cancer Cells, Mol. Cell Proteomics, 2009,8, 1117-1129. 3. Marin-Vicente, C.; Zubarev, R. A. Search engine forproteomics, Fact or Fiction? G.I.T. Lab J, 2009, 11-12, 10-11.(W10-3) Metabolic Biomarkers, MetabolicNetworks, and Pathway AnalysisV. TolstikovUniversity of California Davis Genome Center, Davis, CA,United StatesWe apply well established MS-based Metabolomics platform towardsthe detection, characterization and identification of small moleculebiomarkers. Data acquisition is performed with the assistance of GC/TOF/MS line and LC/MS line. Sophisticated data mining approach usingdifferent algorithms deals with electron impact (EI) generated data as wellas data generated with the unit resolution, high resolution and ultrahighWorkshop SessionAbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 49

Workshop SessionAbstractsresolution hybrid instruments having API sources. These competitiveapproaches deliver exciting results on selection of the predictors’ paneland potential metabolic biomarkers discovery. We here report onefficiency of Metabolic Biomarkers Discovery Project as a platform forcases study: Kidney diseases (RCC and PKD); Pancreatic Cancer (PDAC);Human Embryonic Stem Cells functional characterization. However,Metabolic Networks simulation and Metabolic Pathway Analysis,which could be essential tools for Metabolic Biomarkers validation andconnections with underlying biochemical processes are still difficultand approached not systematically, but rather case wise. Challengesand developments in this field will be further presented and discussed.(W11) The Business of Running a Core FacilityN.P. Ambulos, Jr. 1 , S.A. Bobin 21University of Maryland School of Medicine, Baltimore, MD,United States; 2 Dartmouth School of Medicine, Hanover, NH,United StatesThe success and sustainability of a Core Facility has been dependenton factors which include (1) the ability to maintain state-of-thearttechnology, (2) recruiting and retaining talented technical staff,(3) a strong financial management plan, and (4) strong Institutionalsupport from both end users and the Administration. Historically,most core facilities enjoyed generous subsidies from their InstitutionalAdministration, which have helped defray costs for end users, purchasenew instrumentation, or to hire new staff. In light of the current economy,it is likely that many core facilities are now seeing these subsidiesdrastically cut, or even eliminated. In essence, core facilities are facedwith operating as a self-supporting ‘business’, and at the same time,ensure compliance with federal guidelines. This workshop will presentideas, both conventional and creative, that might provide core directorswith the tools necessary to continue to sustain a successful core facility.This will set the stage for an open discussion with the audience toencourage a free-flow of additional ideas that could benefit the futuresuccess of our cores.(W12) Microarrays: The Reports of My DeathHave Been Greatly Exaggerated(W12-1) Chip or Seq: Helping Clients ChooseD. BaldwinUniversity of Pennsylvania, Penn Molecular Profiling Facility,Philadelphia, PA, United StatesThe Penn Molecular Profiling Facility provides services using microarrayand deep sequencing platforms for a variety of applications. Drawingfrom experiences helping prospective clients to choose one approachor the other, and sometimes both, this portion of the workshop willaddress some of the factors to consider when thinking about a newproject. The budgeting process will be discussed, with particular focuson the use of interactive cost calculators.(W12-2) Microarray Analysis of FluorescenceActivated Cell Sorter-Derived Cells: CreatingHarmony Between TechnologiesS. TigheUniversity of Vermont, Advanced Genome Technology Core,Burlington, VT, United StatesAlthough microarray technology is well-established in both theresearch and clinical fields, it continues to evolve into new areas thatrequire new methods for the successful isolations of nucleic acid fromnon-traditional sources. Because RNA specifically is a labile molecule,special procedures and considerations must be implemented to avoiddegradation from methods such as fluorescence activated cell sorting(FACS) and laser capture microdissection (LCM) to name a few. Thispresentation will discuss specific methodologies to maximize the successof nucleic acid recovery from these approaches including instrumentpreparation, extraction methods, and the use of special reagents todeal with problematic samples.(W12-3) Microarray Futures: Don’t DecommissionYour Scanners Just YetS.D. CrosbyDepartment of Genetics, Washington University School ofMedicine, St. Louis, MO, United StatesThe first attempt to wind down the Washington University MicroarrayFacility was made by Genome Sequencing Center in 2008. It wasclear to most of us involved in the attempt that, with the advent ofNGS, microarrays were headed the way of differential display PCR(remember that!?). The attempt failed, and 2010 was the busiest yearyet for our microarray facility. Sequence remains a bit too expensiveand complex for many to make the leap. In addition, because of therarity of variants and the modest number of bases required to identifya locus, most sequence data is superfluous. Beyond that, as the actualcost of sequence continues to fall, so do the price of arrays, while thedensity of elements of the latter rises. It seems that until the advent ofreagentless sequencing, it is unlikely that the actual cost of sequencingwill be less than the cost of an array that covers the same loci. Whilesequencing technology will remain an important tool for discovery,over time most exons and biologically relevant variants will be capturedin toto on low cost arrays. Hospital or contract labs will (indeed arealready) use small, cheap arrays that capture disease relevant genes andvariants.50 • ABRF 2011 — Technologies to Enable Personalized Medicine

(W13) Institutional Core ManagementS. Meyn 1 , P. Turpen 2 , G.K. Farber 3 , S. Mische 4 ,P. Alexander 5 , J. Auger 61Vanderbilt University Medical Center, Nashville, TN, UnitedStates; 2 University of Nebraska Medical Center, Omaha,NE, United States; 3 National Center for Research Resources,Bethesda, MD, United States; 4 New York University LangoneMedical Center, New York, NY, United States; 5 MorehouseSchool of Medicine, Atlanta, GA, United States; 6 University ofCalifornia San Francisco, San Francisco, CA, United StatesThis workshop session will focus on issues related to InstitutionalCore Management, in response to the national conversation evolvingaround research core facility issues and management. The workshopwill be formatted as an experts’ panel; each participant currentlyplays an important role in supporting and developing research coreresources at an institutional level. Some of the topics to be discussedinclude: (1) Core Consolidation — one size fits all? (2) Bottom-up vs.top-down management, advantages and disadvantages of centrallymanaged cores. (3) Performance metrics and impacts on professionaldevelopment, core infrastructure support and improved operations.(4) Impacts of NIH-NCRR programs on improving access to researchresources, including core facilities. We also plan to highlight the newCore Administrators Network Coordinating (CAN). In responseto an emerging trend to centralize the oversight of research corefacilities, ABRF has fostered development of this network and anew committee: the Core Administrators Network-CoordinatingCommittee (CAN-CC). The committee seeks input and participationfrom scientists, administrators and others with an interest in issuesrelated to the administration of research core facilities which, by thenature of their service role, must interface with multiple constituencieswithin a research enterprise. Today many institutions have establishedadministrative positions designed to assist core facilities withmanagement of economic, regulatory and performance issues. In orderto facilitate greater interaction between and among core scientistsand administrators, the mission of the CAN-CC is to contribute to thecommon interests of core administrators, and promote interactions withcore scientists in a collegial and productive manner. The specific goalsof the Core Administrators Network Coordinating Committee (CAN-CC) are: to identify and reach out to our target community; provideopportunities for networking; and assess goals for program focus anddevelopment.(W14) Next Generation Sequencing Softwarefor Data Management, Analysis, andVisualization(W14-1) Tools for Next Generation Sequencing DataAnalysisK. BodiTufts University School of Medicine, Tufts University CoreFacility, Boston, MA, United StatesAs NGS technology continues to improve, the amount of data generatedper run grows exponentially. Unfortunately, the primary bottleneckin NGS studies is still bioinformatics analysis. Not all researchershave access to a bioinformatics core or dedicated bioinformatician.Additionally, much of the software for NGS analyses is written to runin a Unix / Linux environment. Researchers unfamiliar with the Unixcommand line may be unable to use these tools, or face a steep learningcurve in trying to do so. Commercial packages exist, such as the CLCGenomics Workbench, DNANexus, and GenomeQuest. However,these commercial packages often incorporate proprietary algorithmsto perform data analysis and may be costly. Galaxy provides a solutionto this problem by incorporating popular open-source and communitylinux command line tools into an easy to use web-based environment.After sequence data has been uploaded and mapped, there are avariety of workflows for NGS analyses that use open-source tools. Thisincludes peak-calling analyses for ChIP-Seq (MACS, GeneTrack indexer,Peak predictor), RNA-Seq (Tophat, Cufflinks), and finding smallinsertions, deletions, and SNPs using SAMtools. Any researcher canapply a workflow to his NGS data and retrieve results, without having tointeract with a command line. Additionally, since Galaxy is cloud-based,expensive computing hardware for performing analyses is not needed.In this presentation we will provide an overview of two popular opensourceRNA-Seq analysis tools, Tophat and Cufflinks, and demonstratehow they can be used in Galaxy.(W14-2) GenomeView: Visualizing the Next-Generation of DataT. AbeelBroad Institute of MIT and Harvard, Cambridge, MA, UnitedStates, and VIB Department of Plant Systems Biology, GhentUniversity, Ghent, BelgiumDue to recent advances in sequencing technologies, billions of nucleotidesequences are now produced on a daily basis. A major challenge isto visualize these data, including both whole genome sequence andtranscriptome data, for further downstream analyses. Visualization isoften overlooked and undervalued, but it is an extremely valuable toexplore your data on several levels. A first area where visualization shinesis at the early stages of data analysis to perform sanity checks on yourdata. Eye-balling your data in a visually pleasing way is the best way toget a good feel on what came out of your experiments. Once you havea good idea of what is in your data a good visual representation can beused to generate new hypotheses and to fine-tune analysis parameters.The appropriate image often makes the solution obvious and as such, itreally makes it easier to develop algorithms. The ability to interactivelyexplore gives you insights in large-scale data sets and definitelyaugments our ability to reason about complex data. To this end, wepresent GenomeView, a stand-alone sequence browser specificallydesigned to visualize and manipulate a multitude of genomics data.GenomeView enables users to dynamically browse high volumes ofaligned short read data, with dynamic navigation and semantic zooming,from the whole genome level to the single nucleotide. At the same time,the tool enables visualization of whole genome alignments of dozens ofgenomes relative to a reference sequence. GenomeView is unique in itscapability to interactively handle huge data sets consisting of dozensof aligned genomes, thousands of annotation features and millions ofmapped short reads both as viewer and editor. GenomeView is freelyavailable for academic use as an open source software package athttp://genomeview.org.Workshop SessionAbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 51

(W14-3) Galaxy Next Generation SequencingFunctionality from Sample Tracking to SNP CallingG. Von Kuster 1 , The Galaxy Team 1,21Center for Comparative Genomics and Bioinformatics,Pennsylvania State University, University Park, PA, UnitedStates; 2 Departments of Biology and Mathematics &Computer Science, Emory University, Atlanta, GA, UnitedStatesA new generation of DNA sequencing technologies has enabled a varietyof novel genome-scale experimental techniques. What is perhapsmost unique about this recent data explosion is that it is distributed— relatively inexpensive instruments allow any lab or institution toproduce enormous amounts of data. Yet the infrastructure upstreamand downstream of sequencing instruments is largely undeveloped.In addition to the instrument cost labs, core facilities and sequencingservice providers are forced to earspend thousands on commercialLIMS systems and sequence analysis packages, which are in-turn basedon tools from the public domain. Galaxy provides a robust open-sourcealternative. It’s lightweight sample tracking system is aimed at helpingsmall labs and core facilities managing requests for sequencing runs. Itallows one to track the entire “life-cycle” of sequencing request fromthe initial sample to the resulting dataset. Once the run is completethe user can apply a variety of NGS tools including format converters,mappers, ChIP-seq and transcriptome utilities. Results of these analysescan be visualized, shared, and published. In this presentation we willdemonstrate sample tracking functionality from the moment of samplesubmission to the sequencing facility, through the sequencing run, untilthe sample becomes a dataset and can be analyzed with a variety ofNGS tools.(W15) ETD Workshop Seminar Series: ManualInterpretation of Electron Transfer Dissociation(ETD) Mass Spectra of PeptidesD.F. HuntDepartments of Chemistry and Pathology, University ofVirginia, Charlottesville, VA, United StatesElectron transfer dissociation mass spectrometry is a break-thoughtechnology for sequencing post-translationally modified peptides.In this technique, radical anions of polyaromatic hydrocarbons(fluoranthene and azulene) are employed to transfer electrons tothe carbonyl group in the polyamide backbone of multiply chargedpeptides generated by electrospray ionization. Capture of an electroninto the peptide backbone reduces the positive charge on the ion byone, and forms a carbonyl radical anion that then abstracts a protonfrom a nearby protonated amino group. The resulting carbonyl radicaltriggers cleavage of the adjacent nitrogen-carbon bond to producefragments of type c’ and z•. The purpose of the workshop is toprovide instruction on how to manually interpret peptide ETD massspectra. Following a lengthy tutorial about ion structures, fragmentationpathways, predictable changes in fragment ion isotope patterns, etc.,we will outline a general approach for the manual interpretation ofpeptide ETD spectra, solve the sequence of several post-translationallymodified peptides, assign homework spectra, and reconvene on thesecond day of the workshop to go over the homework problems. Apacket of lecture notes and handouts will be provided. Attendeesshould bring an inexpensive calculator, pad of paper, and a small ruler.Workshop SessionAbstracts52 • ABRF 2011 — Technologies to Enable Personalized Medicine


Research Group Presentation AbstractsResearch GroupPresentation Abstracts(R1) Joint Session: Proteome InformaticsResearch Group (iPRG) & Proteomics StandardsResearch Group (sPRG)(R1a) iPRG 2011: A Study on the Identification ofElectron Transfer Dissociation (ETD) Mass SpectraL. Martens 10 , M. Askenazi 1 , N. Bandeira 2 , R.J. Chalkley 3 ,K.R. Clauser 4 , E. Deutsch 5 , H.H.N. Lam 6 , W.H. McDonald 7 ,T. Neubert 8 , P.A. Rudnick 91Dana-Farber Cancer Institute, Boston, MA, United States;2University of California San Diego, La Jolla, CA, UnitedStates; 3 University of California San Francisco, San Francisco,CA, United States; 4 The Broad Institute of MIT and Harvard,Cambridge, MA, United States; 5 Institute for SystemsBiology, Seattle, WA, United States; 6 University of Scienceand Technology, Hong Kong, China; 7 Vanderbilt UniversitySchool of Medicine, Nashville, TN, United States; 8 NewYork University School of Medicine, New York, NY, UnitedStates; 9 National Institute of Standards and Technology,Gaithersburg, MD, United States; 10 Ghent University and VIB,Ghent, BelgiumThe field of mass spectrometry based proteomics has seen several keyinnovations over the last several years, including novel experimentalmethods, new instruments, and unique fragmentation strategies. Thelatter, in the form of electron capture dissociation (ECD) and electrontransfer dissociation (ETD) have captured the imaginations of manyresearchers, expanding their ability to identify and analyze peptidesand proteins. However, since ECD/ETD spectra differ substantial frommore traditional collision induced dissociation (CID) spectra in boththeir prominent ion series as well as their preferred bond-breakingcharacteristics, the (automatic) interpretation of ECD/ETD spectrarequires novel algorithm optimizations. Efficient identification of ECD/ETD spectra thus remains an active and exciting field of proteomicsinformatics research. In this work, the ABRF Proteome InformaticsResearch Group (iPRG) presents the results of a collaborative studyfocusing on the analysis of an LC-MS/MS dataset from a yeast lysatedigested with Lys-C and enriched for highly charged peptides usingstrong cation exchange fractionation. The data derived from one fractionanalyzed exclusively by ETD was distributed to participants for analysisin several equivalent formats, along with a standardized sequencedatabase derived from the UniProtKB/Swiss-Prot yeast complement,a decoy version of this database, and an applicable spectral library.Participants were free to use any and all methods available to them toidentify this fraction, and results were to be submitted using an Exceltemplate. All participant identities were subsequently anonymized, anda survey was used to collect information about participant experienceand software tools used to produce the submitted analysis. This uniformcollection of data has allowed a thorough comparison of participantresults. A summary, including a comparison of results submitted bymembers of the iPRG, will be presented.(R1b) ABRF-sPRG2011 Study: Development ofa Comprehensive Standard for Analysis of Post-Translational ModificationsA. Ivanov 1 , C. Colangelo 2 , C.P. Dufresne 3 , J.G. Farmar 4 ,D.B. Friedman 5 , C. Kinsinger 6 , K.S. Lilley 7 , K. Mechtler 8 ,B.S. Phinney 9 , S.A. Shaffer 10 , S.T. Weintraub 111Harvard University School of Public Health, Cambridge,MA, United States; 2 Yale University, New Haven, CT, UnitedStates; 3 Thermo Fisher Scientific, West Palm Beach, FL,United States; 4 University of Virginia, Charlottesville, VA,United States; 5 Vanderbilt University, Nashville, TN, UnitedStates; 6 National Cancer Institute, Bethesda, MD, UnitedStates; 7 University of Cambridge, Cambridge, UnitedKingdom; 8 Research Institute of Molecular Pathology,Vienna, Austria; 9 Proteomics Core, University of CaliforniaDavis Genome Center, Davis, CA, United States; 10 Universityof Massachusetts Medical School, Worcester, MA, UnitedStates; 11 University of Texas Health Science Center at SanAntonio, San Antonio, TX, United StatesThe Proteomics Standards Research Group (sPRG) has initiated a studythat focuses on development of a standard that can be used in bothassessment of a laboratory’s ability to detect an array of post-translationalmodifications in a complex proteomic sample and development ofnew approaches for characterization of post-translationally modifiedproteins. The sample that has been generated for the first stage of thisstudy contains a mixture of more than seventy synthetic peptides with avariety of modifications, in a tryptic digest of six proteins. Modificationsrepresented in the sample include acetylation, methylation, nitration,phosphorylation, and sulfation. The individual proteins were purified,digested and analyzed prior to formulation of the sample. Thesynthetic peptides were each analyzed and then mixed with the digests;the mixture was aliquoted and lyophilized in sufficient quantities thatwould permit evaluation by several analytical approaches. The samplewas fully characterized by members of the sPRG. It is planned that in2011, the sample will be distributed to requesting laboratories for alarger study focusing on PTM characterization. The sPRG will presentresults obtained at different stages of preparation and characterizationof the sample. Approaches for analysis of complex proteomic samplescontaining various post-translationally modified proteins will also bediscussed. The sPRG is planning to open the study for sample requestsat the ABRF 2011 conference. From the responses returned by theparticipating laboratories, the sPRG will subsequently report on theeffectiveness of the approaches used for characterization of posttranslationalmodifications in protein digests.54 • ABRF 2011 — Technologies to Enable Personalized Medicine

(R2) Antibodies: Moving Closer to PersonalizedTherapeutics and DiagnosticsB.R. Curtis 1 , R. Umeck 2 , J.E. Harlan 31Blood Center of Whisonsin, Milwaukee, WI, United States;2Meso Scale Discovery, Gaithersburg, MD, United States;3Abbott Laboratories, Abbott Park, IL, United StatesThe possibilities of personalized medicine are largely rooted insuccessfully exploiting the ever-growing genome and proteome-wideunderstanding of human biology. Translating this knowledge base intothe clinic requires, among other things, being able measure the patient’sgenomic and proteomic disease signature in a routine manner. Thus,expanded focus on assays and platforms capable of measuring thesespecific signatures within tens, hundreds or even thousands of complexpatient samples simultaneously is critical. Antibody-based assays andplatforms have become a centerpiece of many technologies pushingthese boundaries because of the antibody’s intrinsic specificity, diverserecognition capacity and ease with which they can be produced.Furthermore, these same characteristics have made them an equallypromising prospect for therapeutic approaches. This seminar trifectawill highlight the pivotal role antibodies have in multiplex biomarkerassay development and therapeutic venues. Specifically: 1. BrianCurtis will discuss a robust multiplex assay platform to detect humanplatelet auto-antibodies and platelet antigen polymorphisms bycombining bead array technologies with flow cytometry. Such asimple, yet high-throughput platform would facilitate routine testingfor platelet disorders like neonatal alloimmune thrombocytopenia,multi-platelet transfusion refractoriness, and post-transfusion purpura;2. Robert Umeck will summarize the challenges in developingantibody-based multiplex assays and how Meso Scale Discoveryutilizes an electrochemiluminescence detection system combinedwith a patterned multi-array format to generate multiplex assays fornearly any application. Several actual examples will be presented todemonstrate how this approach could support personalized medicine;3. John Harlan will introduce and discuss the evolution of therapeuticantibodies. To illustrate where the field is venturing, he will highlightAbbott Laboratory’s new bi-specific antibody format.(R3) Joint Session: Genomic Variation ResearchGroup (GVRG) & DNA Sequencing ResearchGroup (DSRG)(R3a) Evaluation of DNA Whole GenomeAmplification Technologies for GenotypingA. Hutchinson 1 , C. Dagnall 1 , C. Nicolet 2 , H. Escobar 3 ,S. Blake 4 , B. Sanderson 5 , B. Kingham 6 , K. Jonscher 71Core Genotyping Facility, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, United States; 2 Data ProductionFacility, USC Epigenome Center, Los Angeles, CA, UnitedStates; 3 DNA Sequencing Operations, Eurofins MWGOperon, Huntsville, AL, United States; 4 DNA Core Facility,University of Missouri, Columbia, MO, United States; 5 LifeTechnologies, Austin, TX, United States; 6 Director, DNASequencing & Genotyping Center, University of Delaware,Newark, DE, United States; 7 Director, SBCF and TATCProteomics, University of Colorado Denver, Denver, CO,United StatesThe evolution of genomic technologies is occurring rapidly and oftenrequires large amounts of source DNA. There is also an expandeddesire to analyze smaller numbers of cells for higher resolution studiesas well as to take advantage of large numbers of archived samples (e.g.FFPE, serum, etc.). To provide enough material for the newest genomictechnologies, whole genome amplification (WGA) has reemerged as animportant and necessary technique. With some new WGA productson the market, we have evaluated the quantity and quality of WGAproducts generated as well their performance on some of the currentgenomic applications. The GVRG has completed a benchmarkingstudy evaluation of 6 commercially available WGA kits using severalgenotyping assays. Utilizing 6 samples, the different WGA kits weretested following the manufacturer’s protocols. The samples includedCoriell DNA from a trio of CEPH individuals, 2 FFPE DNAs, bothfrom the same individual (1 newly extracted and the other extracted2 years ago), and 1 sample from the Coriell trio that was fragmentedprior to amplification for a total of 6 samples tested. The WGA kitsincluded those from Sigma, NuGen, Qiagen and GE Life Sciences. The6 samples were amplified as blind duplicates at several GVRG memberlab sites resulting in a total of 78 amplified products. Quality metricswere performed on the 78 amplified products using several differenttechniques in order to provide measurements on yield, fragment size,and concentration. Several widely used genotyping methods wereused to evaluate the amplification products, including Illumina HumanOmni1 Quad Beadchip, TaqMan copy number and SNP genotypingassays, and STR genotyping. Data will be presented on genotypingconcordance and loss of heterozygosity checks across the differentplatforms where possible.Research GroupPresentation AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 55

Research GroupPresentation Abstracts(R3b) DNA Sequencing Research Group (DSRG)(R3b-1) Comparison of Custom Target EnrichmentMethods; Agilent vs. NimblegenA. Perera 4 , K. Bodi 1 , P.S. Adams 2 , D. Bintzler 3 , K. Dewar 5 ,D.S. Grove 6 , J. Kieleczawa 7 , R.H. Lyons 8 , T. Neubert 9 ,A.C. Noll 1 , S. Singh 10 , R. Steen 11 , M. Zianni 121Tufts University, Boston, MA, United States; 2 TrudeauInstitute, Saranac Lake, NY, United States; 3 DNA Analysis,Inc., Cincinnati, OH, United States; 4 Stowers Institute, KansasCity, MO, United States; 5 McGill University, Montreal, QC,Canada; 6 Pennsylvania State University, University Park,PA, United States; 7 Pfizer Research, Cambridge, MA, UnitedStates; 8 University of Michigan, Ann Arbor, MI, UnitedStates; 9 New York University, New York, NY, United States;10University of Minnesota, Minneapolis, MN, United States;11Harvard Medical School, Cambridge, MA, United States;12Ohio State University, Columbus, OH, United StatesOver the last four years, we witnessed the tremendous advances inNext Generation Sequencing (NGS) that have dramatically decreasedthe cost of whole genome sequencing. However, the cost of sequencinglarger genomes is still significant. In addition and depending on thegoal of study, whole genome sequencing creates a large amount ofadditional/auxiliary data that complicates data analysis. There areseveral commercial methods available for isolating subsets of genomesthat greatly enhance the efficiency of NGS by allowing researchersto focus on their regions of interest. For the 2009-11 DSRG study,we compared products from two leading companies; Agilent andNimblegen that offer custom enrichment methods. Both companiesobtained the same genomic DNA stock and performed DNA captureon the same specified regions. Following capture, the Illumina GenomeAnalyzer IIx system was used, in two different laboratories, to generatethe sequence data. We present our data comparing in terms of cost,quality, reproducibility and most importantly completeness and depthof coverage. Acknowledgements: We would like to thank Agilent,Illumina and Nimblegen for all their support in making this studypossible.(R3b-2) A Methodology Study for MetagenomicsUsing Next Generation SequencersS. Singh 1 , D. Grove 21Biomedical Genomics Center, University of Minnesota,Saint Paul, MN, United States; 2 Genomics Core Facility, HuckInstitutes for Life Sciences, Pennsylvania State University,University Park, PA, United StatesMetagenomics is one of several genomics applications, which hasbenefited immensely from the high throughput and cost efficacy ofNext generation sequencers. And although hundreds of studies onmetagenome analysis have been published over the past few years,the methodology for conducting them is still very much evolving.In this DSRG study we will evaluate the influence of various samplepreparation methods, specifically DNA extraction and amplificationapproaches, on data output along with a comparative analysis of Nextgeneration sequencing platforms. We will study the effect of thesedifferent experimental and technical strategies on determination ofsample biodiversity.(R4) Joint Session: Protein Sequencing ResearchGroup (PSRG) & Glycoprotein Research Group(gPRG)(R4a) PSRG 2011 Study Results: SensitivityAssessment of Terminal Sequencing TechniquesUsing an Unknown ProteinJ.J. Walters 1 , W. Sandoval 2 , K. Mawuenyega 3 ,J.S. Smith 4 , H. Remmer 5 , B. Xiang 6 , D. Suckau 7 , V. Katta 2 ,P. Hunziker 81Sigma-Aldrich, St. Louis, MO, United States; 2 Genentech,Inc., South San Francisco, CA, United States, 3 WashingtonUniversity School of Medicine, St. Louis, MO, United States,4University of Texas Medical Branch, Galveston, TX, UnitedStates, 5 University of Michigan, Ann Arbor, MI, UnitedStated, 6 Monsanto Company, St. Louis, MO, United States,7Bruker Daltonics, Bremen, Germany, 8 University of Zurich,Zurich, SwitzerlandEstablishing the N-terminal sequence of intact proteins plays a criticalrole in biochemistry and potential drug development. N-terminalsequence analysis is necessary for quality control of protein biologics,for determining sites of signal peptide cleavage events, as a first stepin elucidating the sequences of genes from uncommon species andfor the characterization of monoclonal antibodies. Automated Edmandegradation has been the method of choice for these analyses. However,alternate methods for N-terminal sequence analysis have emerged. Therecent PSRG studies have established that Edman sequencing and massspectrometry based techniques have varied strengths and weaknessesdepending on several experimental factors and both play an importantrole in terminal sequencing. With this complimentary role realized,the 2011 PSRG study attempts to evaluate the sensitivity limits of thevarious sequencing techniques. The PSRG distributed three samplesets of 3 tubes each, varying by sample format (lyophilized, gel sliceor membrane piece). Each set of three samples contains the samerecombinant protein with increasing amounts of material. The sequenceof this protein is not listed in any database. Participants could requestany one, two, or all three sample sets. Including PSRG committee, a totalof 38 participants requested 74 sample sets. The participants wereasked to determine as many amino acids from both termini by theirmethod of choice, and were encouraged to try multiple methods forsequence elucidation. Study participants were directed to a websiteto anonymously upload sequences and supporting data. Our analysisfocuses on the length and accuracy of the sequence calls reported bythe participants, and how that compares with decreasing amounts ofprotein and the type of sample format analyzed. A comparison of theresults obtained by Edman chemistry and by alternative technologiesas well as information on the type of instruments and protocols isreported.56 • ABRF 2011 — Technologies to Enable Personalized Medicine

(R4b) gPRG: Toward Consensus on Glycan Analysis:Reliable Methods and ReproducibilityJ. Zaia 1 , D. Kolarich 2 , R. Orlando 31Boston University, Boston, MA, United States; 2 Max PlanckInstitute of Colloids and Interfaces, Berlin, Germany; 3 TheUniversity of Georgia Carbohydrate Research Center,Atlanta, GA, United StatesThe field has undertaken over the past few years a series of studies toevaluate methods for glycan analysis. There are several methods thatare widely used at this time for characterization of glycoprotein glycansthat appear to be generally reliable. These include (1) reductiveamination followed by reversed phase chromatography, (2) reductiveamination with matrix assisted laser desorption/ionization (MALDI)mass spectrometry (MS), (3) reductive amination with electrosprayliquid chromatography-MS, and (4) permethylation with tandem MS.The ABRF glycoprotein research group (gPRG) is planning a study inwhich participant laboratories carry out glycan analysis according toprotocols representing best practice in the field. The results will bepresented at the 2012 ABRF meeting. The results will be useful fordemonstrating the reproducibility of glycan analysis using comparableprotocols. They will also highlight the extent to which different methodsbias the glycan analysis results obtained. This workshop will summarizethe history and use of the glycan analysis methods proposed for the2012 study. There will be opportunity for discussion and comment onthese methods.(R5) Joint Session: Nucleic Acids Research Group(NARG) & MicroArray Research Group (MARG)(R5a) Determining miRNA Expression Levels inDegraded RNA Samples Using Real-Time RT-qPCRand Microarray TechnologiesS. Chittur 1 , S. Tighe 2 , J. Holbrook 3 , V. Nadella 4 ,R. Carmical 5 , K. Sol-Church 3 , A.T. Yueng 61State University of New York at Albany, Albany, NY, UnitedStates; 2 University of Vermont, Burlington, VT, United States;3Nemours/A.I. duPont Hospital for Children, Wilmington, DE,United States; 4 Ohio University, Athens, OH, United States;5The University of Texas Medical Branch, Galveston, TX ,United States; 6 Fox Chase Cancer Center, Philadelphia, PA,United StatesThe Nucleic Acid Research Group (NARG) has previously conductedstudies evaluating the impact of RNA integrity and priming strategieson cDNA synthesis and real-time RT-qPCR. The results of last year’s fieldstudy as it relates to degraded RNA will be presented. In continuationof the RNA integrity theme, this year’s study was designed to evaluatethe impact of RNA integrity on the analysis of miRNA expressionusing real-time RT-qPCR. Target section was based on data obtainedby the Microarray Research Group (MARG) and other published datafrom next gen sequencing. These 9 miRNAs represent three groupsof miRNA that are expressed at low, medium or high levels in the FirstChoice human brain reference RNA sample. Two popular RT primingstrategies tested in this study include the Megaplex miRNA TaqManassay (ABI) and the RT2 miRNA qPCR assay (Qiagen/SA Biosciences).The basis for the ABI assay design is a target-specific stem-loopstructure and reverse-transcription primer, while the Qiagen designcombines poly(A) tailing and a universal reverse transcription in onecDNA synthesis reaction. For this study, the human brain referenceRNA was subject to controlled degradation using RNase A to RIN (RNAIntegrity Number) values of 7 (good), 4 (moderately degraded), and2 (severely degraded).These templates were then used to assess bothRT methods. In addition to this real-time RT-qPCR data, the same RNAtemplates were further analyzed using universal poly(A) tailing andhybridization to Affymetrix miRNA GeneChips. This talk will provideinsights into RT priming strategies for miRNA and contrast the qPCRresults obtained using different technologies.(R5b) Microarray Research Group Projects, 2010-11D. Baldwin 1 , N.G. Reyero-Vinas 2 , N. Jafari 31Penn Molecular Profiling Facility, University of Pennsylvania,Philadelphia, PA, United States; 2 Jackson State University,Jackson, MS, United States; 3 Northwestern University,Evanston, IL, United StatesMembers of the MARG will discuss our research projects: Comparisonof microarray and deep sequencing platforms for microRNA profiling,Performance of a synthetic human microRNA reference panel,Participation in the SEQC Sequencing Quality Control consortium, andRNA-Seq profiling of environmental samples exposed to the Gulf oilspill.(R6) Joint Session: Protein Expression ResearchGroup (PERG) & Molecular InteractionsResearch Group (MIRG)(R6a) PERG Research Group Presentation: RefoldingStudyC. KinslandCornell University, Ithaca, NY, United StatesCurrently, the overwhelming majority of protein purification projectsstart with a recombinant protein expressed in a suitable host. For arange of reasons, E. coli is the predominant expression host yet a largepercentage of proteins expressed therein are found in an insolubleform called inclusion bodies. Inclusion bodies have the advantage ofconsisting of relatively homogeneous protein, which can simplify thepurification process. However, this leaves the challenge of solubilizingand refolding the protein into its native and biologically active structure.The conditions for efficient refolding are particular for each protein andthere are a wide range of methods to choose from. For this and otherreasons, many researchers are hesitant to pursue a refolding strategyto obtain a target protein. An overview of refolding methods andstrategies will be presented along with a description of the upcomingProtein Expression Research Group (PERG) refolding study.Research GroupPresentation AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 57

Research GroupPresentation Abstracts(R6b) Conclusions from the MIRG 2010 BenchmarkStudy: Molecular Interactions in a Three ComponentSystem and Presentation of 2011 Survey Results onLabel-Free TechnologiesA.P. Yamniuk 1 , S.P. Yadav 5 , S. Bergqvist 2 , M.L. Doyle 1 ,E. Eisenstein 3 , M.K. Robinson 4 , T. Neubert 61Bristol-Myers Squibb, Princeton, NJ, United States; 2 Pfizer, LaJolla, CA, United States; 3 University Maryland BiotechnologyInstitute, Baltimore, MA, United States; 4 Fox Chase CancerCenter, Philadelphia, PA, United States; 5 Cleveland ClinicFoundation, Cleveland, OH, United States; 6 New YorkUniversity School of Medicine, New York, NY, United StatesCharacterizing the assembly of multi-protein complexes and thecompetition between multiple protein ligands for a given target arecommon challenges faced by core facilities. The MIRG2010 Benchmarkstudy was designed to assess participants’ ability to correctly describethe interactions between two protein ligands and their target proteinusing primarily biosensor technologies such as surface plasmonresonance. Participants were provided with microgram quantities ofthree proteins (A, B and C) and asked to determine if a ternary A-B-Ccomplex can form, or if ligands B and C bind competitively to proteinA. This presentation will summarize the conclusions from the 2010Benchmark Study, and provide perspective on the potential for futureapplication of this system as a reference standard for quantitativecharacterization of protein-protein interactions using biosensortechnologies. The field of label-free biophysical technologies likesurface plasmon resonance (SPR) and isothermal calorimetry (ITC) arebecoming indispensable in translational research and in the discoveryphase of biotherapeutics. Investigators are much more aware about thedevelopments in biomolecular interaction analysis using SPR and ITCand usefulness of these technologies in designing better drugs basedon biomolecules and vaccines. The Molecular Interaction ResearchGroup (MIRG) of ABRF has conducted an on-line survey to capture therecent explosive developments in these technologies. The survey wastargeted to both academia and pharmaceutical industry and the surveydata will be presented during the meeting.(R7) Light Microscopy Research Group (LMRG)(R7-1) Point Spread Functions, Spectral Calibration,and BeyondR. Stack, R. ColeWadsworth Center, New York State Department of Health,Albany, NY, United StatesModern light microscopes are highly evolved opto-electronicmechanicaldevices. Establishing instrument performance is crucial inensuring that reliable and accurate images can be acquired. Imagers, aswell as granting agencies, need to be confident that data collected willbe uniform and quantifiable both temporally and from instrument toinstrument. Last year, in phase-one of our world-wide research studyon instrument performance, we successfully concentrated our effortson three image-based tests: long and short term stability of illuminationsources, uniformity of field illumination, and co-registration acrossvarious wavelengths. A manuscript summarizing the phase-one studyhas been submitted to and accepted by Microscopy & Microanalysis,one of the highest rated imaging journals. In the coming year, phasetwoof our instrument performance study will focus on determining thefollowing: the point spread function of an imaging system, the system’sspectral separation ability and the spectral calibration and resolutionof the detection system. As with the phase-one study, the goal of thisstudy will be neither to compare the performance of different brandsof instruments, nor to ascertain which brand had better performancein a given area. Instead, the goal of our proposed phase-two study iscontinue to focus on determining the current state of modern imagingsystems through straightforward, efficient and robust tests. These testswill aid imagers in the early detection of system problems. Moreover,these tests will continue to help define relative standards that will assistboth core personnel and imagers in maintaining their instruments inoptimal operating conditions.(R7-2) Deconvolution: Core Concepts, Algorithmsand Advanced IssuesB. Northan, N. BeaversMedia Cybernetics, Guilderland, NY, United StatesDeconvolution is a computational technique used to remove blurfrom images. In this presentation image formation in the microscopewill be reviewed and it will be explained what deconvolution doesand why deconvolution is needed as a post acquisition processingstep. The concept of Point Spread Function (PSF) will be introduced.Several approaches to deconvolution and deblurring exist from simplesubtractive methods to complex iterative approaches. The majorapproaches will be examined. It will be explained why in the presenceof noise a statistical approach is required. Blind deconvolution will beintroduced. The concept of Point Spread Function will be examinedin detail. The effect of microscope modality, lens parameters, andspecimen parameters on PSF shape will be discussed. Theoretical PSFcalculation and PSF measurement using beads will be reviewed withexamples. It will be explained why theoretical and measured PSFs candiffer from the true system PSF. Blind deconvolution will be furtherexamined as a tool to deal with the uncertainty of the true form ofthe PSF. Practical guidelines for data acquisition will be reviewed. Therelationship between sampling rate and quality of deconvolutionresults will be explained.58 • ABRF 2011 — Technologies to Enable Personalized Medicine

(R8) Joint Session: Proteomics Research Group(PRG) & Metabolomics Research Group (MRG)(R8a) PRG-2011: Defining the Interaction betweenUsers and Suppliers of Proteomics ServicesD. Hawke 1 , T.M. Andacht 2 , M.K. Bunger 3 , C. Bystrom 4 ,L. Dangott 5 , H. Molina 6 , R.L. Moritz 7 , R.E. Settlage 8 ,C.W. Turck 91University of Texas MD Anderson Cancer Center, Houston,TX, United States; 2 Centers for Disease Control andPrevention, Atlanta, GA, United States; 3 RTI International,Research Triangle Park, NC, United States; 4 QuestDiagnostics, San Juan Capistrano, CA, United States;5Texas A&M University, College Station, TX, United States;6Center for Genome Regulation, Barcelona, Spain; 7 Institutefor Systems Biology, Seattle, WA, United States; 8 VirginiaBioinformatics Institute, Blacksburg, VA, United States; 9 MaxPlanck Institute of Psychiatry, Munich, GermanyOver the last ten years the Proteomics Research Group (PRG) hasundertaken technical studies that have covered a wide range ofissues unique to the rapidly developing field of proteomics and haveincluded a range of qualitative and quantitative experiments. The PRGstudies have resulted in a great deal of attention not only within theABRF community but also outside as is evident from numerous articlesdealing with proteomics methods, procedures and standardization.As the field continues to develop, the diversity of instrumentationand laboratory workflows have grown in tandem. Therefore, in thePRG2011 study it seemed especially useful to perform a survey tohelp the PRG define future studies based on the current blend ofsample types and technologies and obtain a view of emerging trends.A survey was created to ascertain three main insights into core facilityfunction: 1) How labs interact with their clients, 2) The capacity of labsto meet the demands of their clients, and 3) The blend of experimentaltechniques offered to and requested by clients. Survey questions weredesigned to obtain information from both users of core facilities andthe directors and personnel of core facilities. Questions covered suchtopics as the type and age of instruments in use, how data is analyzedand presented to client, sources of funding, and emerging proteomicstrends. Results are compiled en masse and presented without regardto institution. Early results reveal that about 2/3 of the responders arenot ABRF members, and at least one lab still has an operational massspectrometer that was acquired in 1990!(R8b) Metabolomics Research Group 2011 StudyW.R. Wikoff 1 , J.M. Asara 3 , V.V. Tolstikov 1 , P. Aronov 2 ,B. Kesler 4 , V. Shulaev 5 , C.W. Turck 61University of California Davis, Davis, CA, United States;2Stanford University, Stanford, CA, United States; 3 BethIsrael Deaconess Medical Center, Boston, MA, United States;4Thermo Fisher Scientific, Redwood City, CA, United States;5University of North Texas, Denton, TX, United States; 6 MaxPlanck Institute of Psychiatry, Munich, GermanyThe ABRF Metabolomics Research Group (MRG) was formed in2009 and aims to educate research scientists and resource facilitiesin the analytical approaches and management of data resulting fromcomprehensive metabolite studies and to promote the science andstandardization of metabolomic analyses for a variety of applications.Last year the MRG conducted a ‘Survey Study’ on the current use ofmetabolomics technologies and procedures in core facilities. This yearthe MRG is organizing a ‘Research Study’ involving a spiked plasmasample. The study sample consists of a human biofluid as the matrix,replicating a typical small scale metabolomics pilot experiment thateither a core or research laboratory would perform. The sampleconsists of two groups of normal human plasma (NIST plasma ‘StandardReference Material’) with spiked in compounds. There are threebiological replicates in each group (n = 3 design) with different levelsof spiked compounds differentiating the two groups. Participants areasked to determine statistical significance, fold change, and identifycompounds that differ significantly between groups A and B. The designreflects issues encountered in an actual metabolomics experimentconducted with human or animal specimens. The study is compatiblewith many methodological approaches in metabolomics, including,but not limited to LC/MS, GC/MS, NMR, as well as other methods. Aswith any metabolomics profiling experiment, the best results wouldbe expected using a combination of approaches. The study is the firstof its kind in the field of metabolomics and is expected to produceimportant information on the strengths and limitations of the variousplatforms and technologies that are commonly used for comprehensivemetabolite analyses.Research GroupPresentation AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 59

Poster Session Abstracts**ABRF Poster Award Semifinalists102 Designing an Institutional Web-basedCore Facility Management SystemPoster Abstracts101 Center for Genome Research andBiocomputing at Oregon StateUniversityC. Rosato, M. Dasenko, A. Girard, M. Peterson,C. Sullivan, J.C. CarringtonCenter for Genome Research & Biocomputing,Oregon State University, Corvallis, OR, UnitedStatesThe Center for Genome Research and Biocomputing (CGRB) CoreLab at Oregon State University provides services for fee in genomictechnologies (DNA sequencing, DNA genotyping (fragment analysis))and in functional genomic technologies (microarray). We manage aZeiss LSM510Meta confocal microscope as a multi-user instrument;training is required and assistance is available, both for fee. Sequencingis provided both for traditional Sanger sequencing on an AB 3730 andultra high throughput sequencing on the Illumina Genome AnalyzerGAIIX. With the most recent software and reagent upgrades, 2 x150bp runs are now supported with up to 40 million reads per lanepassing filter. DNA genotyping (fragment analysis) runs on the AB3730. Our microarray services include Affymetrix and NimbleGenplatforms, and sample labeling, hybridization and scanning areoffered. We purchased ArrayStar microarray analysis software, whichis accessible to our researchers through our Biocomputing clusternetwork. The Agilent BioAnalyzer service offers High Sensitivity DNAand RNA applications. Our Biocomputing capabilities have expanded.This past year we reorganized the computing resources into a cloudarchitecture. GENOME Cloud is at 507 Processors, 1.5TB Memoryand 300+TB Storage. We process around 20,000 jobs / day on thecloud. We have increased access to our computational resources,decreased analysis turn-around time, and we have added new toolsto monitor computational resources. The Biocomputing changes toour High Throughput Sequencing service include removing the IPARunit from the network and simplifying data pathways both for datamanagement and researcher analysis capabilities. The CGRB Core Labalso maintains multi-user instruments available to researchers. Theseinclude a Zeiss LSM510Meta confocal microscope, an AB 7500 FASTqPCR instrument includes High Resolution Melting (HRM) capability, aStorm 820 Phosphoimager, a Nanodrop, an Invitrogen Qubit, a GenetixQ-Pix colony picker, an Axon Genepix 4200A microarray scanner and afluorescent plate reader.A. Hagen 1 , D. Tabarini 2 , S. Clisham 3 , D. John 31iLab Solutions, LCC, Cambridge, MA, UnitedStates; 2 Memorial Sloan-Kettering Cancer Center,New York, NY, United States; 3 Institute for SystemsBiology, Seattle, WA, United States; 4 Dana-FarberCancer Institute, Boston, MA, United StatesThe authors and their four institutions collaborated to (i) identify the keychallenges to core facility management; (ii) identify the requirementsfor an effective core facility management system; (iii) design, test anddeploy such a system. Through a series of interviews with all participantsin the core work flow (customers, core staff, administrators), the teamidentified a number of key challenges, including: (i) difficulty forresearchers in identifying available services; (ii) inconsistent processesfor requesting services; (iii) inadequate controls for approving servicerequests; (iv) inefficient processes for tracking and communicating aboutproject processes; (v) time-consuming billing practices; (vi) incompleterevenue capture; (vii) manual reporting processes. The team identifiedthe following requirements for a system to address these challenges:(i) ability to support a broad range of core business practices suchas complex quote generation and project management; calendaring/equipment reservation management; sample tracking; complex forms;and import of usage data from hardware; (ii) ability to offer servicesfor both internal and external customers, including flexible pricingand off-site access; (iii) ability to interact with institutional financialsystems (e.g. SAP, PeopleSoft, Lawson, SunGard Banner) and identifymanagement systems (e.g. Microsoft Active Directory, LDAP, and otherSAML 2.0-compliant services). The team developed and deployed thissystem across the collaborative partners, as well as other major researchinstitutions.103 RI-INBRE Centralized Research CoreFacilityA. Ahmed, N. NousUniversity of Rhode Island, Kingston, RI, UnitedStatesThe RI-INBRE Centralized Research Core Facility inaugurated in July2003, is being supported by the Rhode Island IDeA Network ofBiomedical Research Excellence (RI-INBRE) grant from NCRR /NIHand by the participating institutions that include: University of RhodeIsland, Brown University, Rhode Island College, Providence College,Salve Regina University, and Roger Williams University. This facility islocated in the College of Pharmacy at the University of Rhode Island’sKingston campus. It is equipped with instrumentation in biomedical,pharmaceutical and biotechnological research. The Core Facility isproviding access to research instrumentation and training supportto RI-INBRE participants as well as other scientists affiliated withacademic institutions and the private sector throughout the state ofRhode Island. Instrument reservation and scheduling are available,through the core facility’s website (wwww.uri.edu/inbre/corelab).All new users are supported with operator assisted access to theequipment. Independent access to the equipment is also available to60 • ABRF 2011 — Technologies to Enable Personalized Medicine

all trained users. In addition, full service access via sample submissionis provided particularly in ICP-MS, LC/MS/MS and N-terminal proteinsequencing. Technical staff members manage the laboratory and areavailable to assist in the operation of various instruments and to analyzesamples on a fee-for-service basis. (Supported by NIH-NCRR Grant #1P20RR16457)104 Implementation of QualityManagement in Core ServiceLaboratoriesB. Hicks, T. Creavalle, J. Dickens, K. Haque,C. Raley, M.W. SmithGenetics and Genomics Group, AdvancedTechnology Program, SAIC-Frederick, NationalCancer Institute at Frederick, Frederick, MD, UnitedStatesThe Genetics and Genomics group of the Advanced TechnologyProgram of SAIC-Frederick exists to bring innovative genomic expertise,tools and analysis to NCI and the scientific community. The SequencingFacility (SF) provides next generation short read (Illumina) sequencingcapacity to investigators using a streamlined production approach.The Laboratory of Molecular Technology (LMT) offers a wide rangeof genomics core services including microarray expression analysis,miRNA analysis, long read (Roche) next generation sequencing,transgenic genotyping, Sanger sequencing, and clinical mutationdetection services to investigators from across the NIH. SF and LMT areworking together to bring online the third generation Pacific BioscienceSMRT sequencing platform. As the technology supporting thisgenomic research becomes more complex, the need for basic qualityprocesses within all aspects of the core service groups becomes critical.The Quality Management groupworks alongside members of theselabs to establish or improve processes supporting operations control(equipment, reagent and materials management), process improvement(reengineering/optimization, automation, acceptance criteria for newtechnologies and tech transfer), and quality assurance and customersupport (controlled documentation/SOPs, training, service deficienciesand continual improvement efforts). Implementation and expansion ofquality programs within unregulated environments demonstrates SAIC-Frederick’s dedication to providing the highest quality products andservices to the NIH community.105 Cornell University Life Sciences CoreLaboratories CenterG. Grills, J. VanEe, P. Schweitzer, S. Zhang,R. Williams, J. Pillardy, Q. Sun, W. Wang, Y. Li,D. Betel, T. Stelick, J. Spisak, L. Cote, R. Cameron,H. Wroblewski, B. Hover, L. Zhang, J. Mosher,Y. Xin, G. Westby, J. Busuttil, S. Monni,R. Sherwood, A.C. Ptak, W. Chen, J. McCardle,C. Bayles, J. Dela Cruz, M. Riccio, R. Bukowski,L. Ponnala, C. Myers, H. Singh, M. Howard,J. Flaherty, A. Manocchia, E. Dodge, K. Smith,C. Aquadro, A. Melnick, T. Brenna, W. Zipfel,A. Clark, A. Siepel, L. Carr, J.K.C. RoseCornell University, Ithaca, NY, United StatesThe Cornell University Life Sciences Core Laboratories Center (CLC)provides an array of genomics, proteomics, imaging and informaticsshared research resources and services to the university communityand to outside investigators. The CLC includes fee-for-service research,technology testing and development, and educational components.The Center has seven core facilities, including genomics (DNAsequencing, genotyping, and microarrays), epigenomics, proteomicsand mass spectrometry, microscopy and imaging, bioinformatics, bio-IT, and advanced technology assessment. The CLC is part of a NewYork State designated Center for Advanced Technology in Life ScienceEnterprise. The mission of the CLC is to promote research in the lifesciences with advanced technologies in a shared resource environment.Use of the CLC resources and services is steadily increasing due to thegrowth in the number and types of cores in the center, to the expansionof existing services and the implementation of new core technologies,and to the coordinated integration and synergy of services betweenthe CLC cores. Multidisciplinary support for multi-functional instrumentplatforms is implemented by integrated operations of the CLC corefacilities. Investigators are offered coordinated project consultationswith the directors and staff of all relevant cores during the design,data production and analysis phases of their projects. The CLC isinvolved in establishing and supporting multidisciplinary researchprojects that involve both intercampus initiatives and multi-institutionalcollaborations. With a concentration of advanced instrumentation andexpertise in their applications, the CLC is a key resource for life sciencesresearch.106 Searchable Core Facility Database:Building Resource BridgesB. Fleming, T. HunterVermont Genetics Network, University of Vermont,Burlington, VT, United StatesThe VGN Searchable Core Facility Database (http://vgn.uvm.edu/corefacilities) is a directory of Core Facilities primarily focused on NorthAmerica but with entries from around the world. It is a tool intendedto foster collaboration and assist cores in growing their user base andproviding networking opportunities. It is populated with Core Facilitiesthat have voluntarily listed themselves and would like to be contactedby researchers and other core facilities for potential collaborations.Benefits: Allows researchers to locate resources needed for their studies;Provides a channel for facilities to collaborate; and Facilitates cores toreach financial sustainability. Researchers are able to perform searchesonline by service offerings, location, association, and key phrases to finda facility that will best meet their needs. Information listed for individualcores include: short description of core, contact name, email, address,services offered, hyperlink to website, equipment, and date of lastrevision of information. The data can be exported to an excel readableXML file. The database currently lists 292 cores, representing 39 statesplus DC, 104 institutions, and 10 associations.107 Dartmouth Genomics SharedResourcesJ. Hamilton, H. Trask, W. Taylor, C. TomlinsonDartmouth Medical School, Norris Cotton CancerCenter, Hanover, NH, United StatesIn order to carry out an accurate diagnosis, prognosis, and/ortherapeutic assessment for a disease; high-throughput approaches toexamine the whole genome and transcriptome are now a necessity formodern research. Furthermore, to more fully understand the underlyingcauses of disease, high-throughput genomics are required to examinePoster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 61

Poster Abstractsglobal gene expression, regulation, and interactions. To meet theseand the future needs of the research community at Dartmouth, thelatest technologies in deep sequencing and microarrays are offered. Inaddition, we offer the following services: Specialized, expensive, highendinstrumentation; Expert staffing; Cost-effective for individual labs;Competitive pricing and services with outside sources; On site NorrisCotton Cancer Center facility; Free experimental design consultations;Competitive fee for service charges for all high-throughput approaches;and Close proximity to Biostatistics and Bioinformatics shared resources.108 A Collaborative Life Cycle Process forthe Bioinformatics Core FacilityS. Lin, W.A. KibbeNorthwestern University, Evanston, IL, United StatesThe operation of the bioinformatics core facility is constantly challengedby increasing data volume, emerging technologies, and limited budget.We discuss a Collaborative Life Cycle (CLC) process as a businessmanagement model for this challenging environment. Unlike thetraditional involvement at the last stage of data analysis, the CLC processengages the bioinformatics core facility throughout the project with thePis and the wet lab core facilities. Various tasks of the bioinformaticscore during the project’s life cycle include: 1) Planning -study designand statistical power analysis with the PIs 2) Experiment-core wet labsample tracking and data management 3) Data analysis -data qualitycontrol and interpretation in the biological context. Collaborationsthroughout the project life cycle are critical because the multiple-phaseprocess involves numbers of professional disciplines with many skills,tools, and procedures. The CLC process will help the bioinformaticscore facility align the multiple goals, adjust the expectations, mitigatethe potential risks, and improve the end results.109 Promoting Diversity in the CoreFacilityM. PersonThe University of Texas at Austin, Austin, TX,United StatesDiversity is encouraged to counter historical bias against a varietyof groups and has emerged as a positive attribute of the workplace.However, among pressing economic and scientific outcome pressures,issues not tied to measurable rewards are often overlooked in the corefacility. This poster will present an overview of diversity issues andstrategies relevant to core facilities. The variety of types of diversityis discussed, including sex, race, ethnicity, nationality, socioeconomicbackground, and sexual orientation. Institutional efforts to encouragediversity are described with regards to organizational mandates andimplementation at the level of core facilities. Contrast between writtenpolicies and real world practices are noted. A valuable tool availableto the core facility director is self-examination, actively counteringbarriers that unconscious bias and learned prejudice have created.Systematic analysis can identify areas of diversity strength and weaknessin the facility. Strategies for increasing diversity are presented for thehiring process. Practices in the workplace that accommodate diversepopulations and foster career development are discussed. Finally,identifying rewards for diversity promotion encourages consistentprogress in the face of competing demands.110 A Web Based Tool for EfficientManagement and Use of ResearchCore FacilitiesP. Turpen, L. Wilkie, L. MillerUniversity of Nebraska Medical Center, Omaha,NE, United StatesThe need to efficiently and effectively manage research resourcesis an area of growing concern to both grantor agencies and granteeinstitutions. Central administration of core facilities is one way toaddress some of those concerns, but managing the needs of disparatecores can be a challenge. All research core facilities, irrespective oftheir emphasis, need to be able to contract for services, bill for thoseservices and provide documentation of facility use. While commercialsystems are a welcome recent development, most concentrate onsample management data, LIMS, with invoicing and utilization metricsas secondary functions. The shortcoming in these types of offerings isthat they do not readily capture the institution’s or grantor agencies’needs for use reporting without considerable customization andcorresponding associated costs. We have chosen to create a web basedtool that can facilitate the scheduling/contracting of services, invoicingand fee collection for those services, and address the complex reportingneeds of the institution.111 XSQ: A New Binary Output Format of5500/5500XL SystemsC. Yang, J. Zhang, D. Thomas, P. SuriLife Technologies, Carlsbad, CA, United StatesThe XSQ (eXtensible SeQuence) format was designed to accommodatedifferent run types (standard SOLiD sequencing and exact chemistrycall (ECC) runs), simplify indexing samples workflow and support newdata types such as ECC in the new 5500 sequencing instrument; thesedata cannot be stored in any existing formats (e.g. csfasta & QV.qual) asthere are more than one call per position. Other problems with existingformats are the file size, I/O demand, and lack of pairing of reads. TheXSQ format uses information packing to reduce the file size by 60%,resulting in reduced storage needs and reduced transfer times. It alsohas integrated pairing of reads, so mapping and pairing can happentogether much more quickly, rather than having the two reads of a pairmapped separately and then merged subsequently. The new XSQ fileallows users to perform very efficient indexing reassignment and suchreassignment only introduces minimal impact to downstream analysisinstead of reanalyzing all indexing samples. The hierarchical data formatalso provides a basic level of partitioning and indexing within the file sothat subsets of the data can be retrieved without reading through theentire file.112 Microsoft Biology Initiative: .NETBioinformatics Platform and ToolsB. Diaz AcostaMicrosoft Biology Initiative: .NET BioinformaticsPlatform and ToolsThe Microsoft Biology Initiative (MBI) is an effort in Microsoft Researchto bring new technology and tools to the area of bioinformatics andbiology. This initiative is comprised of two primary components,the Microsoft Biology Foundation (MBF) and the Microsoft BiologyTools (MBT). MBF is a language-neutral bioinformatics toolkit built62 • ABRF 2011 — Technologies to Enable Personalized Medicine

as an extension to the Microsoft .NET Framework—initially aimed atthe area of Genomics research. Currently, it implements a range ofparsers for common bioinformatics file formats; a range of algorithmsfor manipulating DNA, RNA, and protein sequences; and a set ofconnectors to biological web services such as NCBI BLAST. MBFis available under an open source license, and executables, sourcecode, demo applications, documentation and training materials arefreely downloadable from http://research.microsoft.com/bio. MBT is acollection of tools that enable biology and bioinformatics researchersto be more productive in making scientific discoveries113 NERLSCD: A Model for RegionalNetworking of Life Sciences CoreDirectorsG.S. Grills 1 , M. Detwiler 2 , T. Thannhauser 3 ,T. Hunter 4 , P.S. Adams 5 , S.A. Bobin 6 ,K. Sol-Church 7 , P. Spatrick 8 , S. Perkins 81Cornell University, Ithaca, NY, United States;2Roswell Park Cancer Institute, Buffalo, NY, UnitedStates; 3 United States Department of Agriculture/Agricultural Research Service, Beltsville, MD, UnitedStates; 4 University of Vermont, Burlington, VT,United States; 5 Trudeau Institute, Saranac Lake,NY, United States; 6 Dartmouth Medical School,Hanover, NH, United States; 7 Nemours COBRECenter for Pediatric Research, Wilmington, DE,United States; 8 University of Massachusetts MedicalSchool, Worcester, MA, United StatesThe Northeast Regional Life Sciences Core Directors (NERLSCD)annual meeting is a forum that provides an exceptional opportunityfor life sciences core facility directors and managers to network withcolleagues, to learn about biotechnology advances and applications,and to discuss the challenges and results of regional implementation ofshared research resources. The NERLSCD meeting was established in2006 as a grass roots effort by core directors. The meeting was held lastyear at the University of Massachusetts Medical School in Worcester,MA. The sixth annual NERLSCD meeting will be held at CornellUniversity in Ithaca, NY, Nov. 9-11, 2011. The goal of the meetingis to facilitate regional networking and sharing of resources and tohelp reduce regional duplication of costly biotechnology researchinfrastructure. The meeting provides a structured yet informal settingfor addressing the operational and technical challenges of life sciencescore laboratories. There are plenary sessions, workshops and discussionforums on financial and management issues facing biotechnology corelaboratories. There are technical workshops on genomics, functionalgenomics, proteomics, imaging, bioinformatics, bio-IT, and othertechnologies. A core facility poster session offers an opportunity forlearning about regional life sciences shared resources and services.The number and diversity of attendees reflects the broad appeal andusefulness of this meeting for core directors at many institutions in theregion. The meeting supports a diverse array of core directors andmanagers who play a crucial role in facilitating advances in knowledgeand understanding in a wide variety of life sciences research areas. Thisregional networking meeting for life sciences core facility directorscan serve as an example for the organization, funding, structure, andcontent of regional meetings for core facility directors in other regionsof the United States and in other countries.114 ABRF Affiliates and ChaptersS.F. Jennings 1 , G. Grills 2 , M. Detwiler 31University of Arkansas at Little Rock, Little Rock,AR, United States; 2 Cornell University, Ithaca,NY, United States; 3 Roswell Park Cancer Institute,Buffalo, NY, United StatesThe mission of the ABRF is to advance life sciences core facilities andbiotechnology laboratories through research, communication, andeducation. To facilitate this mission, the ABRF has implemented ABRFAffiliates and Chapters and the ABRF Affiliates and Chapters Committeefor the following purposes: a) To encourage the establishment, supportthe operations, and facilitate the coordination of new regional andspecial interest groups that have goals related to those of the ABRF insupport of life sciences shared resources; b) To establish partnershipsand collaborate with other existing organizations that have goals relatedto those of the ABRF in support of life sciences shared resources; c)To promote the technologies, research support and administration ofbiomolecular resource facilities; d) To promote the development andapplications of biotechnologies as shared research resources and tofacilitate the advancement of life sciences research; e) To play a leadershiprole in networking core laboratories, researchers, and students,matching those with similar and complementary interests and skills; e)To enhance communication on the regional, national and internationallevel regarding ABRF activities; to enhance the visibility of the ABRFin the scientific community; to educate the scientific community aboutthe value of the ABRF; and to broaden the number and diversity ofcore laboratories and biotechnology laboratories that take advantageof the ABRF research group studies and ABRF membership networkingopportunities; and f) To enhance the visibility of the ABRF with fundingagencies that support the development, acquisition and applicationof core facility shared research resources. ABRF Chapters are specialinterest groups which may be formed based on common interestsand/or geographical boundaries and support grassroots networks ofindividuals who wish to help advance ABRF goals and promote themission of shared resource facilities and biomolecular resources. ABRFAffiliates are special interest organizations that are autonomous fromthe ABRF, have common and complementary interests with the ABRF,and have the goal of developing a collaborative relationship with theABRF. Please join us for the ABRF Affiliates and Chapters Open MicSession from 6:00 pm to 6:45 pm on Sunday.115 The Midwest Association of CoreDirectorsW. Hendrickson 1 , P. Hockberger 21University of Illinois, Chicago, IL, United States;2Northwestern University, Evanston, IL, UnitedStatesThe Midwest Association of Core Directors (MWACD) was organized in2010 by a group of scientists at 6 different institutions to foster closerinteractions among directors and managers of core facilities throughoutthe Central Plains. The organization shares the same goals as ABRF,and it has applied to become a chapter of ABRF. The MWACD differsfrom ABRF only in that its focus is on regional matters rather than onissues of national concern. Towards this goal, the first annual meetingof the MWACD took place on October 21-23, 2010, at the CrownePlaza Hotel in Chicago. The goal of the meeting was to provide anopportunity for networking among core directors and managers, toenable interactions with colleagues, sharing of technical advice, andPoster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 63

Poster Abstractsdiscussions of continuing challenges associated with the operation ofshared research resources and technologies. Keynote presentationswere delivered by leaders of NIH-NCRR and FASEB, and there werepanel discussions on networking, bioinformatics, and informationmanagement systems. There was a poster session, vendor exhibits, andbreakout sessions on 8 different core-related topics. The meeting wasattended by 120 researchers and supported by 17 corporate and notfor-profitorganizations.116 Automated Isolation of Genomic DNAfrom Large Volumes of Whole BloodE. Vincent 1 , S. Krueger 1 , J. Kennedy 2 , S. Lee 2 ,C. Helt 1 , A. Bonk 2 , C. Cowan 11Promega Corporation, Madison, WI, UnitedStates; 2 Hamilton Robotics, Reno, NV, United StatesA key source for genomic DNA(gDNA) is blood drawn into a standard10mL Vacutainer® tube. The Promega ReliaPrep Large Volume HTgDNA Isolation System integrated on the Hamilton MICROLAB®STARplus liquid handling workstation provides a unique anddependable system for isolating genomic DNA from large volumes(3 mL–10 mL) of blood. The novel chemistry and instrumentationresolve many challenges encountered when processing large-volumesamples in a high-throughput format such as: loss of sample pelletsduring decanting of fluids, transport of full 50 mL tubes to variouslocations on a liquid handling robot, and manual re-suspension offinal DNA pellets. Liquid handler resource constraints were removedby creationof a new accessory, the ReliaPrep HSM 32 LV instrument,which provides heating, shaking and magnetization of samples atone deck position. The combination of this device, the MICROLABSTARplus workstation and the ReliaPrep Large Volume HT gDNAIsolation System allows automated recovery of pure gDNA from up to96 ten milliliter blood samples within 8 hours. We present verificationstudies demonstrating automated system performance. Comparisonsbetween the ReliaPrep Large Volume HT gDNA Isolation System anda standard precipitation-based method were made for duplicate bloodsamples from multiple donors. Yield, purity, and integrity of extractedgDNAwere assessed using UV absorbance spectroscopyand gelelectrophoresis. Genomic DNA yields from normal 10 mL whole bloodsamples were 200 400 µg (depending on white blood cell count) in aneluted volume of 1mL. Recovered DNA exhibited good purity withA260/A280 ratios greater than 1.7 and A260/A230 ratios between 1.8and 2.2. Isolated DNA was suitable for storage and was used in manydownstream analysis applications. Results of genomic DNA purificationfrom frozen (hemolysed) blood samples and blood collected usingcommon anticoagulants (EDTA, heparin, citrate)are also comparedtodemonstrate the efficacy of the new system.117 Overview of the Agilent TechnologiesSureSelect TM Target EnrichmentSystemJ. Ong 1 , A. Giuffre 1 , S. Joshi 1 , H. Ravi 1 ,C. Pabón-Peña 2 , B. Novak 2 , M. Visitacion 2 ,M. Hamady 2 , F. Useche 2 , B. Arezi 3 , B. Buehler 3 ,E. Lin 2 , S. Hunt 2 , D. Roberts 2 , S. Happe 1 ,E. Leproust 21Agilent Technologies, Cedar Creek, TX, UnitedStates; 2 Agilent Technologies, Santa Clara, CA,United States; 3 Agilent Technologies, La Jolla, CA,United StatesNext-generation DNA sequencing has revolutionized the discoveryof rare polymorphisms, structural variants, and novel transcripts. Tomeet the demand for fast, cost-effective, and accurate genome analysismethods from small scale studies to large sample cohorts, AgilentTechnologies has developed the SureSelectTM Target EnrichmentSystem. Available for the Illumina, SOLiD, and 454 NGS sequencingplatforms, SureSelect is a highly robust, customizable, and scalablesystem that focuses analyses on specific genomic loci by in-solutionhybrid capture. In addition, Agilent has introduced SureSelect XTfor Illumina and SOLiD, which combines gDNA prep, library prep,and SureSelect Target Enrichment reagents in one complete kit. BothSureSelect and SureSelect XT demonstrate high performance, asmeasured by capture efficiency, uniformity, reproducibility, and SNPdetection. We highlight the utility of the SureSelect system across awide range of target sizes and genome complexity using pre-designedcatalog libraries targeting cancer gene sets, sequences encoding thekinome, and both human and mouse All Exon content. In addition,user-defined custom content can be easily developed using theAgilent eArray software with candidate variant coordinates as input.User-defined content can be manufactured on-demand as a customSureSelect kit, or combined with pre-defined Agilent catalog contentusing the Plus option. We propose a novel approach for variantdiscovery - using SureSelect catalog designs to uncover candidatevariants, followed by the design of smaller focused custom libraries forSNP validation and region profiling. By pooling many samples togetherper lane or slide, SureSelect multiplexing kits for Illumina and SOLiDenable validation across large sample cohorts with substantial costsavings. Accurate post target enrichment pooling is facilitated by theAgilent Bioanalyzer and QPCR NGS Library Quantification kits whichensure equal representation across samples. Further efficiencies arerealized using the Bravo Automated Liquid Handling Platform to meetthe need for parallel preparation of multiplexed libraries.118 Gene Synthesis: A Cost-EffectiveAlternative to Traditional MolecularCloningA. Liao, M. Schwartz, H. Lo, J. Zhou, P. YangGENEWIZ, Inc., South Plainfield, NJ, United StatesGene synthesis is the process of synthesizing a gene in vitro without theneed for initial template. Contrary to what many researchers’ beliefs,commercial gene synthesis service is quickly evolving to become a costeffectivealternative to traditional cloning and other molecular biologyprocedures. The main reasons include: 1) Time savings: Traditionalcloning involves a multi-step process that includes cloning strategydesign, primer synthesis, PCR, gel extraction, bacteria transformation,and other complex steps. This process requires considerable amount of64 • ABRF 2011 — Technologies to Enable Personalized Medicine

time and human resource that gene synthesis does not. 2) Cost savings:In most cases, it costs less to order a synthetic gene than it does to orderoligos, cloning kits, and DNA sequencing services. 3) Enhanced DNAperformance: Gene synthesis allows for codon optimization whichhas been proven to increase the efficiency of protein expression. 4)Convenience: Without the need for a physical template and withoutdesign restrictions associated with the traditional cloning process, aresearcher can get a gene of his/her choice by simply supplying thenucleotide sequence or amino acid sequence. GENEWIZ is a globalCRO that provides a wide range of DNA services, including genesynthesis. GENEWIZ’s gene synthesis service features a 2-3 weekturnaround and expert technical and project management support.This informational poster will present case studies of how GENEWIZ’sgene synthesis service benefited researchers who had previously reliedon traditional molecular cloning for plasmid construction.119 Nucleotide-Level Variant Analysis ofNext-Generation Sequencing DataUsing a Cloud-Based Data AnalysisPipelineB. Ganter, G. Asimenos, A. SundquistDNAnexus, Palo Alto, CA, United StatesTo demonstrate the flexibility of a cloud-based solution for analyzingdisparate sets of next-generation sequencing data, we looked at carefullychosen samples across different populations from the 1,000 GenomesProject (www.1000genomes.org) and conducted an extensive analysison two Chinese populations, the “Chinese in Beijing” (CHB) and the“Chinese in metropolitan Denver” (CHD), each consisting of 28 exomes.Each dataset was uploaded into the system using raw data files acquiredfrom the 1,000 Genomes Project. Using these data and a cloud-baseddata analysis pipeline, we performed a nucleotide-level variant analysiscombined with a population allele frequency analysis across all samplesfor the two populations . To identify alleles that are significantlydifferent across the two populations, a Pearson’s chi-square test wasapplied, which resulted in a total of 1.5 Mio SNPs, of which 84 werenonsynonymous with a p-value of less than 0.01. Interestingly, the genesassociated with nonsynonymous variants of the Chinese in metropolitanDenver population were enriched for biological annotations such asendocrine system disorder, metabolic disease, cardiac fibrosis, andinflammation (includes ZNF264, RPS6KA2, ROBO2, CRK, MUSK, CBL,CRK, and others). Furthermore, genes usually associated with liverinjury were also identified for this population, suggesting the liver isexposed to toxic agents more so in this population compared to theCHB population. The observed genomic differences in these twodifferent Chinese populations living in different parts of the world hinttowards a potential link between nutrition and different diseases (e.g.heart disease or metabolic diseases). Using this analysis as a case study,we will demonstrate how a scalable computational infrastructure canprovide researchers and sequencing service providers alike, a costeffectiveand secure cloud-based computing platform as a powerfuland collaborative technology solution for large scale sequence dataanalysis and management.120 Genome Technology Center at theNYU Langone Medical Center: NewSupport for Clinical and TranslationalScienceJ. Zavadil, S. MischeNew York University Langone Medical Center, NewYork, NY, United StatesTo significantly enhance support for clinical and translational researchwithin the framework of its CTSI, the NYU Langone Medical Centerconsolidated the Microarray and DNA Sequencing Cores into a newGenome Technology Center, a shared resource overseen by the Officefor Collaborative Science. The GTC’s team of 4 technical personneland one faculty level director assists >120 NYULMC laboratories intheir basic, clinical and translational research. The Sequencing Unitoperates 2 Illumina GAIIs, and a HiSeq sequencer will be added in Q12011. The GAII capacity is applied to research applications (ChIP-seq,small-RNA-seq and RIP-seq) and to identification of disease-relatedgenome-level structure changes and correlates (e.g. RNA-seq of cancertranscriptomes). GTC also has a Roche GS FLX System (454) used forde novo sequencing of microbial species and for amplicon sequencingin clinical genetics, patient microbiome diversity, etc. The MicroarrayUnit operates Affymetrix GeneChip system and high-capacity QPCR(ABI 7900HT) with automated plate setup and loading for gene andmicroRNA profiling and for SNP genotyping in clinical genetics. TheGTC cooperates closely with the newly established Center for HealthInformatics and Bioinformatics (CHIBI) supported by the NIH/NCRRCTSA Award. CHIBI provides an HPC facility for sequencing andmicroarray data storage and offers a full range of informatics services.The GTC is committed to regional and nationwide collaborations withother Cores. GTC participates in the activities of the Genomic Analysisand Technology Excellence (GATE) Working Group of the Academy forMedical Development and Collaboration (AMDeC), particularly in thesections of Core Facility Directors, Funding Strategy and Bioinformatics.It also contributes to the AMDeC Facilities Instrumentation ResourcesServices Technologies (FIRST), a real-time online database ofbiomedical research technology and resources available in the NewYork City area and throughout Northeastern US. Key services of theGTC are offered to external clients.121 Integrated Core Facility Support andOptimization of Next GenerationSequencing TechnologiesG. Grills, P. Schweitzer, Q. Sun, J. Pillardy,W. Wang, T. Stelick, R. Bukowski, L. Ponnala ,J. VanEeCornell University, Ithaca, NY, United StatesNew DNA sequencing technologies present an exceptional opportunityfor novel and creative applications with the potential for breakthroughdiscoveries. To support such research efforts, the Cornell UniversityLife Sciences Core Laboratories Center has implemented the IlluminaHiSeq 2000 and the Roche 454 GS FLX platforms as academic corefacility shared research resources. We have established sample handlingmethods, LIMS tools and BioHPC informatics analysis pipelines insupport of these new technologies. Our genomics core laboratory,in collaboration with our epigenomics core and bioinformatics core,provides sample preparation and data generation services andboth project consultation and analysis support for a wide range ofPoster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 65

Poster Abstractspossible applications, including de novo or reference based genomeassembly, detection of genetic variation, transcriptome sequencing,small RNA profiling, and genome-wide epigenomic measurements ofmethylation and protein-nucleic acid interactions. Implementation ofnext generation sequencing platforms as shared resources with multidisciplinarycore facility support enables cost effective access andbroad based use of these technologies.122 Better Outcomes for Our Patients:Moving Science Forward in theBiomolecular Core Laboratory at theNemours Center of Pediatric ResearchJ. Holbrook, D.L. Stabley, E.C. Hitchens, P. Geller,K. Sol-ChurchNemours/A.I. duPont Hospital for Children,Wilmington, DE, United StatesNemours Biomedical Research has a long-standing commitment toscholarly and scientific endeavors directed towards improving thediagnosis and treatment of pediatric medical conditions. Establishedin 2004 with the support of a COBRE grant from the NIH s NationalCenter for Research Resources, the Nemours Center for PediatricResearch provides clinical and translational researchers with the tools aswell as training they need to perform their research. The BiomolecularCore Laboratory (BCL), located at the Alfred I. duPont Hospital forChildren in Wilmington, Delaware, is supported by the Center ofPediatric Research. We provide a unique opportunity for Nemoursclinicians, research staff, and affiliates at University of Delaware andThomas Jefferson University to develop competitive research projectsin genetics as well as provide essential services in molecular biologyand genomics. The mission of the BCL is to facilitate, through state-ofthe-artsservices and stewardship, discoveries that begin at a molecularlevel and move rapidly from patient-oriented research to the bedside.Working in close collaboration with those engaged in research activities,our goal is to enable fast, effective and productive top quality servicesas well as to match BCL s strengths with our clients needs. This posterhighlights the services the Biomolecular Core lab offers, includingmolecular biology training, a listing of our instrumentation, and corebusinesses including DNA sequencing, genotyping, gene expressionand methylation analysis. We will also highlight our newly establishedmicroarray service utilizing the Affymetrix Microarray platform. Thedevelopment of this vital core allowed investigators, who previouslyused extramural services and technologies, to move those projects inhouse, thus saving both time and money for our clients.123 Critical Reagent AnalyticalCharacterization Program atGenentechS. Chamberlain, C. Williams, A. Meier, C. Lu,P. MotchnikGenentech, Inc., South San Francisco, CA, UnitedStatesAt Genentech, Critical Reagents are internally prepared, recombinantlyproducedproteins used in GLP/GMP assays in support of regulatoryfilings. These proteins are typically extracellular domain constructions,growth factors, antibodies and novel hybrid constructions producedin CHO transient or stable cell lines, E. coli or Baculovirus. Genentech’sCritical Reagent System (CritRS) is used to document the processof ordering, manufacturing, characterization, and delivering acritical reagent. The CritRS was put in place in 2004 at Genentechto ensure quality, consistency, and traceability of critical reagentpreparations. Critical reagent analytical characterization is performedusing four platform assays to assess the molecule’s identity andpurity/heterogeneity. Identity is confirmed using Edman N-terminalsequencing and MALDI-TOF peptide mass fingerprinting. Purity/heterogeneity is assessed by size exclusion chromatography (SEC)and SDS-PAGE. The four platform assays provide sufficient identityand purity information for most critical reagents. However, in somecases additional characterization methods have to be used due tounexpected results, such as observed N-terminal sequence is differentfrom expected sequence, more than one N-terminal sequences arepresent, SDS PAGE shows unexpected band pattern, and SEC can notseparate the high and low molecular species of the reagent. In thisposter we’ll present typical analytical characterization results as well assome unusual findings.124 Progress towards Becoming a CLIA-Certified Core FacilityM. McMillan 1 , M. Gray 1 , M. Sandoval 1 ,S. McCann 1 , S. Gorda 21University of Southern California, Norris CancerCenter/Keck School of Medicine, Los Angeles, CA,United States; 2 University of Southern California ,College of Letters, Arts and Sciences, Los Angeles.CA, United StatesThe USC Norris DNA Core Facility (formerly known as the MicrochemicalCore) was created in 1984. It is equipped with an AB3730 DNAsequencer and an AB3900 oligonucleotide synthesizer. It is affiliatedto the Molecular Genomics Core in the Epigenome Center. Our goal isto establish the DNA Core Facility as a CLIA-certified laboratory. Therationale is that for Norris Cancer Center clinicians to use our DNAsequencing data in the diagnosis, treatment and prognosis of cancerpatients, the facility must be CLIA (Clinical Laboratory ImprovementAmendments)-certified. We will present our progress towards this goalincluding qualified personnel, identifying genes of medical interest,the establishment of appropriate records and the purchase of CLIAcompliantequipment. We plan to file for CAP (College of AmericanPathologists) accreditation and apply for a Clinical Laboratory License(State) as well as submitting a CLIA application (Federal) to theCalifornia Department of Health Services.125 Trudeau Institute Molecular BiologyCore FacilityP. Adams, A.M. Moquin, J.J. HoffmanTrudeau Institute, Saranac Lake, NY, United StatesCentralization and merging of core facilities is a favorite topic in thecurrent economy. The Molecular Biology Core Facility (MBCF) at theTrudeau Institute has provided a variety of services to the investigatorsat the institute while recovering the majority of its expenses since itsinception 12 years ago. The MBCF survives by offering an assortmentof services that are specifically tailored to the Institute missionand by personalizing each service to the individual investigator.Shared resource equipment and work areas optimize space andinstrumentation. Chargeback fees are designed to recover costsaccording to OMB Circular A-21. The MBCF recovers ~ 80% of costs.Services provided include DNA sequencing, spectratyping of the T-cellrepertoire (fragment analysis), production, purification and labeling66 • ABRF 2011 — Technologies to Enable Personalized Medicine

of Major Histocompatibility (MHC) Class I and Class II tetramers forFluorescent Activated Cell Sorter (FACS) analysis, RNA and Proteinanalysis using “Lab on a Chip” technology, real-time PCR measurementof gene expression and viral loads, knock-out mouse and Mycoplasmascreening, DNA haptenation and recombinant protein expression/purification for antibody detection, monoclonal and polyclonalantibody production and in vivo immunization. A variety of simpleservices such as primer design, primer ordering, stock primers, peptideordering and Taq production save the investigators time, effort andmoney. Education and training are provided for all techniques andfor using shared MBCF instrumentation, such as spectrophotometers,real-time PCR equipment, tissue prep and image capture/analysisequipment. Not only does the MBCF provide the service, the personnelassist in the planning and analysis to maximize proper usage of thetechnique. Services not available in the MBCF are outsourced to otherCore facilities in the area to be sure the quality of service is the highest.The MBCF at Trudeau Institute survives through versatility, imaginationand customization.126 Molecular Resource Facility, UMDNJ-New Jersey Medical SchoolR. Donnelly, S. Kuppasani, K. DhawanUniversity of Medicine & Dentistry of New Jersey,New Jersey Medical School, Newark, NJ, UnitedStatesThe Molecular Resource Facility of the New Jersey Medical School wasestablished in April 1995 to enhance the resources available to theresearch community within the medical school. It serves to provideservices to the research community and as a source of informationon molecular techniques and research strategies involving molecularbiology. Our facility has developed through the years to be an “allinclusive” nucleic acid facility with limited protein analysis capabilities.We provide services in many areas including DNA sequencing, proteinsequencing, peptide synthesis, qPCR and others. In 2009 we beganoffering High Throughput DNA Sequencing on the SOLiD® instrument.Our services are available to any laboratory requiring these services.127 United States Department ofAgriculture/Agricultural ResearchService (USDA-ARS) Eastern RegionalResearch Center Core TechnologiesD. Needleman, A. Nuñez, G. Strahan, D.S. Soroka,W. DamertUnited States Department of Agriculture/Agricultural Research Service (USDA-ARS) EasternRegional Research Center Core Technologies,Wyndmoor, PA, United StatesUnited States Department of Agriculture. The CT unit is comprisedof four research related components: genetic analysis, proteomicsbiopolymersmass spectrometry, electron microscopy, and magneticresonance spectroscopy (NMR). In addition, the Research Data Systems,the information pipeline of the CT, provides the means to facilitate datadistribution to researchers, stakeholders, and the general public. Theavailability of integrated resource laboratories assures professional anddependable support to the goals of the ARS community.128 NYULMC Office of CollaborativeScience Cores - Enabling PersonalizedMedicine through TranslationalTesearchC. Curchoe, T. Winner, J. Salcedo, S. Mische,D. LevyNew York University, Langone Medical Center,New York, NY, United StatesThe New York University Langone Medical Center (NYULMC) hascommitted $15 million to ensure that researchers have access to cuttingedge enabling technologies. The Office of Collaborative Scienceenables access to expertise and technology through the centralizedadministration of Shared Resource Centers. In partnership with theNYU Cancer Institute, Center for AIDS Research and Clinical andTranslational Science Institute, we strive to increase collaboration amongclinical, translational and basic scientists to support novel science andto classify and treat diseases not just by their phenotype, but also bytheir molecular profiles. Here we highlight several Core Resource Labsthat support translational research at NYULMC. Human biospecimensare essential validating mechanistic insights into biological processesgleaned from cell lines, animal models of disease, and epidemiologicalstudies. The availability of well-annotated human biospecimensis a critical link between basic science and translational research.The NYULMC Tissue Acquisition and Biorepository Core providesinvestigators with freshly acquired frozen, normal, and diseased tissueat the time of surgery, as well as pathology-archived, formalin fixed,paraffin-embedded (FFPE) specimens, from appropriately consentedpatients. The Immunohistochemistry and Histopathology Coresclosely interface with the Tissue Biorepository, providing the meansfor clinical and basic research collaborations and the microscopicanalysis of clinical research specimens. The Genome TechnologyCenters sequencing and microarray units are dedicated to RNA-seqof cancer patient transciptomes, array expression profiling and SNPgenotyping, enabling translation research as well as the preparation ofnucleic acids for molecular profiling, mutational analysis and microRNAscreens. The RNAi Core provides an integrated, state-of-the-art, RNAinterference (RNAi) based high-throughput screening (HTS) andoffers siRNA/dsRNA screening libraries for the cross-species functionalcharacterization of whole genomes in a systematic, comprehensive, andcost effective manner.Poster AbstractsThe Core Technologies (CT) unit, located at the Eastern RegionalResearch Center (ERRC), is a centralized resource of specializedinstrumentation and technologies. Its objective is to providesupplementary research data processing, interpretation, analysis andconsultation for a broad range of research programs approved by theAgricultural Research Service (ARS), the in-house research arm of theABRF 2011 — Technologies to Enable Personalized Medicine • 67

Poster Abstracts129 Bravo Automated Liquid HandlingPlatform for SureSelect TargetEnrichmentA. Giuffre 1 , M. Visitacion 2 , J. Karbowski 3 ,B. Novak 2 , H. Ravi 1 , J. Ong 1 , S. Joshi 1 ,C. Pabón-Peña 2 , E. LeProust 2 , D. Roberts 2 ,S. Happe 11Agilent Technologies, Genomics, Cedar Creek, TX,United States; 2 Agilent Technologies, GenomicsDivision, Santa Clara, CA, United States; 3 AgilentTechnologies, Automation Solutions Division, SantaClara, CA, United StatesAgilent’s SureSelect Target Enrichment products have met the need fora robust and cost-effective approach for systematic resequencing ofcandidate regions in the human genome and other species. SureSelectis an in-solution method of targeting only the user defined regionsof interest of a genome to identify genetic variants and mutationsassociated with disease.With the advent of SureSelect Target EnrichmentMultiplexing kits, the ability to achieve targeted enrichment on multiplesamples in a single sequencing lane is maximized, saving time and moneywithout sacrificing performance. However, the handling of numeroussamples can be cumbersome and the possibility for the introduction ofsample processing errors greatly increases. Agilent has expanded theSureSelect Target Enrichment System to allow for higher throughput ofnumerous samples with the Bravo Automated Liquid Handling Platform.The SureSelect method selects user defined target regions of interestfrom SureSelect XT prepped genomic DNA libraries by hybridizationto in-solution biotinylated cRNA probes; these enriched libraries arethen compatible for sequencing using the llumina GAII and HiSeqplatforms. The Agilent automation solution for SureSelect enablesprocessing of up to 96 samples per run in much less time than canbe obtained manually. Our SureSelect automation workflow includesSureSelect XT genomic DNA library construction, hybridization setup,and automated capture and wash steps. Each of these steps can berun with minimal user intervention and provide more uniformity thanmanual methods. We demonstrate here the throughput potential andconsistency in performance across entire 96-well plates of multiplexedsamples. In summary, SureSelect Automation solution provides an easyto use, automated gDNA library preparation and target enrichmentsystem that is a cost-effective approach to analyzing discrete genomicregions with unprecedented depth, accuracy and throughput.130 Modification of the Transplex WTA2Amplification Product for NextGeneration SequencingK. Heuermann 1 , B. Ward 1 , D. Fenoglio 21Biotech R&D, Sigma Aldrich Corporation, St. Louis,MO, United States; 2 Biotech Marketing, Sigma-Aldrich Corporation, St. Louis, MO, United StatesTransplex Whole Transcriptome Amplification (WTA2)ä exponentiallyamplifies RNA producing a double-stranded cDNA library whileprecisely maintaining differential levels of individual transcripts in testand reference samples. Though originally designed to amplify nanogramquantities of RNA, Transplex WTA2 has been shown to be exceedinglyeffective for amplification from damaged RNA template (FFPE and lasercaptured tissue samples) and single-cell input quantities (picograms).The efficacy of Transplex WTA2 amplification for downstreamapplications, primarily qPCR and expression microarray analysis, iswell-documented. It follows that the utilization of next-generationsequencing for gene expression research and diagnostics would bewell served by Transplex amplification of RNA isolated from samples ofseverely restricted quantity or quality. Strategies for the integration ofTransplex WTA2 with next-generation sequencing are examined, withparticular emphasis on elimination of the characteristic fixed primersequence associated with each amplicon in the amplification library.Removal of these sites will allow direct entry of the resulting productinto the sequencing workflow. Methods under consideration will enablethe WTA2 amplicon to feed into the current sample prep protocols forthe Illumina GA and GAII, SoLiD 5500/5500xl, and Roche-454 GS FLX/Junior platforms.131 Robotic Scripts, Methods, Reagents,and Devices for High ThroughputAutomated Production of NextGeneration Sequencing DNAFragment LibrariesJ. Bishop, M. Allen, K. Poulter, W. Zhang,M. Landers, D. Mandelman, B. Laubert, A. Harris,R. BennettLife Technologies, Carlsbad, CA, United StatesRecent technological advances have greatly increased both the speedand throughput of genome and transcriptome sequencing. The paceof sequencing has further increased with target-enrichment andlibrary barcoding techniques that allow multiplexing of sequencingruns. However, current manual methods for creating libraries do notscale well, limiting the practical investigation of large numbers ofsamples. To ease the library-creation bottleneck, we describe here aset of protocols, robotics scripts, bulk reagents and instrumentationdeveloped to automate the production of up to 96 DNA fragmentsequencing libraries at once. Current protocols for creating sequencinglibraries are lengthy, laborious, and not amenable to automation.We describe here new magnetic bead based methods that producelibraries with yield, purity, and size-selection comparable or superiorto current column and gel-based protocols. Using these bead-basedmethods we developed a unique combination of optimized adaptorconcentrations and clean-up techniques which increase the yield oflibraries from small amounts of DNA by several fold. We also describerobotic scripts for producing 1-96 libraries simultaneously on twocommonly used robotic platforms. These protocols accept 10-8000 ngof sheared DNA, calculate and dilute barcode adaptors for each libraryas necessary, automate all intermediate processing steps, and deliverpurified libraries ready for amplification off-instrument. An additionalscript for post-PCR purification of the libraries is also provided. Wefurther describe the Library BuilderTM System, comprising a benchtopdevice and kits of plastic tips, tubes and sealed cartridges prefilledwith reagents necessary for producing 1 to 13 DNA fragment librarieseither with or without size selection. Our analysis of sequencing datashows that libraries produced by all of the above methods are free ofexcess adaptors which interfere with quantitation, are unbiased, of highcomplexity, and free from cross-contamination. The protocols hereinare provided to the community for use or customization.68 • ABRF 2011 — Technologies to Enable Personalized Medicine

132 Improvements in SOLiD TM WholeTranscriptome Library PreparationWorkflow to Enable Low Input RNAAmountsC. San Jose HinahonLife Technologies, Austin, TX, United StatesThe SOLiD TM Total RNA-Seq (STaR-Seq) kit from Life Technologiesprovides a complete workflow for generating directional, randomprimed, whole transcriptome libraries from total RNA and fractionatedRNA. Poly(A) selected RNA is commonly used for expression profilingusing short read sequencing on the SOLiD TM instrument. The inputrequirements in the current STaR-Seq protocol is 100-500ng poly(A)selected RNA, input amounts well suited for studies using cell cultureor large tissues. However, in circumstances where the available sampleis limited, such as in the case of clinically derived material or the needto focus the analysis on specific cell populations, there is often too littleRNA available to perform the cDNA library analysis. Improvements tothe STaR-Seq workflow have been developed and validated pushingthe starting input to 5ng of poly(A) selected RNA, while still maintaininghigh concordance with the current method. Improvements includeoptimizations in: 1. Reducing RNA fragmentation time to increase RNAfragments within the suitable size range; 2. Altering purification methodsafter RNA fragmentation to minimize sample loss; 3. Adoption of abead purification and size selection method to reduce sample loss dueto gel size selection; 4. Modifying PCR conditions to maximize cDNAlibrary yields without biasing expression profiles. These improvementsenable STaR-Seq to be a viable option for samples with small quantitiesof total RNA. As the field of next generation sequencing advances, it isnecessary to meet the need for decreased sample requirements, whilemaintaining strand specificity, and accuracy.133 RNA-Seq Analysis with NextGENeSoftwareJ. McGuigan, Y. You, C. LiuSoftGenetics, LLC, State College, PA, United StatesRNA sequencing is a powerful tool for interrogating the entiretranscriptome at once. It allows identification of novel isoformsincludinggene fusions and alternative splicing- and expression levelanalysis across several orders of magnitude. NextGENe’s new RNA-seqapplication can be used to analyze data from any of the three main2nd generation sequencing systems including Roche GS FLXTM andFLX Titanium, Illumina Genome Analyzers, and Applied BiosystemsSOLiDTM Systems. Variant detection, expression level analysis, andisoform identification are all performed in one analysis. The tool employsa hybrid approach, combining the use of an exon junction referencesequence with the de novo detection of exons. This allows discovery ofpreviously un-annotated genes in addition to highly accurate alignmentto known transcripts. All detected transcripts are reported in additionto the information provided by NextGENe’s variant and expressionreports. Results are shown in the NextGENe Viewer which provides ahigh level of visualization for review and editing.134 GeneMarker® Genotyping Software:Tools to Increase the Statistical Powerof DNA Fragment AnalysisC.S. Johathan Liu, D. Hulce, X. Li, T. Snyder-LeibySoftGenetics, LLC, State College, PA, United StatesThe discriminatory power of post-genotyping analyses, such askinship or clustering analysis, is dependent on the amount of geneticinformation obtained from the DNA fragment/genotyping analysis. Thenumber of microsatellite loci amplified in one multiplex is limited by thenumber of dyes and overlapping loci boundaries; requiring researchersto amplify replicate samples with 2 or more multiplexes in orderto obtain a genotype for 12-15 loci. AFLP is another method that islimited by the number of dyes, often requiring multiple amplificationsof replicate samples to obtain more complete results. Traditionally,researchers export the genotyping results into a spread sheet, manuallycombine the results for each individual and then import into a thirdsoftware package for post-genotyping analysis. GeneMarker is highlyaccurate, user-friendly genotyping software that allows all of these stepsto be done in one software package, avoiding potential errors fromdata transfer to different programs and decreasing the amount of timeneeded to process the results. The Merge Project tool automaticallycombines the results from replicate samples processed with differentprimer sets. Replicate animal (diploid) DNA samples were amplifiedwith three different multiplexes, each multiplex provided informationon 4 -6 loci. The kinship analysis using the merged results provideda 1017 increase in statistical power with a range of 108 when 5 lociwere used versus 1025 when 15 loci were used to determine potentialrelationship levels with identity by descent calculations. These samesample sets were used in clustering analysis to diagram dendrograms.The dendrogram based on a single multiplex resulted in three branchesat a given Euclidian distance. In comparison, the dendrogram that wasconstructed using the merged results had eight branches at the sameEuclidian distance.135 Hotspot-Mutation Analysis of theEGFR/KRAS/BRAF Pathway UsingMutation Surveyor® SoftwareJ. Liu, D. Hulce, K. LeVan, N. ShouyongSoftGenetics, LLC, State College, PA, United StatesHotspot-mutation analysis of the EGFR/KRAS/BRAF pathway (orother clinically relevant pathway) can quickly genotype patients ascandidates who may respond favorably to specific drug treatmentsand therapies or into other groups where treatment options arelimited and less favorable. Sanger sequencing analysis using MutationSurveyor software provides high-throughput, high-sensitivity variationdetection. Increased efficiency can be achieved using flexible andcustomizable reporting-sequencing results can be organized bypatient identifiers, variation type (reported or unreported, pathogenicor benign or drug sensitive), by gene/exon/amplicon, or qualitymetrics, and other options. GenBank sequence files from NCBI forEGFR exons 18, 19, 20, and 21; KRAS exons 2 and 3; and BRAF exon15 were edited to contain reported variations. These reportedvariations included polymorphisms from dbSNP (downloaded withthe GenBank file), pathogenic and drug-sensitivity variations for EGFR(obtained from http://www.egfr.org/), activating mutations for KRAS,and constitutive mutations for BRAF. Bidirectional sequencing datafor twelve, simulated (mutations obtained from sequencing reportsin the scientific literature), patients were developed and comparedPoster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 69

distribution. Additionally, we show that the controls can be used toevaluate performance of various normalization and scaling methodsfor RNA-Seq count data. This analysis suggests that traditional scalingmethods based on the total number of reads result in a significant lossof accuracy and more robust methods are often required. This analysisreveals the importance of using standardized reagents and controls as aroutine part of the RNA sequencing workflow.139 Software Systems for ClinicalResearchT. Smith 1 , N.E. Olson 1 , D. Smith 2 , C. Mason 31Geospiza, Inc. Seattle, WA, United States; 2 MayoClinic, Rochester, MN, United States; 3 Weil CornellMedical College, New York, NY, United StatesBy the end of 2011 we will likely know the DNA sequences for 30,000human genomes. However, to truly understand how the variationbetween these genomes affect phenotype at a molecular level, futureresearch projects need to analyze these genomes in conjunction withdata from multiple ultra-high throughput assays obtained from largesample populations. In cancer research, for example, studies thatexamine 1000s of specific tumors in 1000s of patients are neededto fully characterize the more than 10,000 types and subtypes ofcancer and develop diagnostic biomarkers. These studies will usehigh throughput DNA sequencing to characterize tumor genomes andtheir transcriptomes. Sequencing results will be validated with nonsequencingtechnologies and putative biomarkers will be examined inlarge populations using rapid targeted assay approaches. Geospiza istransforming the above scenario from vision into reality in several ways.The Company’s GeneSifter platform utilizes scalable data managementtechnologies based on open-source HDF5 and BioHDF technologiesto capture, integrate, and mine raw data and analysis results from DNA,RNA, and other high-throughput assays. Analysis results are integratedand linked to multiple repositories of information that includevariation, expression, pathway, and ontology databases to enablediscovery process and support verification assays. Using this platformand RNA-Sequencing and Genomic DNA sequencing from matchedtumor/normal samples, we were able to characterize differential geneexpression, differential splicing, allele specific expression, RNA editing,somatic mutations and genomic rearrangements as well as validatethese observations in a set of patients with oral and other cancers.140 Hybrid-Core Computing for High-Throughput BioinformaticsG. VacekConvey Computer Corporation, Richardson, TX,United StatesAdvanced architectures can deliver dramatically increased throughputfor genomics and proteomics applications, reducing time-tocompletionin some cases from days to minutes. One such architecture,hybrid-core computing, marries a traditional x86 environment witha reconfigurable coprocessor, based on field programmable gatearray (FPGA) technology. Application-specific instructions executedby the coprocessor appear as extensions to the x86 instruction setarchitecture. This integrated approach provides users familiar C, C++and FORTRAN development environments without the complexity ofnon-standard dialects or programming models. Thus the performanceof application-specific hardware is achievable with the familiarprogrammability and deployment of a commodity server. In addition tohigher throughput, increased performance can fundamentally improveresearch quality by allowing more accurate, previously impracticalapproaches. This presentation will discuss the suitability of hybridcoreservers’ advanced architecture and compiler technology forsequence alignment and assembly applications. For example, the Smith-Waterman alignment algorithm is 172 times faster on Convey’s HC-1than the best software implementation on a commodity server. Suchperformance speeds research, while reducing energy consumption,floor space, and management effort. Most bioinformatics applicationsare similarly well suited for this architecture because they have lowdata interdependence, which greatly increases performance throughhardware parallelism. Furthermore, small data type operations (fournucleotides can be represented in two bits) make more efficient use oflogic gates than the data types dictated by conventional programmingmodels. Bioinformatics applications that have random access patternsto large memory spaces, such as graph-based algorithms, experiencememory performance limitations on cache-based x86 servers. Convey’shighly parallel memory subsystem allows application-specific logic tosimultaneously accesses 8192 individual words in memory, significantlyincreasing effective memory bandwidth over cache-based memorysystems. Many algorithms, such as Velvet and other de Bruijn graphbased, short-read, de novo assemblers, greatly benefit from this typeof memory architecture.141 Implementation of GeneSifterImproves Workflow Managementwithin a Core FacilityA. McCary, J. Boland, J. Bacior, V. LonsberryNational Cancer Institute, Bethesda, MD, UnitedStatesAs throughput within sequencing facilities increases, LaboratoryInformation Management Systems (LIMS) will become necessary toallow laboratory managers and staff to organize workflows, samplesand sample data in a repeatable and reliable manner. At the NCI’s CoreGenotyping Facility, we have successfully implemented Geospiza’sGeneSifter TM Lab Edition, a commercially available LIMS, for use with ourRoche 454 GS FLX sequencer processes. Our current setup consists ofseven different sample preparation techniques, (six target enrichmentmethods and one whole genome method), which have been dividedinto a series of modular workflows that are arranged in the properorder for each process. Each workflow is then further broken down intospecific protocol steps, which are used as guides for the techniciansduring protocol performance. Additionally, mastermix preparations,reagent lot numbers, file uploads and other pertinent information areinput into the system at all relevant protocol steps. Each sample’s datais stored on our internal GeneSifter TM server, with the ability to recalland report it at a later date. Therefore, by developing an extensiveand interconnected setup of workflows in GeneSifter TM , the NCI’s CoreGenotyping Facility has successfully increased reliability in sampletracking while decreasing the need for paper- or Excel-base loggingmethods. This project has been funded in whole or part with federalfunds from the NCI, NIH under contract HHSN261200800001E.Poster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 71

Poster Abstracts142 Comparison of Data EnvelopmentAnalysis Models to Identify PotentialCancer Biomarker Genes for ColonCancerP. Diaz-Candelas, M.E. Sanchez-Pena, C.E. Isaza,M. Cabrera-RiosBio IE Lab at University of Puerto Rico, Mayagüez,Puerto RicoThe identification of cancer biomarkers is critical to characterize,detect and understand the illness. In our research group, a novelmethod based on multiple criteria optimization has been proposedto detect potential cancer biomarker genes through the analysis ofmicroarray data. More precisely, the multiple criteria optimizationproblem is approached through a technique called Data EnvelopmentAnalysis (DEA). Given a number of genes characterized by at least twoperformance measures, DEA is able to find those genes that are thebest compromises among these measures. When using microarray dataand quantifiers of significant differences of gene expression betweenhealthy tissues and cancer tissues, those genes identified through DEAhave been shown to have a high potential to be biomarkers. DEA isknown to be efficient due to its structure based on linear optimization.Our studies have also shown that DEA can be very effective for thestated purpose. There are, however, several different DEA formulationsthat can be used. In this work, a comparison of these formulations ispresented in terms of number and quality of the selection of genes fortwo publicly available colon cancer microarray databases. The resultswill provide some light on the different solutions that can be arrivedto in multiple criteria optimization using DEA, to then establish what ismost adequate to find cancer biomarkers.**143 Comparing Protein and mRNAAbundances to Protein ExpressionRegulationC. Vogel 1,2 , J.M. Laurent 2 , T. Kwon 2 , S.A. Craig 2 ,D.R. Boutz 2 , H.K. Huse 2 , K. Nozue 3 , H. Walia 3 ,M. Whiteley 2 , P.C. Ronald 3 , S. Abreu Rde 4 , D. Ko 4 ,S.Y. Le 5 , B.A. Shapiro 5 , S.C. Burns 4 , D. Sandhu 4 ,L.O. Penalva 4 , E.M. Marcotte 21New York University, New York, NY, United States;2University of Texas at Austin, Austin, TX, UnitedStates; 3 UC Davis, CA, United States; 4 Universityof Texas at San Antonio, San Antonio, TX, UnitedStates; 5 NCI-Frederick, Frederick, MD, United StatesTranscription, mRNA decay, translation and protein degradationare essential processes during eukaryotic gene expression, but theirrelative global contributions to steady-state protein concentrations inmulti-cellular eukaryotes are largely unknown. Using measurementsof absolute protein and mRNA abundances in cellular lysate from thehuman Daoy medulloblastoma cell line, we quantitatively evaluate theimpact of mRNA concentration and sequence features implicated intranslation and protein degradation on protein expression. Sequencefeatures related to translation and protein degradation have an impactsimilar to that of mRNA abundance, and their combined contributionexplains two-thirds of protein abundance variation. mRNA sequencelengths, amino-acid properties, upstream open reading framesand secondary structures in the 5’ untranslated region (UTR) werethe strongest individual correlates of protein concentrations. In acombined model, characteristics of the coding region and the 3’UTRexplained a larger proportion of protein abundance variation thancharacteristics of the 5’UTR. Further, we used data from human and sixother organisms (bacteria, yeast, worm, fly, and plant) and establishedthat steady-state abundances of proteins show significantly highercorrelation across these diverse phylogenetic taxa than the abundancesof their corresponding mRNAs (p=0.0008, paired Wilcoxon). Thesedata suggest strong selective pressure to maintain protein abundancesduring evolution, even when mRNA abundances diverge. The absoluteprotein and mRNA concentration measurements for >1000 humangenes and for other organisms represent one of the largest datasetscurrently available, and reveal both general trends and specificexamples of post-transcriptional regulation.144 The Application of Targetedand Exome Sequencing for theIdentification of Spontaneous andInduced Mutations in MiceD. Hinerfeld, E. Antoniou, S. Daigle, Y. Ding,W. Zhang, L. Reinholdt, M. Barter, L. RoweThe Jackson Laboratory, Bar Harbor, ME, UnitedStatesThe Jackson Laboratory has established a large collection ofspontaneous and N-ethyl-N-nitrosourea (ENU) induced mousemutants with a wide variety of medically relevant phenotypes. Whilespontaneous mutations can be quite complex, including singlenucleotide polymorphisms (SNPs), transposon insertions, deletions, orinversions, ENU induced mutations are typically SNPs. The traditionalmethod of identifying the causative mutation through genetic mappingand Sanger sequencing of candidate genes has been effective but is timeconsuming, requires large populations of mice and can be expensive.In addition, it is particularly challenging when the mutation is not ina coding sequence. With a size of over 3 GB, it is still too expensiveto sequence the genomes of the many mutant strains of interest. Thecombination of array capture for targeted resequencing and/or exomecapture followed by high-throughput sequencing on the Illumina GAIIXhas greatly accelerated the pace at which mutations have be identified.In deciding what approach should be employed to identify a specificmutation, a number of factors must be considered; 1) Is the mutationspontaneous or ENU induced; 2) Have traditional mapping approachesbeen exploited, and if not, should they be; 3) If there is mapping data,what is the size of the genetic interval; 4) What data analysis tools will berequired; This approach has led to the identification of many mutationsthat result in disease-relevant phenotypes including craniofacialdisorders, neurodegeneration, neuromuscular dysfunctions, cholesterolbiosysthesis and reproduction.145 Single-Cell Copy Number AnalysisUsing the Illumina Genome AnalyzerE. Kamberov, J. Langmore, T. Kurihara,J. M’Mwirichia, T. Tesmer, D. OldfieldRubicon Genomics, Ann Arbor, MI, United StatesCNV, SNP, and mutation analyses of singles cells are important tocharacterize cancer, stem, and embryo cells. We used PicoPlex WholeGenome Amplification kits to sequence single human cancer cells,single-copy mouse chromosomes, and single bacterial cells using the72 • ABRF 2011 — Technologies to Enable Personalized Medicine

Illumina Genome Analyzer. PicoPlex is a 1-tube, 3-hr, 4-step methodto convert a single cell into cluster-station ready DNA. High qualitysequences with very little background were obtained (0.7% mismatchrates, 90+% mapped reads, 98% concordance betweenSureSelect re-sequencing results and previously determined genotypeis observed. Lastly, we introduce the SureSelect XT kit for preparationof samples for multiplex sequencing using the Illumina GAII or HiSeq.The SureSelect Multiplexing kit provides the ability to combinetargeted enrichment with multiplexing, thus maximizing the number ofsamples that can be sequenced at one time, providing optimum timeand cost savings without sacrificing performance.148 Comparison of SYBR Enzymesand Standards in Illumina LibraryQuantificationK. Thai, S.S. LevineMIT BioMicro Center, Massachusetts Institute ofTechnology, Cambridge, MA, United StatesPoster AbstractsAccurate quantification of libraries generated for Illumina nextgenerationsequencing is critical to prevent under- and overclusteringof the flowcell. Success of qPCR library quantifications depend onthe accuracy and reproducibility of standards used in addition to theability of DNA polymerase to efficiently amplify all adaptor-flankedlibraries. We compared the (i) Roche SYBR Green and (ii) KAPASYBR Fast polymerases and the PhiX standards and KAPA pre-dilutedstandards to determine the relative importance of these two factorsin library quantification by qPCR. We evaluated the success of eachmethod by comparing variation in the number of reads and absoluteABRF 2011 — Technologies to Enable Personalized Medicine • 73

Poster Abstractsyield generated from sequencing. Our results indicate that the DNApolymerase from the KAPA SYBR Fast kit is better suited to libraryquantification applications while the Roche SYBR Green kit showedsignificant variability, possibly related to sample type or insert size.No differences were observed between the two types of standardsused, making DNA polymerase the determining factor in the successfulamplification of each library.**149 A New Sequencing Primer andWorkflow Increase 5’ Resolution andThroughput on HLA SequencingJ. Chuu 1 , S.C. Hung 1 , S. Berosik 1 , M. Wenz 1 ,S. Schneider 1 , P. Ma 1 , D. Berchanskiy 2 , D. Dinauer 21Life Technologies, Foster City, CA, United States;2Life Technologies, Brown Deer, WI, United StatesHigh quality and high accuracy are the hallmarks of Sanger resequencingprojects. We have developed a new sequencing primer andworkflow that improves 5’ sequence resolution, increases throughput,and reduces hands-on time. The novel sequencing primer chemistryproduces high quality bases from base 1 on POP-7TM polymer thatpreviously only could be resolved on the slower POP-6TM polymer.The new primer chemistry and workflow also eliminates the needfor a separate PCR clean-up step. These improvements reduce theentire workflow from PCR to finished sequence data to under 5hours, compared to 8 hours for the standard workflow. We used ourenhanced sequencing primer and workflow to investigate feasibilityon Human Leukocyte Antigen (HLA) polymorphisms on twelve DNAsamples by using the Invitrogen SeCore® HLA-DRB1 primer set andGroup Specific Sequencing Primers. Sequencing reactions generatedwith the traditional sequencing primer and with the new sequencingprimer were electrophoresed on Applied Biosystems 3500xl GeneticAnalyzer using POP-7TM polymer. For each sequencing primer, wecompared 5’ resolution and basecalling accuracy and quality. Onaverage the traditional primers produced high quality readablebases by base 25 after the sequencing primer while the new primersproduced high quality bases by base 5, and by base 1 in many cases.Because of improved resolution, basecalling accuracy was increased.This simplified process without a separate PCR clean-up step reducedthe overall workflow time by 40%. For HLA genes, obtaining readablesequence within 5 bases of the primer offers improved polymorphismdetection and more efficient use of allele specific sequencing primersfor heterozygous ambiguity resolution. In conclusion, the novel primerchemistry and workflow generates data superior in quality relative toother currently used solutions and offers significant time savings as well.Specific applications of this product are under development and notintended for clinical use.150 ScriptSeq RNA-Seq LibraryPreparation Method: A SimplifiedWork-Flow for Directional NGS RNA-Seq Library Preparation with Whole-Transcript RepresentationA. Khanna 1 , R. Sooknanan 2 , J. Hitchen 2 , A. Radek 21Epicentre BIOtechnologies, Madison, WI, UnitedStates; 2 RiboTherapeutics Inc., Saint Laurent, QC,CanadaRNA sequencing is an emerging revolutionary tool for wholetranscriptomeanalysis that provides information about the structureof transcripts and their expression levels. Current methods for makingsequencer-specific di-tagged DNA fragment libraries for RNA-Seqtypically comprise preparing rRNA-depleted RNAand either (i) RNAfragmentation, 5’ and 3’ adaptor-ligation, size selection, cDNA synthesis,and multiple clean-up steps; or (ii) cDNA synthesis followed by cDNAfragmentation, end-polishing, 5’ and 3’ adaptor-ligation, size selectionand multiple clean-up steps. These methods are generally long andrequire significant hands-on time. We describe a novel protocol thatutilizes a unique Terminal-Tagging technology that simplifies thepreparation of directional RNA-Seq libraries from rRNA-depleted orpoly(A)-enriched RNAin about 3 hours, without the need for adaptorligation, cDNA nebulization or gel purification. The di-tagged cDNAfragments that are generated from this simple, single tube protocolare compatible with different NGS sequencing platforms. Apart fromits simplicity, another major strength of this RNA-seq protocol is itsability to determine the polarity of the RNA transcripts, which is criticalfor the annotation of novel genes. Sequencing results obtained fromlibraries prepared by using RiboZeroTM rRNA-depletion method andScriptSeq mRNA-Seq Library Preparation Method show excellentdirectionality with less than 2 % of the sequence reads that map torRNA sequences (28S, 18S, 5.8S and 5S). This reduction in rRNAsequence reads improves sequence depth and coverage, and increasesthe percentage of uniquely mapped reads. Further, there is a highcorrelation (R2=0.9235) between differentially expressed transcriptsfound in the ScriptSeq RNA-Seq libraries and the MAQC QPCR panelof genes.151 The Agilent Technologies’SureSelect TM All Exon ProductPortfolio: High Performance TargetEnrichment System for Human andMouse Exome Sequencing on Illuminaand SOLiD PlatformsH. Ravi, A. Giuffre, C. Pabón-Peña, B. Novak,S. Joshi, J. Ong , M. Visitacion, M. Hamady,F. Useche, J. Eberle, S. Hunt, S. Happe, D. Roberts,E. LeproustAgilent Technologies, Stratagene Products Division,Cedar Creek, TX, United StatesThe dramatic increase in throughput of sequencing data from nextgenerationsequencing platforms has enabled scientists to study thegenome with unprecedented depth and accuracy. Nevertheless, routinegenetic screens in large numbers of individuals continue to remain costprohibitivethrough these approaches. Agilent Technologies’ SureSelectplatform for targeted exome capture, combined with massively parallelsequencing, provides a more affordable method to gain novel insightsinto the genetic causes of inherited disorders. In addition, identificationof both common and rare polymorphisms implicated in complexdiseases like cancer is greatly facilitated by selectively sequencing theprotein-coding regions of the genome. In collaboration with the Broadand Sanger Institutes, Agilent Technologies has continued to expandthe number of SureSelect target enrichment catalog products in orderto enable a more comprehensive view of the protein-coding regionsin humans and model organisms. We discuss the SureSelectHuman AllExon v2 (44Mb) and SureSelectHuman All Exon 50Mb designs. We alsointroduce the SureSelectMouse All Exon target enrichment system,which improves the ability to study genetic variation between strains ingreater detail, and significantly increases the efficiency of screening forcausative mutations in N-ethyl-N-nitrosourea (ENU)-mutagenized mice.We demonstrate high performance with respect to capture efficiency,74 • ABRF 2011 — Technologies to Enable Personalized Medicine

uniformity, reproducibility of enrichment, and ability to detect SNPs,insertion/deletions, and CNVs across Illumina (Genome Analyzer IIxand HiSeq2000) and SOLiD platforms. We highlight the utility of theSureSelect All Exon product portfolio for a wide variety of applicationsprimarily due to the high specificity and excellent cross-platformsequence coverage. SureSelect All Exon designs also provide a meansfor standardization, consistency of performance, and reliability acrossmultiple laboratories.152 PCR-Free Nextera Di-TaggedDNA Library Preparation for NGSApplicationsC. Kinross, H. Grunenwald, B. Baas, I. Goryshin,N. Caruccio, M. MaffittEpicentre Biotechnologies, Madison, WI, UnitedStatesThe Nextera TM technology for generating libraries of di-tagged DNAfragments is rapidly becoming the preferred method for massivelyparallel DNA sequencing. Despite rapid advances in sequencinginstrument throughput, classic library preparation by step-wise ligationof adaptors is a time-intensive and throughput-limiting bottleneck.PCR amplification of libraries prior to cluster generation is also amajor concern because of its possibility to reduce library complexity,particularly in regions of extreme G+C content (high or low), therebyproducing uneven genome coverage and confounding mapping andassembly. In this study, we describe novel modifications of the Nextera TMlibrary preparation system to address such library preparation biasby eliminating PCR amplification. Sequencer-ready libraries can beobtained from as little as 200 ng of genomic DNA in 3 hours with90-minutes of hands-on time. Deep sequencing of genomic librariesindicates that this system reduces coverage bias and GC bias, as well asimproves library diversity.153 Semiconductor Sequencing for LifeJ. Myers, J. RothbergIon Torrent, South San Francisco, CA, United StatesIon Torrent has invented the first device-a new semiconductor chipcapableof directly translating chemical signals into digital information.The first application of this technology is sequencing DNA. The deviceleverages decades of semiconductor technology advances, and in justa few years has brought the entire design, fabrication and supply chaininfrastructure of that industry-a trillion dollar investment-to bear on thechallenge of sequencing. The result is Ion semiconductor sequencing,the first commercial sequencing technology that does not use light, andas a result delivers unprecedented speed, scalability and low cost. Allof these benefits are a result of applying a technology that is massivelyscalable, as proven by Moore’s Law, to a task that has traditionally usedoptics-based solutions, which work in a linear fashion: increasing capacityrequires increasing the number of signals that must be read resultingin longer run times, higher capital costs and ever more sophisticatedoptics. By contrast, Ion Torrent semiconductor technology can provideincreases in chip capacity without impacting capital costs or runtime.Ion Torrent sequencing uses only natural (label-free) reagents andtakes place in Ion semiconductor microchips that contain sensors whichhave been fabricated as individual electronic detectors, allowing onesequence read per sensor. We will show how the technology has scaledin just a few months from ~1 million sensors in the first-generationIon 314 chips to ~7 million sensors in the second-generation Ion 316chips-all while maintaining the same 1- to 2-hour runtime. We will alsodemonstrate that Ion semiconductor sequencing provides exceptionalaccuracy, long read length and scalability on a single, affordable benchtopsequencing platform.154 A Comparison of Post-DNASequencing Dye-Terminator RemovalProtocolsM. Zianni, A. McCoyThe Ohio State University, Columbus, OH, UnitedStatesCapillary electrophoresis, a method for separation of ions based upontheir size to charge ratio, remains in high demand for DNA sequencing.In the process of dideoxynucleotide terminator sequencing,unincorporated nucleotides and other contaminants remaining in thereaction mixture can cause multiple issues in the electropherograms,such as unincorporated dye peaks, missed base calls, decreased signalstrength or a complete lack of data as a result of blocked capillaries. Avariety of dye-terminator removal protocols exists to clean and purifythe sequencing reaction extension products. To determine their qualityand reproducibility, six protocols were tested with one large volumecontrol reaction aliquoted into a 96-well PCR plate. The protocolsincluded ethanol precipitation, gel filtration, and 4 solid phasereversible immobilization procedures with 1 utilizing the surface of aplate and the other 3 utilizing magnetic beads. The cleaned and purifiedsequencing reactions were processed on the 3730 DNA Analyzer(Applied Biosystems), and the contiguous read lengths, QV20+ scores,and signal strengths of the resulting sequences were analyzed withSequence Scanner v1.0 (Applied Biosystems). Based on the results fromtwo replicate rounds of testing, the gel-filtration protocol provided thelongest contiguous read lengths and highest QV20+ scores.155 Simplified Reagents and Workflowsfor Robust Sample Preparation ofDNA, mRNA, and Small RNAF.J. Stewart, C.L. Hendrickson, L.M. Apone,D.B. Munafo, C.R. MeyerNew England Biolabs, Inc., Ipswich, MA, UnitedStatesAs yields of data generated by the Illumina, SOLiD, and 454sequencing platforms increase, NGS users have transitioned fromperforming multiple sequencing runs per sample to multiple samplesper sequencing run. As a result, the bottleneck in sequencing labs hastransitioned from data generation to sample preparation, necessitatingthe development of streamlined library construction workflows. Wehave developed a series of reagents to facilitate the easy preparationof numerous samples in parallel, compatible with both manual andautomated pipelines. These reagents reduce the amount of laborrequired, minimize error in reaction set up, and increase the stability ofenzymes used in the construction of libraries for DNA, mRNA and SmallRNA sequencing. As a result, these reagents enable the developmentof robust workflows for both individual and high throughput samplepreparation.Poster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 75

156 Comparison of Custom TargetEnrichment Methods: Agilent vs.NimblegenK. Bodi 1 , P.S. Adams 2 , D. Bintzler 3 , A. Perera 4 ,K. Dewar 5 , D.S. Grove 6 , J. Kieleczawa 7 , R.H. Lyons 8 ,T. Neubert 9 , A. C. Noll 1 , S. Singh 10 , R. Steen 11 ,M. Zianni 121Tufts University, Boston, MA, United States;2Trudeau Institute, Saranac Lake, NY, UnitedStates; 3 DNA Analysis, Inc., Cincinnati, OH, UnitedStates; 4 Stowers Institute, Kansas City, MO, UnitedStates; 5 McGill University, Montreal, QC, Canada;6Pennsylvania State University, University Park,PA, United States; 7 Pfizer Research, Cambridge,MA, United States; 8 University of Michigan, AnnArbor, MI, United States; 9 New York University, NewYork, NY, United States; 10 University of Minnesota,Minneapolis, MN, United States; 11 Harvard MedicalSchool, Cambridge, MA, United States; 12 OhioState University, Columbus, OH, United StatesOver the last four years, we witnessed the tremendous advances inNext Generation Sequencing (NGS) that have dramatically decreasedthe cost of whole genome sequencing. However, the cost of sequencinglarger genomes is still significant. In addition and depending on thegoal of study, whole genome sequencing creates a large amount ofadditional/auxiliary data that complicates data analysis. There areseveral commercial methods available for isolating subsets of genomesthat greatly enhance the efficiency of NGS by allowing researchersto focus on their regions of interest. For the 2009-11 DSRG study,we compared products from two leading companies; Agilent andNimblegen, that offer custom enrichment methods. Both companiesobtained the same genomic DNA stock and performed DNA captureon the same specified regions. Following capture, the Illumina GenomeAnalyzer IIx system was used, in two different laboratories, to generatethe sequence data. We present our data comparing in terms of cost,quality, reproducibility and most importantly completeness and depthof coverage. Acknowledgements: We would like to thank Agilent,Illumina and Nimblegen for all their support in making this studypossible.157 A Methodology Study forMetagenomics Using Next GenerationSequencersD. Grove 1 , I. Albert 1 , D. Bintzler 2 , K. Bodi 3 ,M. Bruns 1 , K. Dewar 4 , G. Gloor 5 , T. Johnson 6 ,J. Kieleczawa 7 , R.H. Lyons 8 , T. Neubert 9 ,A.G. Perera 10 , S. Singh 6 , R. Steen 11 , M. Zianni 121Penn State University, University Park, PA, UnitedStates; 2 DNA Analysis, Inc., Cincinnati, OH, UnitedStates; 3 Tufts University, Boston, MA, UnitedStates, 4 McGill University, Montreal, QC, Canada;5University of Western Ontario, London, ON,Canada; 6 University of Minnesota, Minneapolis,MN, United States; 7 Pfizer Research, Cambridge,MA, United States; 8 University of Michigan, AnnArbor, MI, United States; 9 New York University, NewYork, NY, United States; 10 Stowers Institute, KansasCity, MO, United States; 11 Harvard Medical School,Cambridge, MA, United States; 12 Ohio StateUniversity, Columbus, OH, United StatesMetagenomics is one of several genomics applications, which hasbenefited immensely from the high throughput and cost efficacy ofNext Generation sequencers. And although hundreds of studies onmetagenome analysis have been published over the past few years,the methodology for conducting them is still very much evolving.In this DSRG study we will evaluate the influence of various samplepreparation methods, specifically DNA extraction and amplificationapproaches, on data output along with a comparative analysis of NextGeneration sequencing platforms. We will study the effect of thesedifferent experimental and technical strategies on determination ofsample biodiversity.158 What Your Blood Has to Say:Amplifying Blood RNA for theAffymetrix GeneChip® PlatformN. Supunpong Hernandez, T. Barta, C. Willis,R. Conrad, P. Whitley, K. BramlettLife Technologies, Austin, TX, United StatesPoster AbstractsGene Expression measurements from human blood RNA havebecome an increasingly important research area of focus. A reliableand consistent workflow to obtain RNA measurements from bloodwould serve to open the clinical arena to gene expression studies aspotential diagnostic indicators. The challenge lies in isolating highquality RNA from human whole blood at room temperature withoutcompromising gene expression profiles. To address this challenge, wereport a workflow using newly developed MagMax TM RNA isolation kitsfor Tempus TM stabilized blood and PAXgene® stabilized blood. Thesetwo stabilization methods are currently the most widely used methodsfor blood collection and stabilization in clinics in the United States.High quality RNA generated from these two RNA isolation processeswas amplified using the Ambion MessageAmp TM Premier RNAAmplification Kit and hybridized to Affymetrix GeneChip® microarrays.This platform and experimental tools are used, in combination, todemonstrate high quality gene expression profiles from human bloodRNA. The reported experimental design includes human whole bloodobtained from two donors collected and stabilized in either Tempus TMPAXgene® tubes. RNA was isolated using new MagMax TM RNA isolation76 • ABRF 2011 — Technologies to Enable Personalized Medicine

kits for Tempus TM and PAXgene® stabilized blood. Isolated total RNAwas then processed through the GLOBINclear TM -Human kit and thenamplified with the MessageAmp TM Premier kit creating a library forhybridization to the Affymetrix Human U133A 2.0 expression arrays.Affymetrix GeneChip® microarray analysis showed parameters withinnormal limits for expressed genes. Reported resultsof this controlledexperiment, support a validated gene expression workflow for bloodon Affymetrix expression arrays using a combination of commercial kitsfor RNA purification from stabilized blood and library amplificationoptimized for hybridization and analysis on expression microarrays.159 Performance Comparison of FourMethods Utilized for the Purificationof Enzymatically-Digested,Fluorescently-Labeled PCR FragmentsGenerated During T-RFLP AnalysisM. Zianni, J. Panescu, P. KumarThe Ohio State University, Columbus, OH, UnitedStatesThe fluorescently-labeled Terminal Restriction Fragment LengthPolymorphism (T-RFLP) assay based on amplified ribosomal DNAfrom bacteria is an inexpensive, widely accessible, effective and wellestablishedmolecular technique for the identification and comparativequantification of bacterial species in metagenomics. In order tofacilitate the detection of a large proportion of species in a givensample, it is necessary to maximize the recovery of the fluorescentlylabeled,restriction enzyme-digested PCR fragments generatedduring the process. The post-digestion purification method used tofacilitate fragment separation is a critical step in the retention of DNAfragments. Four methods for post-digestion purification were testedin an effort to characterize their integrity, effectiveness, ease of useand potential biases: solid phase reversible immobilization (AMPure TMand CleanSEQ TM ), contaminant capture (BigDye® XTerminator TM ),and dialysis (Millipore TM Nitrocellulose Membranes). Samples werecollected from four different oral sites in each of 16 patients. Eachsample was divided equally and the DNA was isolated with two distinctmethods. PCR reactions were carried out with two paired universalprimers for the 16S gene that were labeled with VIC and FAM, purifiedwith AMPure TM and digested separately with HhaI and MspI restrictionenzymes. From each of the digestions, identical aliquots were purifiedwith the four different methods. The DNA fragments were analyzedon the Applied BioSystems TM 3730 DNA Analyzer using identicalconditions, followed by data analysis with GeneMapper® v4.0. Basedon preliminary data analysis, CleanSEQ TM is the superior purificationmethod because it resulted in the most numerous peaks recovered persample with a wide distribution of sizes from approximately 50 to 1200bp.160 Electrochemical Simulationof Covalent DNA AdductFormation Monitored with LiquidChromatography/Mass SpectrometryJ. Powers 2 , S. Plattner 1 , R. Erb 1 , F. Pitterl 1 ,J.P. Chervet 2 , H. Oberacher 11Institute of Legal Medicine, Innsbruck MedicalUniversity, Innsbruck, Austria; 2 Antec, Palm Bay, FL,United StatesDNA adduct is a piece of DNA covalently bond to chemicals. Adductsactivate repair processes and, unless repaired prior to replication, maylead to nucleotide substitutions, deletions, and other rearrangements.As alterations of the genetic material can cause severe diseasesincluding cancer, inflammation, and neurodegenerative disorders,tests on mutagenicity/genotoxicity have become an integral part of riskassessment during the development of any new chemical. A number of invitro tests using different cell lines and in vivo tests mainly using rodentsare available to identify hazardous compounds. The majority of thesetests are time-consuming, labor-intensive and hardly automatable. So insearch for alternative methods, the usefulness of electrochemistry (EC)-liquid chromatography (LC)-mass spectrometry (MS) was evaluated.Generally, EC represents a powerful tool to study in vitro biologicaloxidation processes of a number of different compounds, includingdrugs, peptides and proteins. Here, we used EC to initiate adductformation. The obtained reaction products were separated by LC anddetected by MS. Tandem MS experiments were used for structuralconfirmation. In a proof of principle study acetaminophen was selectedas model compound. Covalent adduct formation was observed forelectrochemical activated mixtures of acetaminophen and guanosine.Mechanistic studies revealed that adduct formation will only start atelectrochemical potentials sufficiently high to initiate oxidation of bothacetaminophen and guanosine. The stringent necessity of coactivationsheds a new light on the mechanism of adduct formation, becauseaccording to the generally accepted theory activation of the adductformingagent should be sufficient. Chromatographic separationenabled the differentiation of four isomeric forms. Their connection toacetaminophen was proven in dose-response experiments. EC/LC/MSrepresents a fast, simple, convenient and functional tool to study DNAadduct formation which has great potential to complement the existingbattery of mutagenicity/genotoxicity tests.**161 Quantitative miRNA ExpressionAnalysis Using Fluidigm MicrofluidicsDynamic ArraysJ. Jen, J. Sung Jang, V.A. Simon, R.M. Feddersen,F. Rakhshan, D.A. Schultz, M.A. Zschunke,W.L. Lingle, C.P. Kolbert.Mayo Clinic Micorarray Shared Resource andBiospecimens Accessioning Processing SharedResource, Rochester, MN, United StatesPoster AbstractsMicroRNA (miRNA) is a small non-coding RNA that can regulate geneexpression in both plants and animals. Studies showed that miRNAs playa critical role in human cancer by targeting messenger RNAs that arepositive or negative regulators of cell proliferation and apoptosis. Here,we evaluated miRNA expression in formalin fixed, paraffin embedded(FFPE) samples and fresh frozen (FF) samples using a high throughputqPCR-based microfluidic dynamic array technology (Fluidigm). Wecompared the results to hybridization-based microarray platformsABRF 2011 — Technologies to Enable Personalized Medicine • 77

Poster Abstractsusing the same samples. We obtained a highly correlated Ct valuesbetween multiplex and single-plex RT reactions using standard qPCRassays for miRNA expression. For the same samples, the microfluidictechnology (Fluidigm 48.48 dynamic array systems) resulted in a leftshifttowards lower Ct values compared to those observed by standardTaqMan (ABI 7900HT, mean difference, 3.79). In addition, as little as10ng total RNA was sufficient to reproducibly detect up to 96 miRNAsat a wide range of expression values using a single 96-multiplexingRT reaction in either FFPE or FF samples. Comparison of miRNAsexpression values measured by microfluidic technology with thoseobtained by other array and Next Generation sequencing platformsshowed positive concordance using the same samples but revealedsignificant differences for a large fraction of miRNA targets. The qPCRarraybased microfluidic technology can be used in conjunction withmultiplexed RT reactions for miRNA gene expression profiling. Thisapproach is highly reproducible and the results correlate closely withthe existing singleplex qPCR platform while achievingmuch higherthroughput atlower sample input and reagent usage. It is a rapid, costeffective, customizable array platform for miRNA expression profilingand validation. However, comparison of miRNA expression usingdifferent platforms requires caution and the use of multiple platforms.162 Apollo 200 Fully Integrated DNAHID System: Multi Channel Results inUnder 2 HoursS. Jovanuvich, O. El-Sissi, H. Franklin, B. Nielsen,S. Pagano, R. Belcinski, G. BogdanIntegneX, Inc, Pleasanton, CA, United StatesThe ideal solution to meet the requirements of modern humanidentification is an automated, DNA-based hu man identificationsystem that processes samples rap idly and at low cost. IntegenX Inc.will describe the Apollo 200 DNA HID System. The Apollo200 isthe first fully automated sample-to-answer system for STR based HID.The system is based on integration of the company’s proprietary andpatented technologies as well as its rapid in-house microfluidic chipprototyping. Reagents in disposable cartridges are loaded on thesystem with up to four buccal swab samples, the sample processing isinitiat ed and a CODIS compatible profile is ready in less than two hourswith no further user interaction. The Apollo 200 integrates all of thesample handling steps starting from buccal swab(s) or blood, throughcell lysis, DNA extraction, amplification, separation, and detection.Further, the system uses rapid PCR chemistry, on-board capillaryelectrophoresis, and integrated la ser induced fluorescence detection.Initial data from testing conducted by IntegenX and customers will beshown. **Apollo 200 is a trademark of IntegenX, Inc.163 Transcriptome Analysis Using Next-Generation Sequencing TechnologyK. Bramlett, K. Lea, L. Qu, P. Whitley,J. Schageman, J. GuLife Technologies, Carlsbad, CA, United StatesHigh throughput RNA sequencing (RNA-Seq) is becoming increasinglyutilized as the technology of choice to detect and quantify known andnovel transcripts. Multiple next-generation sequencing (NGS) platformsare available that enable transcriptome profiling through RNA-Seqworkflows. Demonstrations of the power of RNA-Seq to profile thewell annotated transcriptome and also identify novel transcribedregions, gene fusions, and even identify novel classes of RNA arerapidly increasing in the field of RNA research. Our aim has been todevelop library preparation methods and tools that aid in the reliablegeneration of libraries for next generation sequencing from total RNA.Reported here are results from the development of the Ambion®RNA-Seq Library Construction kit optimized for sequencing on theIllumina® next generation sequencing instruments. We show resultsfrom two protocols utilizing the same reagents that allow generationof RNA-Seq libraries targeting either the small RNA fraction of totalRNA, or the whole transcriptome which includes transcripts larger than100 base pairs. Results are reported from Illumina® Genome AnalyzerII sequencing of both small RNA and transcriptome libraries with afocus on mapping to the miRBase and RefSeq references respectively.We also demonstrate the use of External RNA Control Consortium(ERCC) transcripts as spike-in controls for transcriptome librariesthat aid in quality control of the library generation procedure andaid in downstream data analysis. The library construction technologyembedded in the Ambion® RNA-Seq Library Construction kit enablesresearchers to analyze the transcriptome of their research samples ina precise, sensitive and robust manner while maintaining informationregarding the genomic DNA strand to which the RNA transcript mapsutilizing the Illumina® Genome Analyzer II sequencing platform. Theworkflow and results reported here demonstrate new commerciallyavailable options for library construction enabling small RNA andtranscriptome profiling and novel discovery using next-generationsequencing technology.164 A Case Study: Molecular Profiling ofBreast Cancer from Formalin-Fixed,Archival Material — Gene ExpressionProfiles from FFPE Samples withImproved RNA DecrosslinkingTechnologyR. Jaggi 1 , S. Quabius 2 , G. Krupp 31University Bern, Bern, Switzerland; 2 UKSHUniversity Klinik, Kiel, Germany; 3 AmpTec GmbH,Hamburg, GermanyWe have developed a novel demodifaction/decrosslinking protocolfor RNA recovery from archival (FFPE) material. The resulting FFPERNA quality is superior to RNA obtained with other commercialFFPE RNA isolation kits: larger RNAs can be recovered, and RTqPCRdata demonstrate less variability and lower Cq values. ThisFFPE RNA is suitable for differential gene expression measurementby qPCR, high concordance with parallel RNA samples from freshfrozentissues was observed. Prognosis of breast cancer is determinedby clinicopathological and molecular factors. We developed andvalidated molecular scores reflecting the hormone status (ER, PGR,HER2 scores) and the proliferation status (PRO score) of breast cancercells. The scores can be combined to an overall RISK score. Molecularscores are independent prognostic parameters, they were validated inpostmenopausal patients with estrogen receptor positive breast cancer.Multivariate analysis revealed that PRO and RISK scores outperformconventional parameters (histological grading and Ki-67 labelingindex). Molecular scores are based on routine pathological material,testing can be implemented easily into routine diagnosis.78 • ABRF 2011 — Technologies to Enable Personalized Medicine

165 Benchmarking miRNA ExpressionLevels in Degraded RNA SamplesUsing Real-Time RT-qPCR andMicroarray TechnologiesJ. Holbrook 5 , S.V. Chittur 1 , S. Tighe 2 , V. Nadell 3 ,R. Carmica 4 , K. Sol-Church 5 , A.T. Yeung 61State University of New York at Albany, Albany,NY, United States; 2 University of Vermont,Burlington, VT, United States; 3 Ohio University,Athens, OH, United States; 4 The University ofTexas Medical Branch, Galveston, TX, UnitedStates; 5 Nemours/A.I. duPont Hospital for Children,Wilmington, DE, United States; 6 Fox Chase CancerCenter, Philadelphia, PA, United StatesThe Nucleic Acid Research Group (NARG) has conducted experimentsto determine the impact of RNA integrity and priming strategies oncDNA synthesis and Real-Time RT-qPCR. As a continuation of the RNAintegrity theme, this year’s study was expanded to evaluate the impactof RNA integrity on priming strategies for the analysis of nine miRNAtargets using Real-Time RT-qPCR. The nine targets were selectedbased on data obtained by the Microarray Research Group (MARG)and represent groups of miRNAs that are expressed at low, medium, orhigh levels in the First Choice human brain reference RNA sample. Thetwo RT-qPCR priming strategies tested in this study include the miRNATaqMan assay (Megaplex) of ABI and the RT2 miRNA qPCR assay ofQiagen/SA Biosciences. The basis for the ABI assay design is a targetspecificstem-loop structure and reverse-transcription primer, whilethe Qiagen design combines poly (A) tailing and a universal reversetranscription in one cDNA synthesis reaction. For this study to assessboth RT methods, samples that were used as templates were humanbrain reference RNA that has been subjected to controlled degradationusing RNase A to RIN (RNA Integrity Number) values of 7 (good), 4(moderately degraded), and 2 (severely degraded). In addition tothis Real-Time RT-qPCR data, the same RNA templates were furtheranalyzed using universal poly (A) tailing followed by hybridizationto Affymetrix miRNA GeneChips. We present some insights into RTpriming strategies for miRNA and contrasts qPCR results obtained usingdifferent technologies.168 MicroRNA Analysis Using RNAExtracted from Matched Formalin-Fixed Paraffin-Embedded (FFPE) andFresh Frozen Samples on SOLiD TMSystemK. Lea, J. Gu, E. Zeringer, S. Heater, J. Schageman,C. Mueller, K. BramlettLife Technologies, Carlsbad, CA, United StatesArchived formalin-fixed paraffin-embedded (FFPE) specimensrepresent excellent resources for biomarker discovery, but it has beena major challenge to study gene expression in these samples due tomRNA degradation and modification during fixation and processing.MicroRNAs (miRNAs) regulate gene expression at post-transcriptionallevel and are considered as important regulators of cancer progression.Next generation sequencing technologies such as SOLiD TM providean ideal method for measuring the abundance of miRNA moleculesin different cancer stages and provide insightful information ontumorigenesis. However, currently there is no good method tosystematically study miRNA expression in FFPE samples on nextgeneration sequencing platforms. We have designed and developeda ligation-based miRNA detection method to capture small RNAsequences in FFPE samples and convert them into templates suitablefor sequencing on the SOLiD TM System. Total RNA was isolated frommatched lung adenocarcinoma FFPE and snap frozen tissues using anAmbion RecoverAll TM kit. A PureLink TM miRNA Isolation kit was usedto enrich the small RNA fraction in these total RNA samples. Librarypreparation using a SOLiD TM Total RNA-Seq kit with modified protocolwas performed on the enriched RNA followed by sequencing onSOLiD TM system. Our results show that small RNA extracted from FFPEsamples was successfully converted to small RNA libraries. Very similarmapping statistics were obtained from matched FFPE and fresh-frozensamples after SOLiD TM sequencing. A good correlation of miRNAexpression pattern was also observed. This suggests that miRNAmolecules are less affected by sample degradation and RNA-proteincrosslink. This study provides a foundation for miRNA expressionanalysis on SOLiD TM system using FFPE samples in cancer and otherdiseases.**169 Structural Insights into theMechanism of microRNA ModulatedViral TranslationS. Patel 2 , E.A. Pham 1 , P. Pang 1 , M.A. Winters 1 ,M. Eckart 2 , J.S. Glenn 1,31Department of Medicine, Stanford UniversitySchool of Medicine, Stanford, CA, United States;2Protein and Nucleic Acid Core Facility, StanfordUniversity School of Medicine, Stanford, CA, UnitedStates; 3 Palo Alto Veterans Administration MedicalCenter, Palo Alto, CA, United StatesMicroRNAs (miRNAs) are small non-coding regulatory RNAs that controla vast array of cellular processes by repressing mRNA translation. LiverexpressedmiR-122 is a miRNA that has been co-opted by hepatitis Cvirus (HCV) to enhance viral translation. Recently, miR-122 antagomirtherapy in non-human primates has been shown to suppress HCVviremia; this proof-of-concept study demonstrates the considerablepotential of this novel antiviral strategy. The mechanism by which miR-122 modulates HCV translation, however, is unclear. To examine thestructural changes that miR-122 exerts on the HCV internal ribosomalentry site (IRES), we developed an advanced Selective 2’-HydroxylAcylation analyzed by Primer Extension (SHAPE) method of analyzingRNA architecture. SHAPE determines RNA secondary structure atsingle-nucleotide resolution, with an accuracy far superior to othermapping methods. Using the above strategy, we show that binding ofmiR-122 to one of its target sites within the 5’ UTR of HCV induces aconformational shift in the HCV IRES at the distant AUG translation startsite. Surprisingly, binding of miR-122 to its second target site in HCVis mediated by a number of non-canonical base-pairings. Mutation ofthe 3’ half of miR-122 (tail) disrupted these non-canonical interactionsand its ability to induce a conformational shift at the AUG start site.We also observed that, in vitro, the first miR-122 target site in HCVis part of a putative triple-strand RNA motif. These results providethe first demonstration that the tail of this liver-encoded miRNA candirectly alter the RNA conformation of the HCV IRES, and therebyprovide new insights into the mechanism by which miR-122 influencesviral translation. We also show that the 5’ UTR of HCV contains a triplexstructure important for viral translation.Poster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 79

Poster Abstracts170 Development of ERCC RNA Spike-InControl MixesL. Qu, A. Lemire, K. Lea, D. Batten, S. Jian Gu,P. Whitley, K. BramlettLife technology, Carlsbad, CA, United StatesThe National Institute of Standards and Technology (NIST) hostedExternal RNA Control Consortium (ERCC) has been working since2003 to generate a common set of RNA controls that can be used ingene expression measurements. These controls have been designedto mimic natural eukaryotic mRNA sequences with the ability to beused across multiple measurement platforms. These controls open-upthe possibilities for technical evaluation of multiple gene expressionsystems including real-time quantitative PCR, one-color and two-colormicroarray systems, next generation sequencing platforms, as well asserve as an in-process quality control check for library amplificationprocesses. The long standing history of Ambion® as the RNACompany creates a great opportunity to formulate the ERCCs controlsinto useful mixes that can be spiked into RNA after isolation. Each mixcontains 92 of the ERCC controls in a mixture that spans over 6 logs ofdynamic range. The 92 transcripts in each mix are further divided into4 sub-pools that can be utilized to evaluate fold-change measurementsof gene expression between the two mixes. TaqMan® Gene Expressionassays targeting the 92 transcripts are utilized to monitor progressthrough the transcriptome library preparation steps required tocreate a next generation sequencing (NGS) library from RNA. Wedemonstrated the use of TaqMan® assays to inform a researcherof library quality prior to continuing with an expensive and timeconsumingnext generation sequencing experiment. These controlsare platform agnostic and provide informative data for multiple librarygeneration methods targeting various gene expression measurementplatforms from RNA. ERCC RNA spike-in control mixes from theNIST traceable ERCC plasmids has great potential in the hands of ourcustomers to open-up new capabilities in understanding variabilityin RNA preparation, RNA library preparation, determining detectionlimits of measurement systems, and informing downstream analysis. Assuch, we report here the first in a potential line of RNA control productsleveraging the certified plasmid sequences from NIST.171 Effects of Particle Porosity on theSeparation of Larger MoleculesR. Freeman, D. DiFeo, H. Jurgen Wirth, A. GooleySGE Analytical Science, Austin, TX, United StatesThe pore structure of a chromatographic stationary phase accountsfor the vast majority of the surface area responsible for the separation.The pore diameter influences the overall surface area and with it thecapacity of the column but also limits the size of the analyte the columncan or should be used for. In adsorption chromatography the limitingeffect of the pore diameter is further enhanced by adsorbed analytemolecules partially blocking the pore structure. In the analysis of largemolecules pore diffusion becomes a crucial parameter in the efficiencyof the column. A number of models deal with hindered mass transfer inporous systems. Effects of particle porosity on the separation of largermolecules are discussed and examples for the separation of small,medium and large analytes on various pore size stationary phases aregiven. Pore size is shown to be an important parameter when analyzinglarger molecules. By selecting the right pore size for a task the capacityand the mass transfer trade-off can be optimized to achieve the bestpossible separation. For tryptic digests a 200 Å stationary phase issuperior to a 300 Å due to its two-fold increase in surface area while1000 Å are required for the separation of proteins.172 Biological Application of ConstrainedPeptidesE. Murage, M. Castro, H. FazeliBiosynthesis, Inc., Lewisville, TX, United StatesPeptides are attractive therapeutic agents for many diseases. However,poor cell permeability, short half life in vivo due to rapid enzymedegradation has long been a major drawback in their clinical application.In order to overcome this, modification of peptides backbone hasbeen of most interest. In particular, synthetic cyclic peptides are beinginvestigated as potential drug candidates due to their improved cellpermeability, enzyme stability, high receptor affinity and selectivity. Forinstance, hydrocarbon stapled peptides has been shown to bind andinhibit the NOTCH1 transcription factor. NOTCH proteins have beenshown to play a pivotal role in cellular differentiation, proliferationand apoptosis. Mutations in NOTCH1 have been linked with diseaseslike T-cell acute lymphoblastic leukemia.1 Thus constrained peptideassemblies would provide a good opportunity to explore the proteinproteininteractions due to the increased binding affinity comparedto the linear counterparts. As a result of the attractive biologicalproperties offered by these modified peptides there is a growingdemand for constrained peptides in the current drug discoveryresearch. To support the unmet need for custom made constrainedpeptides, Biosynthesis Inc. is now offering stapled peptides and lactambridge constrained peptides. 1. Raymond E. M., Melanie C., Tina N. D.,Cristina Del Bianco, Jon C. A., Stephen C. B., Andrew L. K., D. Gary G.,Gregory L. V., James E. B., Nature 2009, 462, 182-188.173 The University of Texas at Austin— Protein and Metabolite AnalysisFacilityM. Gadush, H. Lo, F. Geigerman, M. Mercado,M. PersonUniversity of Texas at Austin, Austin, TX, UnitedStatesThe Protein and Metabolite Analysis Facility at the University of Texasat Austin is a joint effort of the College of Pharmacy, Center for Researchon Environmental Disease (CRED), and the Institute for Cellular andMolecular Biology (ICMB). Services and collaborative research areoffered for the detection, characterization and quantification ofbiomolecules. The Facility’s goals are to provide sensitive proteinidentification and modification analyses, to provide custom peptidesyntheses, to offer services for the identification and quantification ofmetabolites, nutrients and xenobiotics, to implement novel analyticalmethods, to improve the sensitivity of existing analyses, to provideconsultation on the selection and implementation of analytical methods,to offer training in the usage and applications of the instrumentation,and to provide technical expertise in support of individual researchgoals. The ICMB portion of the Core contains an ABI Procise 492 cLCprotein sequencer, a Protein Technologies Inc. Symphony peptidesynthesizer, two Bio-rad Duoflows and a GE Heathcare AKTA proteinpurification systems, two Beckman System Gold HPLC systems, aBerthold Technologies Mithras luminescence and fluorescence detector,an Invitrogen gel electrophoresis set-up, an Art Robbins Instruments80 • ABRF 2011 — Technologies to Enable Personalized Medicine

Phoenix crystallography robot and a LC-MALDI-TOF/TOF (an ABI 4700with a LC Packings Ultimate Nano-LC system with a Probot spottingrobot). In the College of Pharmacy, the Core has an Applied Biosystems4000 Q-trap LC MS/MS system with ESI, APCI and nanospray sourcescoupled with a Shimadzu LC-20AD HPLC system, ThermoFinnigan LCQion trap mass spectrometer with ESI, APCI and microspray interfacescombined with a Michrom Magic 2002 HPLC system, a ThermoFinniganTrace MS GC-quadropole with EI positive, negative CI and selected ionmonitoring (SIM), an ABI Voyager-DE Pro MALDI-TOF and a Bio-radBioplex 200 fluorescent microbead array system.174 Microscopy and Imaging Facility,Cornell UniversityR. Williams, C. Bayles, J. Dela Cruz, M. Riccio,R. Doran, W. ZipfelCornell University, Ithaca, NY, United StatesThe Microscopy and Imaging Facility of the Cornell University LifeSciences Core Laboratories Center (CLC) provides an array of sharedresearch resources and services relating to optical microscopy,fluorescence, and whole animal imaging. The mission of the facility isto provide cutting edge technologies and high quality services that willsignificantly contribute to life sciences research, training and educationprograms.175 Utilization of Flow Cytometry inPersonalized MedicineP. LopezNew York University, New York, NY, United StatesFlow cytometry is a multiparametric data-gathering tool with anenormous, well-established array of applications for intact cells,organelles, functionalized beads, lysed cell contents or supernatants.Multiparametric analysis using state-of-the-art cytometers has thepotential to yield a distribution of cellular phenotypes with highdimensionality (up to 20 parameters per cell simultaneously) specificto an individual disease state or system-wide perturbation. Purifiedpopulations of atypical and/or cells representative of the disease statecan be collected as the first step towards biomolecular characterization.Flow cytometers may be outfitted with multiwell-plate accessories toprovide single-cell capture of specified cells with correlation to eachcell’s individual phenotype (indexed sorting), or rapid sampling frommicrotiter plates (plug-flow). An exciting new cytometric innovationmay allow determining the full signature of biomarkers from individualcells . The massively multiparametric mass cytometer analyzer makesuse of the high sensitivity and resolution of mass spectroscopy torecord the elemental composition as well as metal-tagged biomarkerdistribution of individual cells at a rate of up to 1000 cells per second.176 Cell Imaging and Analysis Network(CIAN) - Multi-Platform Resourcesand Servicesof six scientific platforms: Cell Imaging (confocal and fluorescencemicroscopy; walk-up), Proteomics (2-D, DiGE and fluorescent proteinanalysis; walk-up), Automation and High throughput screening(Pinning robot and liquid handler; full service), Protein expression andantibody production (in collaboration with local animal facilities; fullservice), Genomics (real-time PCR; walk-up), and Data storage/analysis(cluster, server and workstations). Users get in-depth consultationfor proposed projects, and can obtain training in any of the walk-upaspects of the facility, or take advantage of the full-service platforms.CIAN is designed to facilitate training, enhance interactions, as well asshare and maintain resources and expertise.177 Danforth Center: Proteomics & MassSpectrometry Core FacilityL. Hicks, S. Alvarez, B. Zhang, Z. Liu, H. Wang,J. FazlicDanforth Center, St. Louis, MO, United StatesThe Proteomics & Mass Spectrometry Facility at the Donald DanforthPlant Science Center (http://www.danforthcenter.org/pmsf/) isequipped with state-of-the-art technologies for the detailed study ofa wide range of biomolecules. The facility provides both full- and selfservicecapabilities to both internal and external clients at competitiverates. The facility offers fast, high quality specialized analytical servicesincluding: protein extractions, liquid chromatographic separations; highresolution 1D/2D gel electrophoresis; gel image analysis and proteinexpression analysis; high-throughput protein spot excision; in-solutionand in-gel protein digestion; high-throughput protein identification;accurate protein molecular weight analysis; protein covalent/noncovalentcomplex analysis; biomolecule interactions (surface plasmonresonance); small molecule separation/structure determination; andprotein post-translational modification analysis. Major instrumentationincludes: LTQ Orbitrap Velos (Thermo Scientific), QSTAR XL Q-TOF,two 4000QTRAPs, two 6520 Q-TOFs (Agilent), 5975C GC-MS(Agilent), TriVersa NanoMate (Advion), Chip Cube (Agilent), two 1200HPLCs (Agilent), 3 nanoflow HPLCs (LC Packings/Eksigent), SystemGold HPLC (Beckman Coulter), two Shimadzu HPLCs (Shimadzu),UPLC (Waters), Biacore2000,3100 OFFGEL fractionator (Agilent), 1Dand high resolution2D gel electrophoresis systems (BioRad/AmershamBiosciences), Typhoon 9410 (Amersham Biosciences), GelPix (Genetix),and MultiProbe II (Perkin-Elmer). Protein intact mass, identification andcharacterization are a few of the many applications that the facilityperforms. For proteomics applications,two Q-TOFs andthe LTQOrbitrap Velos instruments are well-suited for analyzing both smallpeptides and large proteins. The LTQ Orbitrap Velos can be set upwith the TriVersa to automate direct infusion of samples to performexact mass measurements for molecular formula determination orfacilitate targeted analysis of modifications. The LTQ Orbitrap Velosand the 6520 Q-TOF are also used for online LC-based quantitativeproteomics (iTRAQ and label-free). The 4000 QTRAP systems serveas powerful instruments for targetedmetabolite analyses (e.g. planthormones, pABA, etc.), and the GCMS and one LC-QTOF are used formetabolomics profiling initiatives.Poster AbstractsE. Küster-Schöck, J. Lacoste, G. Lesage,S. Bunnell, H. HanMcGill University, Montreal, QC, CanadaThe Cell Imaging and Analysis Network (CIAN) provides servicesand tools to researchers in the field of cell biology from within oroutside Montreal’s McGill University community. CIAN is composedABRF 2011 — Technologies to Enable Personalized Medicine • 81

178 Proteomics and Mass SpectrometryApplications in Biomedical ResearchC. Diaz, M. Chow, R. Zheng, C. Silva-Sanchez,J. Koh, S. ChenUniversity of Florida ICBR Proteomics Facility,Gainesville, FL, United StatesBiomolecular interaction analysis (Surface Plasmon Resonance – Biacore).Each of our services is staffed and supported by highly experiencedand dedicated scientists. Beyond merely making available facilities andservices, the PAN facility also enables methods development, and newapplications development, designed to meet the needs of the researchcommunity requiring the services. We will present research exampleswhere the PAN facility played a significant role in the application ofthese technologies to basic science projects.Poster AbstractsProteomics and mass spectrometry have provided unprecedentedtools for fast, accurate, high throughput biomolecular separation andcharacterization, which are indispensable towards understandingthe biological and medical systems. Studying at the protein levelallows researchers to investigate how proteins, their dynamics andmodifications affect cellular processes and how cellular processes andthe environment affect proteins. The mission of our facility is to provideexcellent service and training in proteomics and mass spectrometryto UF scientists and students. Here we present our capabilities inproteomics and other analytical services. The tools include a gel-based2D-DIGE (Two Dimentional Difference Gel Electrophoresis) and gelfreeiTRAQ (Isobaric Tags for Relative and Absolute Quantitation).Along with our capacity of separating thousands of proteins andcharacterizing differential protein expression, we have a suite of stateof-the-artmass spectrometers available for biomedical sciences andadvanced technology research, including a tandem time-of-flight (4700Proteomics Analyzer, AB), quadrupole/time-of-flight (QSTAR XL, AB),and hybrid quadrupole-linear ion-trap (4000 QTRAP, AB). Theseinstruments are mainly used for protein identification, posttranslationalmodification characterization and protein expression analysis (e.g., MassWestern). Our facility is also set up to provide Edman de novo N-terminalprotein sequence analysis and Biacore biomolecule interaction analysis.We are fully set up to synthesize and purify peptides and have a goodtrack record with this service as well. Proteomics and mass spectrometryare useful in large-scale suvey of proteome for hypothesis generation aswell as in detailed analysis of target proteins for hypothesis testing. Ourservices also include accurate molecular weight analysis, MRM-basedprotein screening and targeted metabolite profiling. To ensure successand maximize productivity, the facility offers education, consultation,data processing and reporting, and support of grant application.179 The Protein and Nucleic Acid (PAN)Facility at Stanford UniversityS. Patel, M. Eckart, N. Kosovilka, A. Sanchez,Y. Tran, P. Walker, R. Winant, E. ZuoBeckman Center, Stanford University, CA, UnitedStatesThe Protein and Nucleic Acid (PAN) Facility (http://pan.stanford.edu) atStanford University’s Beckman Center is a multifaceted biotechnologyfee-for-service laboratory providing services to the Stanford scientificcommunity, other non-profit and biopharmaceutical organizations. TheFacility’s mission is to be adaptable and responsive to the changingneeds of biomedical research by providing basic science investigatorscontinued access to key tools and applications in an efficient and costeffective manner. The Facility offers a diverse array of instrumentationand technical capabilities in Molecular Genetics and Protein Analytics.In Molecular Genetics the services include Gene Expression andGenotypring (Affymetrix microarray), quantitative methylation andmutation analysis (PyroMark), qPCR, DNA sequencing and fragmentanalysis, Nucleic Acid QC (Agilent Bioanalyzer) and Oligonucleotidesynthesis. For protein characterization the laboratory offers ProteinSequencing, Peptide Synthesis, Protein Identification (MALDI) and180 University of Nebraska MedicalCenter Mass Spectrometry andProteomics Core FacilityM. Wojtkiewicz, P. CiborowskiUniversity of Nebraska Medical Center, Omaha,NE, United StatesThe UNMC Mass Spectrometry and Proteomics Core Facility offers abroad range of services, such as ESI and MALDI protein identificationusing Mascot and Sequest Algorithms, iTRAQ-based quantitativeproteomics, MRM protein quantitation, phosphoproteomics profilingand molecular weight determination for proteins, peptides and smallmolecules. The facility is equipped with LTQ Orbitrap ETD, LTQ Velos,4800 MALDI TOF-TOF, 4000 Q TRAP, all with supporting nano-LCsystems. Although the majority of users are investigators from UNMC,we also provide services for other outside academics and corporations.For further information, visit our website: www.unmc.edu/mspcf.181 UCLA Molecular InstrumentationCenter- Proteomics and MassSpectrometry FacilitiesM. Sondej, W. Yan, R.J. Alvarado, G.A. Khitrov,G. Czerwieniec, J. StrouseUniversity of California at Los Angeles, Los Angeles,CA, United StatesThe UCLA Molecular Instrumentation Center (MIC) is a state-of-theartcampus-wide facility dedicated to enabling the use of moderninstrumentation in molecular characterizations (www.mic.ucla.edu). TheUCLA Molecular Instrumentation Center is housed within and managedthrough the Department of Chemistry and Biochemistry and is composedof five divisions: J.D. McCullough Laboratory of X-ray Crystallography,Magnetic Resonance Facility, Materials Characterization lab, W. M.Keck Proteomics Center and Mass Spectrometry (MS) Laboratory.The MIC operates as an open access center where qualified users areencouraged to perform their own sample analysis under the trainingand guidance of the MIC personnel and is available to researchers atUCLA, other academic institutions and commercial enterprises. TheUCLA Proteomics Center and Mass Spectrometry Laboratories havefive Ph.D. level staff memberswho are experts in sample preparation,2-D gel and other electrophoresis techniques, bioinformatics, andmass spectrometry. The equipment for the UCLA Proteomics Centerincludes Bio-Rad electrophoresis cells for running 1- and 2-D gels; Bio-Rad Fx Fluorescence Imager and GS-800 Densitometer for imaging;DIGILAB Genomic Solutions ProPicII spotcutter; Thermo LTQ FT MSwith Eksigent NanoLC-2D HPLC; Thermo LTQ Orbitrap XL MS withEksigent NanoLC-2D HPLC and a Bruker SolariX-hybrid Qq-FTMSequipped with a 15 Tesla Magnet System. Our Bioinformatics resourcecenter hosts a number of qualitative and quantitative software for 2-Dgel and mass spectrometry data analysis and hardware such as a linuxcluster and servers for MS database searching and data storage.The MS82 • ABRF 2011 — Technologies to Enable Personalized Medicine

Laboratory is located next door and works closely with the ProteomicsCenter. Their mass spectrometers includes Applied Biosystems-MDSSciex 4000 Q Trap with Autosampler; Applied Biosystems Q-Star EliteQuad-TOF Hybrid LC/MS/MS system; Applied Biosystems Voyager-DE STR MALDI-TOF; Thermo Finnigan LCQ Deca Ion Trap MS withAutosampler and PDA; Agilent 6890-5975 GC-MS with Autosamplerand Waters LCT Premier with ACQUITY UPLC and Autosampler.184 New MAbPac Phases for MonoclonalAntibody (MAb) Variant AnalysisG. Gendeh, S. Rao, Y.X. Hou, X. Liu, Y. Agroskin,C. PohlDionex Corporation, Sunnyvale, CA, United States182 Microscale Thermophoresis:Interactions of Proteins, SmallMolecules, Nucleic Acids, and VesiclesS. Duhr, P. BaaskeNanoTemper Technologies GmbH, Munich,GermanyThis work gives an overview on a new Technology for the measurementof biomolecule interaction that is termed Microscale Thermophoresis(MST). The term Microscale Thermophoresis refers to the directedmovement of molecules in optically generated microscopic temperaturegradients. This thermophoretic movement is determined by the entropyof the hydration shell around the molecules. Almost all interactionsbetween molecules and virtually any biochemical process related toa change in size, stability and conformation of molecules alters thishydration shell and can be quantified. Such changes allow quantificationof binding affinities of proteins, nucleic acids and small molecules aswell as measurement of enzymatic activities with MST. In additionalso functional studies of small molecule inhibitors are possible. Themicroscopic temperature gradient is generated by an IR-Laser, whichis strongly absorbed by water. The readout method of the interactionanalysis is based on fluorescence: intrinsic fluorescence of proteins canbe used as well as proteins expressed with GFP/YFP/RFP and also dyelabeled biomolecules. In this presentation we will describe the technicaldetails and the benefits of the Microscale Thermophoresis technologyplatform. We will show examples for interaction measurements rangingfrom protein -ribosome, protein -protein, small molecule -receptorbinding to studies where the interactions between receptor containingvesicles and proteins are analyzed.183 Reduction of Sample Carryover inProteomics LC-MS ExperimentsG. Gendeh, M. Karsten, E.J. Sneekes, R. SwartDionex Corporation, Sunnyvale, CA, United StatesRelevant biomarkers are often present at concentrations near or belowthe detection limit of current analytical methods. Despite this challenge,several biomarker candidates have been identified and moved into thevalidation phase. The increase in sensitivity of analytical methods andmass spectrometry, in particular, over the past years is the reason forthis accomplishment. However, a sensitivity increase alone is insufficientto accurately identify potential biomarkers; carryover reduction is alsoimportant to ensure a marker is actually present in the sample beinganalyzed. Therefore, reduction of carryover has received increasedattention from the proteomics community.MAbs generally exhibit complex heterogeneity including glycosylation,oxidation, phosphorylation, amino-terminal modifications, incompleteprocessing of the C-terminus, and asparagine deamidation. Thesevariations in composition could impact their efficacy, stability, and safety.Monitoring and reporting such variations in therapeutic proteins isrequired by the FDA and other regulatory agencies. Two new MAbPac TMphases were developed to meet these needs. The MAbPac SCX-10 is anewly designed strong cation-exchange column for the characterizationof heterogeneity of MAbs. This is a complementary addition to theexisting ProPac® WCX-10 column that provides high resolution andorthogonal selectivity for MAb charge variant analysis. The MAbPacSCX stationary phase is based on nonporous, highly cross-linked styrenictype polymeric media with a proprietary hydrophilic coating. Sulfonicacid functionality is added through controlled radical polymerizationgrafting. These particles exhibit a wide range of pH stability with highselectivity and minimal band spreading. The MAbPac SEC-1 is a newsize-exclusion chromatography (SEC) column specifically developed forcharacterization of monoclonal antibody (MAb) aggregates, enzymedigested fragments, and other size-based separation applications. TheMAbPac SEC column is based on high-purity, spherical, porous (300Å), 5 μm silica covalently modified with a proprietary diol hydrophiliclayer. This stationary phase can handle both high- and low- salt eluentsas well as mass spectrometry compatible eluents. The MAbPac columnis packed into a nonmetallic, biocompatible PEEK TM column housingto eliminate metal contamination from the column hardware that cancompromise MAb separations. The stationary phase is designed tominimize undesired nonspecific interactions between proteins and thestationary phase. Various applications with relevant comparisons alongwith a demonstration of the ruggedness of these new phases are shownin this poster.185 The VGN Proteomics Module: ATransferable Laboratory Module forUndergraduatesJ. MurrayVermont Genetics Network, University of Vermont,Burlington VT, United StatesThe Vermont Genetics Network (VGN) Outreach Core’s mission isto bring cutting-edge technology and knowledge to undergraduatesat colleges throughout the state of Vermont. The VGN ProteomicsOutreach project initiated in the fall of 2009 exposes undergraduatesin the state of Vermont to proteomics technology using handsonlaboratory experiences. We provide all teaching materials,laboratory materials and if necessary equipment for colleges withinthe state to run the module. All materials become the property of therecipient institution upon completion of the laboratory module. Theundergraduate students learn about this cutting edge technology andgain new skills that we believe will help them with their future scientificcareers. In this transferable module, students learn how proteinexpression in yeast is changed after exposure to oxidative stress oran environmental toxin. Total protein is then harvested and preparedfor 2D gel analysis. Proteins with differential expression are isolatedfrom the 2D gel and prepared for Mass Spectrometry at the UVMPoster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 83

Poster AbstractsProteomics Core Facility. The data is processed and students examinetheir results and use bioinformatics tools to further understand thebiological implications of the results. The students than present theirfindings, describe specific proteins that showed differential expression.A hypothesis is presented explaining the biological relevance of theprotein expression change along with a plan for testing this hypothesis.This work was sponsored by Grant Number P20 RR16462, from theIDeA Networks of Biomedical Research Excellence (INBRE) Program ofthe National Center for Research Resources (NCRR), a component ofthe National Institutes of Health (NIH).186 Quantitative Western Blotting withAmersham ECL PrimeM. Winkvist, S. Grimsby, K. Söderquist,A. MarcussonGE Healthcare Bio-Sciences AB, Uppsala, SwedenWestern blotting is a well established technique used to study proteinsfrom a wide variety of sources. The technique is used throughout thelife sciences from basic research to medical diagnostic applications.Western Blot is at best considered as semi-quantitative and hencelimited to studies involving large protein differences.Here wedemonstrate the use of a new ECL TM reagent, Amersham TM ECL Primein a number of typical Western Blotting applications. The resultsdemonstrate that, Amersham ECL Prime can be used for detection oflow abundant proteins, that signals are very stable over time and covera broad dynamic range. These features make Amersham ECL Primehighly suitable for accurate quantitative analysis.187 New Approaches to QuantitativeWestern BlottingM. Winkvist, Å. Hagner-McWhirter,K. Söderquist, S. GrimsbyGE Healthcare Bio-Sciences AB, Uppsala, SwedenFluorescent detection in Western blotting offers high sensitivity, broaddynamic range and stability of signals. This makes it highly suitable forquantitative Western blotting. Here we show how fluorescent Westernblotting can be used for simultaneously detection of up to threedifferent proteins on the same blot at the same time and for detectionof proteins of the same molecular weight without stripping and reprobing.We also demonstrate how fluorescent Western blotting with3 layer probing can be used to increase sensitivity and thereby enablesdetection of very low abundant proteins. Finally we demonstrate thatit is possible to compare the total protein amount to the target proteinby using Deep Purple TM protein staining prior to ECLTM Plex TM Westernblotting.188 Rubicon PicoPlex-NGS Kits Availablefor Sequencing Single Cells Using theIllumina Genome AnalyzerJ. Langmore, T. Kurihara, E. Kamberov,J. M’Mwirichia, T. Tesmer, D. OldfieldRubicon Genomics, Ann Arbor, MI, United StatesRubicon Genomics has released its PicoPlex-NGS kits to prepare singlecells for NGS analysis on the Illumina Genome Analyzer. These kitsenable single eukaryotic or prokaryotic cells to be lysed, DNA extractedand amplified, and adapted for paired-end sequencing in a 1-tube,3-hr, 4-step process. Although the read coverage is poor in a single lane,the reproducibility of the reads allows single cells to be compared forSNP and CNV genotype, mutations. The same amplified samples canbe used for PCR and microarray analysis, including genome-wide SNPgenotyping, mutation, and copy number analysis. PRC amplification ortarget enrichment can be used for high accuracy and coverage singlecellgenomic analysis.189 PRG-2011: Defining the InteractionBetween Users and Suppliers ofProteomics Services/FacilitiesD.H. Hawke 1 , T.M. Andacht 2 , M.K. Bunger 3 ,C.E. Bystrom 4 , L.J. Dangott 5 , H. Molina 6 ,R.L. Moritz 7 , R.E. Settlage 8 , C.W. Turck 91University of Texas MD Anderson Cancer Center,Houston, TX, United States; 2 Centers for DiseaseControl and Prevention, Atlanta, GA, UnitedStates; 3 RTI International, Research TrianglePark, NC, United States; 4 Quest Diagnostics, SanJuan Capistrano, CA, United States; 5 Texas A&MUniversity, College Station, TX, United States;6Center for Genome Regulation, Barcelona,Spain; 7 Institute for Systems Biology, Seattle, WA,United States; 8 Virginia Bioinformatics Institute,Blacksburg, VA, United States; 9 Max Planck Instituteof Psychiatry, Munich, GermanyOver the last ten years the Proteomics Research Group (PRG) hasundertaken technical studies that have covered a wide range of issuesunique to the rapidly developing field of proteomics. These studieshaveincluded a range of qualitative and quantitative experiments. The PRGstudies have resulted in a great deal of attention not only within theABRF community but also outside as is evident from numerous articlesdealing with proteomics methods, procedures and standardization.As the field continues to develop, the diversity of instrumentationand laboratory workflows have grown in tandem. Therefore, for thePRG2011 study it seemed especially useful to perform a survey tohelp define future studies based on the current blend of sample typesand technologies and obtain a view of emerging trends. A survey wascreated to ascertain three main insights into core facility function: 1)How labs interact with their clients, 2) The capacity of labs to meet thedemands of their clients, and 3) The blend of experimental techniquesoffered to and requested by clients. Survey questions were designed toobtain information from both users of core facilities and the directorsand personnel of core facilities. Questions covered such topics as thetype and age of instruments in use, how data is analyzed and presentedto client, sources of funding, and emerging proteomics trends. Resultsare compiled en masse and presented without regard to institution.190 GlycoMaster — Software forGlycopeptide Identification withCombined ETD and CID/HCD SpectraP. Shan, L. XinBioinformatics Solutions Inc., Waterloo, ON,CanadaObjective: To automate the data analysis for the identification ofglycopeptides with combined ETD and CID/HCD fragmentation. Theinputs for GlycoMaster software include the raw mass spectrometry84 • ABRF 2011 — Technologies to Enable Personalized Medicine

data of a Thermo Orbitrap instrument and the list of proteins inthe sample. The list of proteins can be identified from the samemass spectrometry data by using a database search method. Thesoftware uses the signature ions of simple sugars in the HCD spectrato identify the HCD-ETD spectrum pairs of glycopeptides. Thenthe ETD spectrum in each pair is used to identify the glycopeptidesequence, the glycosylation site and glycan mass. The output ofthe software is an excel file that contains information about theidentified glycopeptides, including glycan composition, mass error,glycan components, glycosylation site and glycopeptide sequence.A score is associated with each identification result that can be usedto sort the identifications according to confidence. GlycoMaster wastested with an ETD-CID dataset containing 3561 MS and 2738 MS/MS spectra from tryptic digest of reduced and alkylated 12 standardprotein mixture containing 3 glycoproteins ( human serotransferrin,chicken ovalabumin and ovomucoid) from Sigma. Glycopeptides wereenriched on cellulose column or by using ZIC-HILIC spin columns fromEMD. Purified glycopeptides were analyzed by LTQ Orbitrap VelosETD. PEAKSTM Studio Suite was used for spectral preprocessing andprotein identification. GlycoMaster reported 161 identified HCD-ETDspectrum pairs of glycopeptides. 95 of 161 were manually validated. 68glycopeptides were reported by GlycoMaster, and 40 glycopeptideswere validated.191 Development of an AutomatedMethod for Antibody Purificationand AnalysisG. Gendeh, W. Decrop, R. SwartDionex Corporation, Sunnyvale, CA, United StatesThe screening and analysis of monoclonal antibodies can be anextremely time consuming task, especially as the many of the workflowsused today require many manual steps. This situation is fully remediedby the technology offered by the MAb Analysis platform from Dionex- a single platform can screen a large number of MAbs and, in a secondstep, provide detailed analytical information, including charge variantanalysis and aggregate assessment. This unique technology allowsdrug discovery laboratories to develop MAb therapeutics much morequickly than before, and bring these important new therapeutics tomarket in a shorter time frame.192 Fast QC of Intact MonoclonalAntibodies Using MALDI-Top-DownSequencingA. Resemann, R. Paape, L. Vorwerg, D. SuckauBruker Daltonics, Bremen, GermanyAntibodies are playing an important role in drug development in thelast years. The function of therapeutic monoclonal antibodies (mAbs) isdirectly depending on their structure including terminal modificationslike C-terminal lysine excision and N-terminal pyroglutamylation ofthe heavy chain. Here, we describe a method for very fast and simplevalidation of the terminal sequences of heavy and light chain of intactmonoclonal antibodies using MALDI-Top-Down-Sequencing (MALDI-TDS) using in-source decay (ISD). Intact mAb (IgG) in a concentrationof 1 mg to 5 mg/ml was directly mixed with matrix solution without priorreduction and alkylation or separation of the different chains. We used1,5-diaminonaphtalene (DAN) as matrix because of its reductive andISD enhancing properties. The samples were mixed with matrix solutionand dried at ambient air on the MALDI sample plate. In a secondexperiment, the same samples were prepared using sDHB (super DHB),which is known to be a good ISD matrix but without reduction capacity.The samples in sDHB didn’t generate any ISD fragmentation due to theintact disulfide crosslinks in IgG. In DAN, the same samples generatedrich ISD spectra containing fragments from both the heavy and the lightchain of the antibodies. The ISD spectra permitted reading throughthe cysteine residues in the sequences implicating the disulfide bridgeswere reduced by DAN. Up to 60 residues of the different chains ofvarious mAbs were matched and validated, respectively. A dedicatedsoftware tool allowed a very fast interpretation of the spectra. Overallthe method takes only a few minutes from sample preparation,spectrum acquisition and processing to the result in form of sequenceannotations in the ISD spectra.**193 Developing a Redox-Sensitive RedFluorescent Protein BiosensorI. Magpiong 1 , N. Koon 2 , S.M. Yei 2 , A.J. Risenmay 2 ,K. Kallio 2 , S.J. Remington 21Mount Holyoke College, South Hadley, MA,United States; 2 University of Oregon, Portland, OR,United StatesRedox environments are of particular interest, especially in themitochondria with its highly reducing environment and its role as thecentral processing unit of apoptosis. Monitoring of mitochondrial redoxenvironments is crucial to the study of apoptotic disorders. Reportingof the thiol/disulfide status in live cells was made possible with thedevelopment of redox-sensitive green fluorescent protein (roGFP). Weaim to develop a red version redox-sensitive fluorescent protein (roRFP).Expanding the array of redox-sensitive proteins with a red version willenable simultaneous visualization of multiple reducing intracellularcompartments. mKeima is a monomeric red fluorescent protein thatabsorbs light maximally at 440nm and emits red light at 620nm. Thislarge Stokes shift is dramatically decreased in acidic environments. Byfollowing protocol similar to that used in the development of roGFP,surface residues at key positions were changed to cysteines andrandom mutagenesis was performed on varying excitation species ofmKeima. Mutants were screened and a ratiometric variant of mKeimawas identified (roRFP2) which exhibits changes in its spectral propertiesas a result of changes in the thiol/disulfide equilibrium. Preliminaryfluorescence spectroscopy measurements of roRFP2 indicate a highlyreducing redox potential of -330mV indicating it may be a usefulprobe in reducing subcellular compartments such as mitochondria orin the cytoplasm. By employing vector recombination of shuttle vectorPYX142, we successfully targeted roRFP2 in vivo to the mitochondriaand cytoplasm of Saccharomyces cerevisiae. Expression of roRFP2 wasvisualized using fluorescence microscopy. Thus, through mutagenesisand residue substitution we successfully created a red version redoxsensitivebiosensor that tested effectively as a ratiometric indicatorand expressed in the mitochondria and cytoplasm of S. cerevisiae.Moreover, the redox potential of roRFP2 is significantly more negativethan the widely used roGFPs.Poster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 85

Poster Abstracts194 PeaksDB: New Software forSubstantially Improved PeptideIdentification from Orbitrap ETDMass SpectrometryB. Ma 1 , J. Zhang 2 , L. Xin 2 , B. Shan 2 , W. Chen 21University of Waterloo, Waterloo, ON, Canada;2Bioinformatics Solutions, Inc., Waterloo, ON,CanadaObjective: To substantially improve the peptide identification sensitivityand accuracy from the Orbitrap ETD data with computational methods.Method: The algorithm takes full advantage of the characteristicsof the Orbitrap ETD data, including: (1) high mass resolution of theprecursor ions, and (2) the distributions of different fragment iontypes in the MS/MS scans. For the first characteristic, a pre-search stepis conducted to determine the precursor mass error distribution. Thisdoes not only make the precursor mass more accurate by a softwarerecalibration, but also allows the use of the mass error as an importantfeature in the peptide-spectrum matching score function. For thesecond characteristic, the frequencies of different fragment ion typesat different precursor charge states are statistically learned, and usedin the score calculation. Moreover, the precursor-related ions in theMS/MS spectra are removed. Additionally, the score function makesuse of the similarity between a database peptide and the de novosequencing result. Result: PeaksDB was compared against three othersearch engines: MSGF-DB, Mascot, and ZCore. The same shuffled decoydatabase was appended to the target database and searched togetherto estimate the false discovery rate (FDR) of each individual engine. Thesame search parameters were used for all engines except that MSGF-DB does not support variable PTMs. If no variable PTM is allowed, thenumbers of identified peptides of different engines at 1% FDR are:PeaksDB (2356) > MSGF-DB (2147) > Mascot (1459) > ZCore (1030).If a few common PTMs are allowed, the numbers change to PeaksDB(3501) > Mascot (2677) > MSGF-DB (2147) > ZCore(1125). Conclusion:PeaksDB substantially improved the sensitivity and accuracy of peptideidentifications on Orbitrap ETD data. At 1% false discovery rate,PeaksDB identified 1.3 to 1.6 times as many peptides as Mascot 2.3.195 IDSieve: Protein IdentificationUsing Peptide pI Filtering of MS/MSData for Improved Confidence inIdentificationsN.R. Garge 1 , K.D. West 1 , X. Zhang 1 , J.L. Bundy 2 ,J.L. Stephenson, Jr. 2 , B.J. Cargile 3 , M.K. Bunger 11RTI International, Research Triangle Park, NC,United States; 2 Centers for Disease Control andPrevention, Atlanta, GA, United States; 3 SapientProteomics, Morrisville, NC, United StatesThe main challenge of tandem mass spectrometry based proteomicanalysis is to correctly match the tandem mass spectra produced to thecorrect peptides. However, the large number of protein sequences ina database increases the chances of a false positive identification forany given peptide match. Here we present an automated algorithmcalled IDSieve that utilizes target-decoy database search strategy incombination with pI filtering to allow greater confidence for peptideidentifications. IDSieve considers the SEQUEST parameters Xcorrand ΔCn to assign statistical confidence (false discovery rates) tothe peptide matches. The distribution of predicted pI values forpeptide spectrum matches (PSMs) is considered separately for eachimmobilized pH gradient isoelectric focusing fraction, and matcheswith pI values within 1.5 times inter-quartile range (within pI range)are analyzed independently of matches outside the pI ranges. Wetested the performance of IDSieve and Peptide/Protein Prophet on theSEQUEST outputs from 60 immobilized pH gradient isoelectric focusingfractions derived from mouse intestinal epithelial cell protein extracts.Our results demonstrated that IDSieve produced 1355 more peptidespectrum matches (or 330 more peptides) than Peptide Prophet usingcomparable false positive rate cutoffs. Therefore, combining pI filteringwith the appropriate statistical significance measurements allows for ahigher number of protein identifications without adversely affectingthe false positive rate. We further tested the performance of pI filteringusing ID Sieve when samples were prefractionated using either pHrange 3.5-4.5 or 3-10, and either 24cm or 7cm IPG strips.196 Proteomics-Based Molecular Modelfor Prediction of HeterogenicTreatment Response and Discoveryof New Cell Survival Mechanisms ofBasal-Like Breast CancerT. Hemström, Y. Lyutvinskiy, R. ZubarevKarolinska Institutet, Stockholm, SwedenWe aim to build a proteomics-based model for prediction of drugresponse and discovery of new signaling-related mechanismsexplaining the heterogeneity in treatment response observed in thegroup of basal-like breast cancer (BLBC), which is a group of breastcancer associated with a relatively short survival of patients. A panelof cell lines of BLBC origin will be subjected to conventional as well asmolecular pathway specific anti-cancer drugs followed by MTT-assayviability assessment. The range of drug concentrations will cover themaximal plasma drug concentration of patients. Focus will be on celllines exhibiting a high or low sensitivity to treatment. Drug responses ofsuch cell lines will be further examined in terms of cell cycle distributionand cell death levels and based on these assessments relevant drugconcentrations and exposure times will be chosen. Analysis of proteinexpression in cells exposed to relevant treatment conditions will beperformed by LC-MS/MS. By way of key node analysis of proteinexpression data cellular signaling involved in sensitivity or resistance todrugs will be hypothesized and validated by means of anti body-basedtechniques, experimental modulation of signaling and analysis of cellcycle and cell death. Thereby we will add two more dimensions, whichare the drug response-related changes in activity of signaling pathwaysand functional morphological changes, such as apoptotic cell death, togene expression-based models of treatment response. Drug responsedata in terms of viability, protein expression, and cellular signaling willbe integrated in a computerized model. By focusing on a well definedsubgroup of breast cancer exhibiting clinical heterogeneity in anticancerdrug response we have a reasonable good chance to developa fine-tuned model for prediction of treatment response and identifycurrently unknown drug resistance mechanisms, which may involve newpossible predictive markers and drug targets.86 • ABRF 2011 — Technologies to Enable Personalized Medicine

**197 Multi-Functional SuperparamagneticIron Oxide Particles as CancerTherapeutic AgentsA. Narayanan 1 , P.M. Gannett 1 , J.A. Barr 1 ,R.L. Carroll 2 , S.L. Yedlapalli 21Robert C. Byrd Health Sciences Center,Department of Basic Pharmaceutical Sciences,West Virginia University, Morgantown, WV,United States; 2 C. Eugene Bennett, Department ofChemistry, West Virginia University, Morgantown,WV, United StatesSuperparamagnetic iron-oxide nanoparticles (SPIONs) are magneticnanoparticles may be useful for early detection and treatment ofcancers. SPIONs provide therapeutic drug-loading capabilities andtargeting specificity through the use of antibodies or receptor specifictags. These versatile particles are prime candidates for improvingcurrent means to the treatment of cancer and potentially otherdiseases. This multi-disciplinary project includes various milestonesand multiple aims: i) identify and optimize SPION design parametersthat optimize aqueous stability and maximize amphiphilic character,ii) evaluate SPION drug storage and release characteristics, andiii) demonstrate binding and entry into targeted cells. The specificaim of this project is the coupling of cell-specific targeting agents toSPIONs. An antisense oligonucleotide against Survivin mRNA wassynthesized by solid-phase DNA synthesis with an amino-terminatedlinker on the 3’ end (5’CCCAGCCTTCCAGCTCCTTG-(CH2)6-3’NH2).SPIONs were prepared and then coated with a copolymer containingsurface carboxylic acid groups (-COOH). The carboxylic acid groupswere activated by treatment with N-((3-dimethylamino)-propyl)-Nethylcarbodiimide and coupled to oligonucleotides via the –NH2terminus to the –COOH groups resulting in the formation of an amidelinkage between the –COOH groups of the SPION and the –NH2 ofthe antisense oligonucleotide. Circular dichroism (CD) studies wereperformed to quantify/optimize coupling and to demonstrate antisenseSurvivin duplex formation was not inhibited by the presence of theSPION. CD results were correlated with agarose gel electrophoresisdata and demonstrated oligonucleotides coupling to the SPION andthat the SPION did not significantly alter duplex formation. Futurestudies will target cellular absorption and antisense binding to SurvivinmRNA using confocal microscopy (Supported, in part, from NIHGM081348 grant and the WVU Research Corporation).198 Ultrahigh-Performance Nano LC-MS/MS Analysis of Complex ProteomicSamplesG. Gendeh, E.J. Sneekes, B. de Haan, R. SwartDionex Corporation, Sunnyvale, CA, United StatesDetermination of the proteome and identification of biomarkers arerequired to monitor dynamic changes in living organisms and predictthe onset of an illness. One popular method to tackle contemporaryproteomic samples is called shotgun proteomics, in which proteins aredigested, the resulting peptides are separated by high-performanceliquid chromatography (HPLC), and identification is performed withtandem mass spectrometry. Digestion of proteins typically leadsto a very large number of peptides. For example, digestion of a celllysate easily generates 500,000 peptides. The separation of thesehighly complex peptide samples is one of the major challenges inanalytical chemistry. The main strategy to improve the efficiency ofpacked columns is either to increase column length or to decreasethe size of the stationary phase particles. However, to operate thesecolumns effectively, the LC conditions need to be adjusted accordingly.Naturally, the on-line coupling to MS systems has to be taken intoaccount in the optimization process. Here, the authors report on theperformance of nano LC columns operating at ultrahigh pressure.The effects of column parameters (particle size and column length)and LC conditions (gradient time, flow rate, column temperature)were investigated with reversed-phase (RP) gradient nano LC. HighresolutionLC-MS separations of complex proteomic peptide samplesare demonstrated by combining long columns with 2 μm particlesand long gradients. The effects of LC parameters on performance andthe influence on peptide identification are discussed.**199 Comprehensive Biomarker DiscoveryPlatform Reveals Qualified MRMAssay for Prediction of INF/RibavirinTreatment Response in Hepatitis CPatientsJ.W. Thompson 1 , L.G. Dubois 1 , K. Patel 2 ,J.E. Lucas 1 , J. McCarthy 1 , J.G. McHutchison 2 ,M.A. Moseley 11Duke Institute for Genome Sciences and Policy,Durham, NC, United States; 2 Duke Clinical ResearchInstitute, Durham, NC, United StatesThe current standard of care for chronic Heptatitis C (CHC) results insustained viral response in only half of patients and also has significantside effects. Improved pre-treatment clinical predictors of responseare needed to individualize treatment and select individuals mostappropriate for new therapy. We have identified 10 peptides to 6proteins which can differentiate non-responders from responders withhigh accuracy based on pre-treatment samples. Serum samples wererandomized, immunodepleted (MARS 14), and digested with trypsinprior to LC-MSE or LC-MRM analysis. LC-MSE analyses were performedon a Waters nanoAcquity LC and QToF Premier, while MRM analyseswere performed on nanoAcquity and Xevo TQ. Rosetta Elucidator® wasused to quantify all LC-MSE data. PLGS v2.4 was used to make peptideidentifications. MRM method generation and sample quantitationwas performed with Skyline v0.6. The response signature was initiallyidentified from the analysis of 96 samples (Duke Hepatology ClinicalResearch database) by LC-MSE on a QToF mass spectrometer, usingsparse latent factor regression analysis. This yielded a group of almost400 candidate peptides which were part of 3 metaprotein predictors;these peptides were then curated based on set selection criteria togive 86 target peptides for MRM analysis. MRM analysis of the original96 samples yielded a final group of 10 peptides which maintainedBonferroni-corrected statistical significance for predicting treatmentresponse. The assay has been qualified by a blinded analysis of an allcomersclinical trial sample set (PEDS-C, NIH, n=51) using the MRMmethod, which yielded an AUROC of 0.91 with a sensitivity of 0.828and specificity of 0.786. The analysis of an additional CHC clinical trialcohort (Chariot, n = 243) is ongoing. This presentation will utilize CHCtreatment response as a case study on the critical analytical, statistical,and clinical cohort requirements to successfully perform biomarkerdiscovery and verification.Poster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 87

Poster Abstracts200 Discovery of Yersinia pestis andYersiniophage Peptide Targets forUse in Multiple Reaction MonitoringMethodsL. Luna 1 , S. Bearden 21Division of Vector-Borne Infectious Diseases,Centers for Disease Control and Prevention,Fort Collins, CO, United States; 2 Toxicology andEnvironmental Research and Consulting, The DowChemical Company, Midland, MI, United StatesAims: We present the discovery of functional yersiniophage andYersinia pestis (Y. pestis) peptide targets for monitoring bacteriophagebasedamplification processes and species-specific identification ofYersinia pestis, the etiological agent of plague. Methods: For proteinconfirmation and peptide target discovery, ultra performance liquidchromatography and hybrid tandem mass spectrometry was utilizedto identify proteolytic cleaved peptides generated from digestion ofwhole phage and bacterial lysates. Peptide discovery was accomplishedwith a UPLC NanoAcquity interfaced into a Q-TOF Premier (WatersCorporation) for full scan and MS/MS experiments. Targeteddevelopment utilized the MRM initiated detection and sequencing(MIDAS) workflow design software (Applied Biosystems) to create insilico MRM transitions which optimized collision energies for trypsincleaved peptides from the proteins of interest. The analytical columnutilized was an UPLC bridged ethyl hybrid (BEH) 1.0 x 50 mm reversephase C18 (1.7-µm particle size) interfaced into a 4000 Q TRAP forMRM triggered MSMS. Results: We screened yersiniophage phiA1122,R, V, and Y proteins for conserved peptide targets for utilization inroutine mass spectrometry monitoring of the phage amplificationprocess. We also identified murine toxin and F1 antigen peptide targetsthat are unique to Yersinia pestis, which upon lysis, can be utilized forconfirmatory identification of Y. pestis. Conclusions: Major and minorcapsid protein peptides will serve to enhance quantifiable metricmeasurements of the amplification process for rapid yersiniophagediagnostic capabilities and concomitant determination of the presenceof viable Y. pestis bacilli. Moreover, the targeted murine toxin peptidedirectly confirmed the presence of Y. pestis at 26°C and can be appliedas an additional tool for zoonosis surveillance capabilities in a clinicallaboratory setting. Significance and Impact of Study: Applicationsutilizing these peptide targets identified in this work may be beneficialin emergency response situations requiring pre**201 Protein-Binding Assays inBiological Liquids Using MicroscaleThermophoresisS. Duhr 1 , P. Baaske 1 , C. Wienken 2 ,M.J. Willemsen 2 , D. Braun 21NanoTemper Technologies GmbH, and Centerfor Nanoscience, University Munich, Munich,Germany; 2 University Munich, Physics Department,Munich, GermanyProtein interactions inside the human body are expected to differ fromthe situation in vitro. This is crucial when investigating protein functionsor developing new drugs. In this study, we present a sample-efficient,free-solution method, termed Microscale Thermophoresis (MST), thatis capable of analysing interactions of proteins or small molecules inbiological liquids such as blood serum or cell lysate. The technique isbased on the thermophoresis of molecules, which provides informationabout molecule size, charge and hydration shell. We validated themethod using immunologically relevant systems including humaninterferon gamma and the interaction of calmodulin with calcium. Theaffinity of the small-molecule inhibitor quercetin to its kinase PKA wasdetermined in buffer and human serum, revealing a 400-fold reducedaffinity in serum. This information about the influence of the biologicalmatrix may allow to make more reliable conclusions on proteinfunctionality, will facilitate more efficient drug development, and mayallow for sensitive diagnostics in complex biological samples.202 2-D DIGE Analysis of MulticellularTumor Spheroids in Evaluation ofBreast Cancer TreatmentV. Ruddat 1 , M. Winkvist 2 , S. Grimsby 2 ,A. Monazzam 3 , K. Nyamekye 3 ,Å. Hagner-McWhirter 21GE Healthcare, Piscatway, NJ, United States; 2 GEHealthcare Bio-Sciences AB, Uppsala, Sweden;3Uppsala Applied Science Lab, GE Healthcare,Uppsala, SwedenMany cancers can be diagnosed using positron emission tomography(PET) and PET can also be used to monitor how effective varioustreatments are in individual patients. Tumor spheroids are cancer cellsgrown on agar coated dishes forming a 3D structure. They are widelyused in preclinical cancer research, where the multicellular tumorspheroid model is considered biologically and physiologically similarto in vivo grown tumors. In this study we have used Two-dimensionalDifference Gel Electrophoresis (2-D DIGE) analysis to gain more insightin differences in protein expression as a result of drug treatmentof multicellular tumor spheroids. 2-D DIGE can be used to identifypossible new biomarkers for development of new PET tracers and drugtargets. Combining 2-D DIGE and PET results can be used for improvingdiagnosis and treatment.203 N-Glycosylation of AntibodiesCharacterized by Mass Spectrometry:An Integrated Software ApproachU. Schweiger-Hufnagel, A. Asperger,A. Resemann, D. SuckauBruker Daltonics, Bremen, GermanyAntibodies represent one of the most important classes ofglycoproteins playing a central role in the immune response of livingorganisms. Furthermore, there is a growing interest in recombinantantibodies as potential biotherapeutic agents. The analysis of theN-glycosylation pattern present on antibodies is challenging due to itsheterogeneous structure. The glycan profile is highly dependent onthe process by which a recombinant glycoprotein is generated, suchas host organism and growth conditions. Changes to the glycosylationpattern can significantly alter biological function. To characterize theN-glycosylation pattern of a recombinant antibody, a bottom-upapproach was pursued. Tryptic digests of antibody samples wereseparated by nano-LC and analyzed by MALDI mass spectrometry. Anintegrated software approach allowed a detailed characterization ofthe glycosylation pattern and visualization of the relevant mass spectra.LC-MALDI-TOF/TOF analysis of the digested antibody provided,in addition to the nearly complete sequence coverage of the nonglycosylatedpeptides, a detail-rich picture of the highly complex88 • ABRF 2011 — Technologies to Enable Personalized Medicine

pattern of N-linked glycans in form of the respective N-glycopeptides.Targeted analysis of potential glycopeptides significantly increased theinformation content. Integrated glycoprotein analysis software tools(ProteinScape 2.2) allowed identification of glycan modifications andinteractive result validation. In this process software facilitated theinitial characterization of the antibody as well as the subsequent qualitycontrol tasks.204 Magnetic ZIC-HILIC BeadsEnrichment for Neutral and AcidicGlycopeptidesA. Resemann 1 , J. Wohlgemuth 2 , S. Andrecht 2 ,A. Schneider 1 , U. Schweiger-Hufnagel 1 , D. Suckau 11Bruker Daltonics, Bremen, Germany; 2 Merck KGaA,Darmastadt, GermanyGlycosylation is the most abundant protein posttranslational modificationand is involved in many relevant biological processes and crucial to theunderstanding of many diseases. In depth analysis of glycosylationsites is difficult, however, as glycopeptides exhibit a significant microheterogeneity at glycosylation sites. In addition, ion suppressioneffects require selective methods for glycopeptide enrichment. Massspectrometric analysis of glycopeptides is challenging because boththe peptide as well as the glycan moiety have to be elucidated for a fullstructural understanding. We used Fetuin, Asialo-Fetuin and Alpha-1-Acidglycoprotein to equally representing sialylated and non-sialylatedglycosylic structures. In addition, monoclonal antibodies were analyzedas a dedicated example for pharmaceutical QC. Proteins were digestedwith trypsin and glycopeptides were enriched using a dedicated ZIC-HILIC glycocapture beads in combination with an optimized buffersystem (EMD Chemicals Inc.). The glycopeptides were analyzed usingESI ion trap MS for glycoprofiling and MALDI-TOF/TOF-MS for indepth characterization of the glycopeptides. For database searches,an integrated software approach was used: protein searches of theglycopeptide MS/MS spectra were performed for obtaining the aminoacid sequence of the glycopeptide, and searches in glycan databasesbased on the same glycopeptide MS/MS spectra were carried outto complete the characterization of N-linked glycopeptides. In thecurrent study, two important features of glycoprotein analysis areshown: (1) The employed integrated software approach allowed theglycan identification in a similar way as peptide identification. Theimportant step of interactive result validation was facilitated by a suiteof dedicated data and result viewers. (2) Compared to MS analysis ofnative glycoprotein digests, the enriched samples allowed to detectmore glycopeptides and permitted the acquisition of higher qualityMS/MS spectra. For MALDI-TOF/TOF-MS analysis, linear positive ionmode detection of precursor ions proved to be highly suitable for theanalysis of even multi-sialylated glycopeptides.205 Fragment Analysis of CarbohydratesFollowing Capillary ElectrophoresisT. Snyder-Leiby, D. Hulce, F. Li, X. Li, C.S. LiuSoftGenetics, LLC, State College, PA, United StatesDepending on the macromolecule size and configuration, migrationrates through capillary electrophoresis vary greatly. Internal sizestandards for capillary electrophoresis of the same macromolecule maynot be readily available. The Macromolecule Tool in GeneMarker®aids with analysis of macromolecule fragments without an internallane size standard. Methods included importing raw data files to thesoftware and physically identifying reference peaks in the samplesknown to have the same size. The program uses this information tocalibrate from one capillary to another. Characteristics of the aligneddata (such as relative size, peak height, peak area, peak ratios) wereexported in an excel sheet. Ninety six raw data files from 4 dye capillaryelectrophoresis were analyzed. Peak height, height ratio, area, arearatio, and relative sizes were determined for all samples. These valuescan be used to determine characteristics such as number and relativesize of degradation products or other macromolecules, such as DNAbinding carbohydrates commonly functioning in gene regulation.206 Analysis of Complex Oligosaccharidesfrom Glycopeptides andGlycoproteins Using MSn Spectra andOligosaccharides Spectral LibraryF. XiangShimadzu Biotech, Pleasanton, CA, United StatesGlycosylation is a common post-translational modification to cellsurface and extra cellular matrix proteins as well as to lipids. Unlikeproteins and nucleic acids that are linear polymers of amino acidsand nucleotides respectively, with linkages at only one position,carbohydrates can adopt complex branched structures with individualmonomeric units linked at one of several sites. A detailed analysisof complex carbohydrate structures has been explored with massspectrometric techniques, and still presents challenge for the analyst.This presentation will describe a systematic approach of carbohydratesand glycoconjugates analysis with MSn techniques. Oligosaccharides,cleaved from glycoproteins (by hydrazinolysis or enzymatically), werecharacterized using a hybrid MALDI Ion Trap / TOF mass spectrometer.High mannose, biantennary and triantennary oligosaccharides wereanalyzed using MS, NS2, MS3 and MS4 modes. Intact oligosaccharideswere analyzed in MS mode using a cooling gas to prevent fragmentation.Individual precursor ions were isolated in the trap, subjected tofragmentation with Argon, to provide MS2 data. Product ions wereselected for further fragmentation, which was achieved by increasingthe energy for collisionally induced dissociation. In MS mode, the singlycharged sodium adduct form of these molecules was detected, whichis typical of the analysis on conventional MALDI mass spectrometers.In MS2 mode, high mannose oligosaccharides readily lost the coreN-acetylglucosamine residues, whilst MS3 and MS4 modes were usedto sequentially fragment the product ion corresponding to the residualbranched mannose oligomer. In MS2 mode analysis of biantennaryand triantennary structures also fragmented losing disaccharide units,such as the galactose - N - acetylglucosamine units that define eachantennary branch, or core fucosylated - N - acetylglucosamine units.MS3 of selected MS2 products ions could be used to differentiatefragments generated from either the reducing or non-reducing ends.Cross-ring cleavages were also observed during fragmentation in MS2,and the relevant product ions could be used to differentiate branchedstructures by further fragmentation in MS3 mode. Many tandem massspectrometric experiments have been revealing that oligosaccharidesmight have characteristic signal intensity profiles, depending on theglycosidic linkage and branching structures. In addition to the MSncapability of the platform, there is a library of observational mass spectraacquired from structurally defined oligosaccharides. The presentationwill show the enhancement of carbohydrate identification by utilizingcomparison procedure of the signal intensity profiles of MSn spectrabetween the analyte and structurally defined oligosaccharides in thelibrary.Poster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 89

Poster Abstracts207 A Unique Workflow for GlycoproteinCharacterization from SamplePreparation to MS/MS SpectralInterpretationJ. Albanese 1 , R. Lee 21AB SCIEX, Foster City, CA, United States; 2 ProZyme,Inc., Hayward, CA, United StatesProtein glycosylation is a complex dynamic post-translationalmodification, which is used by an organism to regulate a number ofimportant functions. Variable composition, linkage, branching andanomericity of the constituent monosaccharides in combination withthe general heterogeneity due to the indirect, non-template controlof their biosynthesis are the basis of the structural complexity ofglycoprotein glycans. This poster presents a complete workflow forglycoprotein characterization comprising sample preparation forglycan release, glycan separation using graphitized carbon separationcoupled to MALDI spotting, automated MS and MS/MS analysisMALDI-TOF analysis and spectral interpretation with SimGlycan®Software. The workflow has been first optimized using commerciallyavailable immunoglobulin G from human serum and a glycan libraryof defined bisecting and none-bisecting glycan structures. The IgGsample contains both different protein sequence isoforms (IgG1-3) anddifferent glycan isoforms. The results demonstrated that, the RGSPS kitwith InstantABTM labeling provides a fast and robust glycan releaseand labeling method including sample clean up in less than 2 hoursprior to mass spectrometric analysis. The combination of the LC MALDIWorkflow with the nanoflow hypercarb column enables better glycanseparation and therefore more specificity for isomeric glycans structurescharacterization. High energy CID MS/MS spectral comparison of thereleased and/or labeled glycans with the control samples provides agreater number of MS/MS fragments than electrospray CID MS/MSspectra or MALDI post source decay (PSD) spectra for more detailedinformation. Next, this robust workflow will be tested and expandedon therapeutically interesting antibodies. Studying the heterogeneityof glycosylation patterns obtained from cell culture influences can helpunderstand efficacy, binding affinity, specificity and pharmacokineticproperties.208 Analysis of Intact Proteins inBiotechnologyR. Freeman, D. DiFeo, H.J. Wirth, A. GooleySGE Analytical Science, Austin, TX, United StatesThe demand for separation techniques for intact proteins is increasingwith the introduction of a new generation of high resolution massspectrometers which are able to measure the mass of small to mediumsize proteins very accurately. Liquid chromatography is a valuable toolfor separating these proteins prior to the MS analysis. Intact proteinchromatography is most commonly used in a top-down approach inproteomics or to determine expression levels during recombinantprotein synthesis. The size of the protein molecule results in verylow diffusion coefficients and therefore slow mass transfer in and outof the pore system. A sufficiently large pore diameter is required tominimize the effects of restricted pore diffusion. We show examples ofthe separation of intact proteins on a column packed with 3µm C8 silicawith 1000Å pore size. The molecular weight of the protein examplesreach from ribosomal proteins (

advantage of using two separate 15 cm columns rather than one 30cm column is that sample matrix components that are not retained onthe first column (e.g. salts) can be directed to waste, instead of enteringthe nanospray source/mass spectrometer. We have investigated theeffect of increased resolution through doubling column length on thenumber of peptides/proteins identified in a digested cytosolic fractionof a human cell lysate using various gradient lengths. An ABSCIEXTripleTOF 5600 MS was used with an Information DependentAcquisition method consisting of a TOF MS survey scan at >30 000resolution, followed by 20 MS/MS in a second at >15 000 resolution.All data was processed using ProteinPilot Software 4.0 with integratedfalse discovery rate (FDR) analysis. 1 Remco van Soest*, David W.Neyer; Jia Eng Siow, and Phil Paul, Eksigent Technologies, Dublin, CA;poster ASMS 2008: “Improving resolution in nanoLC separations forproteomics using ultra high pressures”.211 Using 200 µm ID cHiPLC Columnsfor Increased Sample Throughput inPeptide QuantitationR. van Soest 1 , N. Hebert 1 , D.W. Neyer 1 ,J.B. Young 21Eksigent Technologies, Dublin, CA, USA; 2 ABSCIEX, Concord, ON, CanadaNanoflow liquid chromatography coupled with nano-electrospray(nanoLC-MS) is the method of choice for sensitive peptide and proteinanalysis. We recently reported on a new microfluidic platform (cHiPLCnanoflex)for nanoLC-MS applications. In addition to deliveringeasy-to-use, dead-volume-free connections to microfluidic devices,the platform’s cHiPLC columns provide extreme column-to-columnseparation reproducibility. The column ID of choice for best sensitivityis 75 µm, using a flow rate of 200 nl/min. While providing excellentsensitivity, the low flow rate reduces sample throughput becauseof gradient delay in the nanoLC system itself, the autosampler valveand sample loop and the connecting tubing between injection valve,column and nanospray source. One way to address this is to use a largerID column at proportionally higher flow rate. While this will reducesensitivity when the injection volume is kept equal, delay times can begreatly reduced. In this presentation we will show data demonstratingthe throughput increase when using 200 µm vs. 75 µm ID cHiPLCcolumns while reporting as well on the sensitivity trade-off, and theeffect thereof on peptide quantitation.212 Flow Rate, Concentration andSaturation: Investigating theFundamentals of Sample-TrappedColumn InjectionC. Marshall-Waggett, H. Svobodova, A. Berg,G. ValaskovicNew Objective, Inc., Woburn, MA, United StatesSample trapped column injection is an injection strategy commonlyemployed in nanobore LC/MS based analysis of complex peptidemixtures. The approach of sample trapped column injection providesseveral inherent advantages. By effectively desalting and concentratingsamples on-line, sample traps improve analytical column longevityand throughput. Additionally, the ability to load samples onto a trapcolumn at a much higher flow rate than is feasible for a typical 75umID nanobore analytical column is a particularly attractive feature of thisapproach. Duty cycle can be significantly improved when the sampleloading flow rate is decoupled from the gradient flow rate. Here weinvestigate the role sample trap column injection has on analyteretention and overall chromatographic performance. Using a direct flownano-LC pump with the ability to deliver flow rates ranging from 50 nl/min to 20 ul/min coupled to an autosampler and commercially availablepeptide standards and protein digests, we evaluated the relationshipbetween flow rate, analyte concentration and analyte composition todetermine the effect on chromatographic performance.214 Spatial Proteomics: A New LC-MS/MS Tissue Imaging WorkflowProviding Protein Identities and TheirDistribution in TissueC. Lübbert, M. Schürenberg, M.Becker, R. Paape,D. SuckauBruker Daltonics, Bremen, GermanyMALDI-Imaging of proteins in tissue sections has established itself as apowerful new approach to biomarker discovery and histopathologicalresearch in recent years. However, the lack of direct identificationstrategies continues to be an obstacle preventing its broader use inProteomics studies. Initial studies that utilized in situ digestion followedby MALDI-MS/MS analysis typically provided 5-50 peptide IDs of only1-5 high abundant proteins. Here we introduce a novel proteomicstechnology that combines the spatial information with the routineidentification of proteins from tissue sections. Highly resolved proteindigests are generated by applying trypsin onto two subsequent tissuesections by supersonic nebulization. One of the sections is then analyzedby MALDI imaging mass spectrometry at up to 50 µm spatial resolutionyielding a list of 200-2000 peptide signals per image. Peptides areextracted from the other sections entire surface and submitted toroutine LC-MS/MS analysis, in our case using MALDI-TOF/TOF. Theidentified peptide list is then filtered by the peaklist from the image.All matching peaks in the image can then be assigned to a protein andthe co-localization of 2 or more tryptic peptides confirm their proteinassociation. We analyzed rat brain and testis/epididymis using the newSpatial Proteomics approach typically yielding peptide distributions atthe 50-100 µm level. In brain, more than 100 peptides were identifiedand more than 20 proteins localized without the need for MS/MSanalysis directly from the tissue. The intensity, co-localization of 2 ormore peptides and the degeneracy of molecular weight of peptideto-proteinmapping were used as primary validation tools beyondsignificant mascot scores from peptide identification. As an extensionof the established top-down imaging strategy, this bottom-up SpatialProteomics approach may facilitate the identification and simultaneouslocalization of a much greater number proteins than it was previouslypossible.215 Increasing the Sample LoadingCapacity for Peptide Analysis by LC-MS/MS Using 150 µm ID Packed TipColumnsA. Berg, H. Svobodova, C. Marshall-Waggett,G. ValaskovicNew Objective, Inc., Woburn, MA, United StatesA predominant workflow for qualitative proteomics has been “GeLC-MS,” a combination of 1- (or 2-D) gel electrophoresis with reverse-phasenanoflow liquid chromatography mass spectrometry (nLC-MS/MS). Thelimited protein quantity isolated from a single gel band coupled withPoster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 91

Poster Abstractscolumn loading capacity maximums necessitate the use of 75 µm IDpacked columns for optimal sensitivity. However, limitations on sampleinjection volume, gradient and flow characteristics, and excessive delayvolume hinder throughput. Novel methods for fractionating complexbiological samples with higher loading capacities and more efficientrecovery, such as novel solution phase tube-gel fractionation andothers, demand a column format which maximizes on the extendeddynamic range of these emerging techniques. Packed tip columns witha larger ID (150 µm to 200 µm) facilitate higher sample loading capacityand enable higher flow rates for improved cycle time while maintainingthe optimal sensitivity realized in the nanobore packed tip colummformat. Using peptide standards, single protein digests and whole yeastdigests improvements in cycle time and sample loading capacity using150 µm ID packed tip columns are demonstrated.216 Comparing Global and Targeted Lipidand Fatty Acid Shotgun Profiling ofBrain Tissue Extracts by NanoESI-InfusionJ. Albanese 1 , B. Simons 2 , G. Impey 2 , E. Duchoslav 2 ,K. Koisten 3 , K. Ekroos 31AB SCIEX, Foster City, CA, United States; 2 ABSCIEX, Concord, ON, Canada; 3 Zora Biosciences,Espoo, FinlandThe growth in lipidomics research is uncovering a need for completeand comprehensive workflows for identifying and quantifying lipidspecies from biological extracts. In choosing analytical methodsfor lipidomics, different yet complementary mass spectrometryapproaches can provide a more complete and comprehensive dataset leading to a detailed characterization of lipid molecular speciesfrom complex extracts. A preliminary strategy carried out as global“shotgun” tandem mass spectrometry by direct infusion electrosprayionization or LC-MS/MS analysis, uses information dependant MS/MSscanning in both polarities for unbiased lipid profiling. The secondapproach involves multiple lipid-class-specific precursor ion andneutral loss scanning whose resulting spectra can be used directly toidentify and characterize lipids and fatty acids in tissue-derived lipidextracts. The multiple precursor ion scanning (MPIS) methods havebeen published and recently reviewed by Ekroos et al, describing theadvantages of targeted MPIS techniques for generating comprehensivelipid arrays from small sample volumes. Fully characterizing these lipidcomponents by high quality MS/MS for fatty acid chain length anddouble bond positioning is a critical step for understanding theirbiological implications in cell signaling and lipid-initiated diseaseprogression. Taking advantage of the speed, selectivity, and sensitivityof hybrid triple quadrupole technology, whole lipid extracts fromrat brain tissue can be analyzed by direct nanoESI infusion for indepth glycerophospholipid profiling - achieving both qualitative andquantitative data (with the use of synthetic lipid internal standards) invery fast analysis times. Lipid species identification and quantitationis carried out using LipidViewTM Software enabling post acquisitionprocessing of precursor ion, neutral loss, MRM, and MS/MS data via lipiddatabase searching and accurate peak integration. We present robusttargeted and global workflows for the identification and quantitation ofglycerphospholipids in total lipid extracts from rat brain tissue.217 Detection of p53 Phosphorylationand Oligomerization Using ProximityLigation AssayS.M. Chen, K. Huwiler, R. Bruinsma, B. Marks,B. Schweitzer, T. Settineri, M. Shannon, D. RuffApplied Biosystems, part of Life Technologies,Foster City, CA, United StatesThe tumor suppressor protein p53 is one of the most studiedarchitects of transcription control in cells. Recent reports detailcrucial tertiary structural requirements for p53 influence intranscription regulation complexes. These findings indicate thetetrameric assembly of p53 protein is essential for stabilizationand localization of the protein. This event is critical for DNA bindingand transcriptional activation. Post-translational modifications (PTM)have been linked to activation of p53 function in response to genotoxicstress. In particular, phosphorylation of the p53 Ser-15 is a critical eventin the genotoxic pathway cascade. We investigate the interconnectionbetween this PTM and p53 higher order structure formation. Weemploy a novel antibody binding assay scheme to elucidate detailsof the multimeric status of p53 in both recombinant p53 proteinexperiments and in cells undergoing genotoxic responses. Thisapproach utilizes the Proximity Ligation Assay (PLA) to simultaneouslyquery the role of Ser-15 phosphorylation and the multimeric state ofp53. By use of a phospho-Ser-15 specific monoclonal antibody PLAprobe, we quantify proximity of two individual phospho-Ser-15 motifsin experimental samples. Our results demonstrate that phospho-Ser15promotes multimerization of p53 protein both in vitro and in cellsresponding to genotoxic stress. Further studies using PLA to explore thep53 protein-protein interactome should reveal a more complete viewof the downstream interplay facilitated by the tetrameric conformation.218 Investigating the Robustness of a NewMicrofluidic DeviceC. Hughes, J. Langridge, T. McKennaWaters Corporation MS Technologies Centre,Manchester, United KingdomSuccessful assembly of the components used in nanoscale LC requiressome level of expertise in order produce good chromatographywith minimal band broadening.Microfluidic devices, wherechromatographic separation is performed on a chip mounted close toa MS inlet, enable significant reduction in dead volumes and enableoptimal chromatography to be achieved by the novice user. Here, wedemonstrate the robustness and reproducibility of a Waters Nanotilecoupled to a new Time of Flight MS. We made 35 injections of an E.Coli tryptic digest, containing 50fmol spikes of four proteins, onto ananoAcquity UPLC system coupled to a Trizaic source.The Trizaicconsists of a ceramic tile containing a trapping column, analyticalcolumn and emitter. Trapping flow rate was set to 8uL/min for 1.8minsand the analytical reversed phase separation was performed at a flowrate of 450nL/min, changing the acetonitrile composition from 3 to40% over 90minutes, with 30minute re-equilibration. Emitter eluentwas analysed using a ToF MS operating in MSe mode. In this mode, theMS performed alternate low and elevated energy scans, the low energyproviding precursor ion information and the elevated energy providingfragment ion information. Data was processed using ProteinLynx GlobalServer where the ion information was collated using retention timeinformation in order to associate precursors and fragments. Data wasthen searched using a non-redundant database and the internal spike92 • ABRF 2011 — Technologies to Enable Personalized Medicine

allowed absolute quantitation. Comparing chromatograms from variousstages of the 70-hour experiment shows excellent reproducibility. Theaverage number of E. Coli proteins identified was 389±17, in which325 replicated in 20 out of the 35 injections. Moreover, the absolutequantitation measurements using the ADH spike for identified proteinswith >3 peptides is 285±7ng and the quantitation measurement for oneof the other protein spikes, Enolase, shows a CV of 7%.220 Identification of Various Pink-RedPigments Formed by Reacting VariousAmino Acids with Onion (allium cepaL.) Thiosulfinates Using HPLC withDiode Array Detector and TandemMass Spectrometry219 Effective Semi-Automated Extractionof Intact Mitochondria from SolidTissues Using Gentle MechanicalHomogenization and Pressure CyclingTechnologyV. S. Gross 1 , G. Carlson 1 , E. Freeman 2 ,A.R. Ivanov 2 , A. Lazarev 11Pressure BioSciences, Inc., South Easton, MA,United States; 2 HSPH Proteomics Resource, HarvardSchool of Public Health, Boston, MA, United StatesImpaired mitochondrial function has been linked to many diseases,such as stroke, heart disease, cancer, Type II diabetes and Parkinson’sdisease. Mitochondria-enriched preparations are needed forproteomic and metabolomic studies that may provide crucial insightsinto tissue-specific mitochondrial function and dysfunction, andanswer fundamental questions of cell energetic and oxidative stress.Mitochondria extractions from whole tissue samples are typicallyperformed using Potter-Elvehjem homogenizers or similar laborintensivemanual disruption methods that require extensive operatorexperience, and often result in damage to fragile organelles and highsample-to-sample variability. Here we describe a semi-automatedmethod that uses a novel gentle mechanical homogenizer (The PCTShredder) and hydrostatic pressure to release intact mitochondriafrom fresh rat tissues with minimal hands-on time. Pressure CyclingTechnology (PCT)-based tissue homogenization is conducted undercontrolled thermodynamic conditions (time, temperature and pressure)leading to more reproducible results. The quality of mitochondriapreparations was characterized by electron microscopy, 2D PAGE andrespiration assays. Our data demonstrate that mitochondria extractedby the PCT sample preparation system (PCT-SPS) are intact, functional,and exhibit a protein pattern comparable to control samples isolatedusing a conventional Potter-Elvehjem homogenizer. The resultingmitochondria-enriched samples were also subjected to trypsindigestion followed by nanoLC-MS/MS analysis on an LTQ-Orbitrap.Proteomic and pathway profiles of mitochondria samples preparedusing the novel extraction technique were compared to those extractedusing a conventional manual method to demonstrate the purity ofmitochondrial preparations extracted using the novel PCT-SPS method.Y. Rezenom 1 , E. Lee 2 , K. Yoo 2 , D. Russell 1 , B. Patil 21Laboratory for Biological Mass Spectrometry,Department of Chemistry, Texas A&M University,College Station, TX, United States; 2 Vegetable& Fruit Improvement Center, Department ofHorticultural Science, Texas A&M University,College Station, TX, United StatesDuring the processing of onion, pink-red colored pigments are oftenformed. The process is believed to be a multistep process includingenzymatic and non enzymatic reactions. In order to investigate thisprocess, we developed a reaction system, where pink-red pigments(‘pinking’) can be formed by reacting amino acids with onionthiosulfinate formed by reacting an isolated garlic alliinase and (+)-S-1-propenyl-L-cysteine sulfoxide (1-PeCSO) in the natural onion juice. Theunknown pink-red pigments formed during this process were separatedand detected using a high-performance liquid chromatography (HPLC)and a diode array detector (DAD) at 515 nm. Fractions collected fromthis separation were further analyzed using liquid chromatography(LC) and tandem mass spectrometry. Similar head group structure, two3,4-dimethyl pyrrolyl rings that were cross linked by allyl group, wasdetermined for all conjugate-pigments formed from different aminoacids based on the accurate and tandem mass spectrometry. However,the tail group attached to the N-terminal of pyrrole ring differed foreach pink-red pigment depending on the amino acid used. In addition,in most cases more than one pink compound were identified for thesame amino acid used. We presumed that the complexity of the pinkredpigments was due to the involvement of 21 natural amino acids andother derivatives of the products. Finally, we suggest that the pinkingprocess in crushed onion is very similar to the greening process incrushed garlic, emphasizing that thiosulfinates from flavor precursorsand free amino acids are absolutely necessary during the discoloration.221 Improvements in Data-DependentAcquisition for Enhanced ProteinIdentificationC. Miller, N. Kitagawa, W. Tang, J. Roark,J. Satulovsky, P. PerkinsAgilent Technologies, Palo Alto, CA, United StatesPoster AbstractsComprehensive proteome analysis can be very challenging due tocomplexity and range of protein concentrations. Techniques such as2D LC, pI-based fractionation and gel electrophoresis are typicallyemployed to increase separation efficiency as a strategy for obtainingmore peptide MS/MS spectra and thus increasing the number of proteinsidentified. In data-dependent mode, efficient and comprehensiveprotein identification depends on several conditions during theacquisition, among them: the number of precursors examined per unittime, selection of the most promising precursors for fragmentation,and application of the appropriate fragmentation conditions to yieldthe highest quality product ion spectrum during acquisition. This workdescribes changes to the precursor selection and fragmentation stepsto select the precursors most likely to produce good quality peptideMS/MS spectra, and to increase the quality of those spectra.ABRF 2011 — Technologies to Enable Personalized Medicine • 93

Poster Abstracts222 UHR-Q-TOF Analysis Can AddressCommon Challenges in Targeted andUntargeted MetabolomicsA. Barsch 1 , G. Zurek 1 , D. Krug 2 , R. Müller 21Bruker Daltonics, Bremen, Germany; 2 Universitätdes Saarlandes, Saarbrücken, GermanyHere, we present an ESI-UHR-Q-TOF based analysis of myxobacterialsecondary metabolites, which permits to solve several challengesfrequently encountered in metabolite profiling studies. Myxobacteriaare promising producers of natural products exhibiting potentbiological activities, and several myxobacterial metabolites are currentlyunder investigation as potential leads for novel drugs. However, themyxobacteria are also a striking example for the divergence betweenthe genetic capacity for the production of secondary metabolitesand the number of compounds that could be characterised to date.Wildtype and mutant strains were analyzed concerning the productionpatterns of known metabolites and with regard to the discoveryof new metabolites. Sample throughput: Since mass accuracy andresolution of TOF instruments are independent of the acquisition rate,they are perfectly suited for a coupling to UHPLC separations. Thesehyphenations enable a reduction of analysis time in combination witha high chromatographic resolution and therefore permit an increasedsample throughput. The UHR-TOF analysis revealed that an acquisitionrate of up to 20Hz did not compromise the achieved mass accuracy orresolution. Targeted and untargeted metabolite profiling: Acquisition offull scan accurate mass spectra enable the targeted screening for knowncompounds e.g. from the class of DKxanthenes based on very selectivehigh resolution EIC (hrEIC) traces with small mass windows of 1.0 - 0.5mDa. A comparison of several datasets following a “comprehensivefeature extraction” combined with a statistical analysis permits anuntargeted discovery of novel biomarkers using the same data filesas for the targeted analysis. Identification: Even a mass accuracy of 0.1ppm is not sufficient for an unambiguous formula identification for m/zvalues above 500. A combination of accurate mass data and isotopicpattern information in MS and MS/MS spectra can extend this m/z rangefor reliable formula suggestions. Examples for novel metabolites fromMyxobacteria will be shown.223 Metabolic Profiling of aCorynebacterium Glutamicum DeltaprpD2by GC-APCI High ResolutionQ-TOF AnalysisA. Barsch 1 , G. Zurek 1 , M. Persike 2 , J. Plassmeier 2 ,K. Niehaus 21Bruker Daltonik GmbH, Bremen, Germany;2Centrum für Biotechnologie, Universität Bielefeld,GermanyMetabolomics studies based on Gas chromatography -Massspectrometry (GC-MS) are well established and typically employelectron impact (EI) ionisation. Target compounds of interest canbe identified by comparison to commercial or public databases.Unfortunately, many possible biomarkers detected in metabolicprofiling experiments cannot be identified due to the lack of referencespectra for a majority of biologically relevant compounds. Therefore,many possible biomarkers remain “unknowns” up till now. Hyphenatinggas chromatography with high resolution TOF-MS technology with softatmospheric pressure ionisation (APCI) can preserve the molecularion information and delivers accurate mass and isotopic patterninformation. This data enables a sum formula generation for knownand unknown target compounds.Additionally, optionally acquired MS/MS data can extend the capabilities for structural elucidation. Massaccuracy, resolution and isotopic fidelity are independent of the TOF-MS acquisition rate. Therefore, these instruments can be coupled to gaschromatography, which typically delivers narrow peak width requiringfast MS scan speeds. Corynebactrium glutamicum, a gram positive, nontoxicbacterium, is used in the industrial production of amino acids likelysine and glutamate. C. glutamicum can be grown on different carbonsources. Glucose is metabolised via glycolysis and the tricarboxylic acid(TCA) cycle, whereas propionate is catabolised through the methylcitricacid pathway. The prpD2 gene encodes a 2-methylcitrate dehydratasewhich is involved in the degradation of propionate. Metabolic profilingof Corynebacterium glutamicum delta-prpD2 extracts of cells grownon glucose or glucose and propionate analyzed by GC-APCI-TOF-MSrevealed several compounds elevated in cells grown on propionate.Identification of 2-methylcitric acid and alanine using accurate massand isotopic pattern information in MS and MS/MS spectra provided aproof of concept for the identification of target compounds using highresolution MS technology.224 Biomarker Discovery Using NewMetabolomics Software forAutomated Processing of HighResolution LC-MS DataD. Peake 1 , S. Hnatyshyn 2 ; M. Reily 2 ; P. Shipkova 2 ;T. McClure 1 ; M. Sanders 11Thermo Fisher Scientific, San Jose, CA, UnitedStates; 2 Bristol Myers Squibb, Princeton, NJ, UnitedStatesRobust biomarkers of target engagement and efficacy are required indifferent stages of drug discovery. Liquid chromatography coupledto high resolution mass spectrometry provides sensitivity, accuracyand wide dynamic range required for identification of endogenousmetabolites in biological matrices. LCMS is widely-used tool forbiomarker identification and validation. Typical high resolution LCMSprofiles from biological samples may contain greater than a millionmass spectral peaks corresponding to several thousand endogenousmetabolites. Reduction of the total number of peaks, componentidentification and statistical comparison across sample groups remainsto be a difficult and time consuming challenge. Blood samples fromfourgroups of rats (male vs. female, fully satiated and food deprived)were analyzed using high resolution accurate mass (HRAM) LCMS.All samples were separated using a 15 minute reversed-phase C18LC gradient and analyzed in both positive and negative ion modes.Data was acquired using 15K resolution and 5ppm mass measurementaccuracy. The entire data set was analyzed using software developedin collaboration between Bristol Meyers Squibb and Thermo FisherScientific to determine the metabolic effects of food deprivationon rats. Metabolomic LC-MS data files are extraordinarily complexand appropriate reduction of the number of spectral peaks viaidentification of related peaks and background removal is essential. Asingle component such as hippuric acid generates more than 20 relatedpeaks including isotopic clusters, adducts and dimers. Plasma andurine may contain 500-1500 unique quantifiable metabolites. Noisefiltering approaches including blank subtraction were used to reducethe number of irrelevant peaks. By grouping related signals such asisotopic peaks and alkali adducts, data processing was greatly simplifiedby reducing the total number of components by 10-fold. The softwareprocesses 48 samples inunder 60minutes. Principle ComponentAnalysis showed substantial differences in endogenous metabolites94 • ABRF 2011 — Technologies to Enable Personalized Medicine

levels between the animal groups. Annotation of components wasaccomplished via searching the ChemSpider database. Tentativeassignments made using accurate mass need further verification bycomparison with the retention time of authentic standards.225 On-Line Electrochemistry/MS - APowerful Tool for Rapid Prediction ofPhase I and II Drug MetabolismJ. Powers 1 , J. Purkerson 1 , A. Kraj 2 , M. Eysberg 2 ,J.P. Chervet 21Antec, Palm Bay, FL, United States; 2 Antec,Zoeterwoude, The NetherlandsThe use of Electrochemsiry is a complementary approach to traditionalmethods such as in vivo (human, rodent) or in vitro (liver microsomes)metabolism studies, and delivers the oxidative metabolic fingerprint ofa (drug) molecule in a very short time. The acquired mass spectra arepresented in simple 2 dimensional or more illustrative 3 dimensionalplots, so-called MS voltammograms. A MS voltammogram visualizesthe ion abundance versus m/z as a function of applied potential to theelectrochemical cell. With a MS voltammogram the optimal potentialcan be determined for electrochemical generation of the desiredmetabolite for further research, e.g., a phase II metabolism study (i.e.adduct formation). It is a quick method to identify reactive pathwaysof the compound of interest. Additionally, electrochemistry allows totrace the reactive metabolite conjugates with targets (e.g., proteins,glutathione) without matrix interactions in contrary to traditionalmethods. A dedicated software program has been developed toautomate and simplify the MS voltammogram acquisition. The programcontrols the syringe pump, the potentiostat and triggers the acquisitionof mass spectra at the designated cell potentials. The total acquisitiontime needed for recording of a full MS voltammogram can be as shortas 5 minutes. Amodiaquine an anti malaria agent was chosen as one ofthe model drugs to investigate oxidative metabolism using the on-lineEC/MS system with automated MS voltammogram acquisition The easyand fast Electrochemical conversion of Amodiaquine into its 4 majorphase I metabolites will be presented. In a second step Glutathione(GSH) is added to the electrochemically generated metabolites to formthe appropriate GSH-metabolite adducts, mimicking phase II reactions.All known adducts were successfully formed and identified with MS.Additionally, MS voltammograms of other drugs and xenobiotics (e.g.,acetaminophen, amiodarone, irinotecan) are presented. The datademonstrate that hyphenation of electrochemistry with electrospraymass spectrometry provides a versatile and user-friendly platformfor rapid and cost efficient screening of target compounds (drugs,xenobiotics, etc.) in phase I and phase II metabolomics studies.226 Facile Solid-Phase Synthesis ofPeptide-p-Nitroanilide (pNA) AnalogContaining Conjugates Using a NovelWang or Rink Amide ResinX. Wang 1 , C. Po 2 , J. He 2 , A. Hong 21AnaSpec, Fremont, CA, United States; 2 EurogentecGroup, Fremont, CA, United StatesProteases play a key role in literally all biological processes, and are ofgreat interest, especially to the pharmaceutical industry. Colorimetricbased Peptide-p-Nitroanilide conjugates (peptide-pNAs), withabsorbance at approximately 408 nm, have historically been and arestill widely used substrates for the study of protease activity. Thepreparation of peptide-pNA however, presents several technicalchallenges. Firstly, the amino group of pNA has a low nucleophilicproperty due to the electron-withdrawing effect of the nitro group.Secondly, the poor solubility of a p-nitroanilide intermediate and lastly,coupling in solution phase by DCC, azide or active ester, commonlyused techniques are not effective. Here we report the developmentof two novel supports for facile solid phase peptide syntheses,namely, Wang-resin and Rink Amide-resin conjugated with a pNAanalog, 5-amino-2-nitrobenzoic acid (Anb5,2). Based on a paper byHojo, et al. in which they described the introduction of Anb5,2 to ap-methylbenzhydrylamine (MB) resin; we successfully coupled Anb5,2to either Wang or Rink Amide resin using the TBTU method in thepresence of p-dimethylaminopyridine (DMAP). Anb5,2-Wang or RinkRmide resin is then coupled to a Fluorenylmethyloxycarbonyl (Fmoc)containing amino acid. Peptide synthesis can subsequently proceedusing Fmoc synthesis strategy. The use of this pNA analog containingresins circumvents the tehcnical difficulties stated above. These resinsalso greatly facilitates the synthesis of peptide-pNA-like chromogenicsubstrates for protease research.**227 Validation of an Ion MobilityHydrogen/Deuterium Exchange MassSpectrometry SystemK. Fadgen 2 , T. Wales 1 , M. Stapels 2 , M. Eggertson 1 ,J. Engen 21Northeastern University, Boston, MA, UnitedStates; 2 Waters Corporation, Milford, MA, UnitedStatesHydrogen/deuterium exchange mass spectrometry (HXMS) has provento be a useful analytical method for the study of protein dynamics andchanges to protein conformation. Rapid chromatographic separationsat 0°C must be utilized to preserve the deuterium label during LC/MS analysis. Unfortunately, fast, low temperature LC separations arenot very efficient and can cause spectral overlap for large proteins.The addition of a gas phase, ion mobility separation (IMS) into theHXMS workflow inserts an orthogonal separation that occurs on themillisecond timescale after the LC step without causing any detrimentalimpact on analysis of deuterium levels. An improved MS system hasrecently been developed that is capable of higher resolution gas-phasemobility separations. The improved MS platform was combined witha fully automated ultra performance liquid chromatography (UPLC)system developed for HXMS. To evaluate the effect of the new MSplatform on deuterium recovery, HXMS experiments were performedusing glycogen phosphorylase B as a model protein. Replicate studiesof deuterium labeling reactions ranging from 10 seconds to 100minutes were evaluated. No significant deviation in deuterium levelswas observed when using either mobility (HDMSE) or non-mobility(MSE) separations and the standard deviation of deuterium uptakefor replicate analyses, for HDMSE and MSE separations were in goodagreement with one another. Using the newly developed protocol, anHXMS study of a monoclonal antibody was performed to comparedeuterium recovery using a 6 or 12 minute chromatographic separation.Even though chromatographic resolution was reduced with the fastergradient, HDMSE allowed several overlapping peptides to easily beinterrogated. In addition, the shorter analysis time improved deuteriumrecovery for the sample. The application of this additional level ofseparation will be essential in future studies of very large proteins inwhich chromatographic efficiency is expected to be suboptimal.Poster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 95

228 Differential Expression of Proteins inLung Cancer Using Difference in GelElectrophoresis (DIGE)F. Masri 1 , P.Beckett 2 , K.S. Aulak 21University of Kalamoon, Faculty of Pharmacy,Syria; 2 Amersham Biosciences, Cleveland ClinicFoundation, Cleveland, OH, United Statesfocusing patterns between the two conditions is clearly visualized withthe Bioanalyzer protein assay. The combination of OFFGEL and lab-onchipprotein analysis allows separation and identification of isoformsbased on pI and molecular weight. Protein isoforms differing only bya few kDa in apparent molecular weight were successfully separatedand enriched by OFFGEL electrophoresis for further downstreamanalysis by LC/MS-MS. The combination of OFFGEL, Bioanalyzer,and mass spectrometry is thus an efficient combination for detailedcharacterization of recombinant proteins such as mAb’s.Poster AbstractsBackground: Lung cancer remains the leading cause of cancer-relatedmortality worldwide. Early detection of lung cancer is problematic dueto the lack of a marker with high diagnosis sensitivity and specificity.The purpose of this study was to develop techniques to identify thedifferential expression protein profiles between tumor and tumorfreeof lung cancer tissues. Methods: 2-dimensional differential ingelelectrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) wereused to analyze four samples of lung cancer tissue (3 replicates each).Results: From optimized 2DE image, A total of 2561 spots were detectedand 427 spots of these were differentially expressed (p

MuDPIT, and off-gel electrophoresis (OGE) ahead of LC-MS/MS.We have evaluated an alternative method that does not requireadditional sample fractionation on additional equipment, meanwhileminimizing alterations introduced by analytical techniques. For bothwhole human cells and isolated mitochondria, the proposed methodinvolved exposure of the pelleted cells or organelles to sequencegradetrypsin in order to obtain peptides and proteins cleaved fromthe cell and organelle surfaces. The “stripped” cells and mitochondriawere also lysed and their contents digested. Additionally, total lysatesof cells and mitochondria not exposed to trypsin ahead of lysis weregenerated and tryptically digested. The obtained proteomic profilingresults were compared to those acquired using commonly employedfractionation techniques (OGE and SDS-PAGE). Resulting digests fromall protocols were analyzed using LC-MS/MS, and all were analyzedfor the proportion of unique and differentially enriched proteinsachieved with each. Our results showed that the inexpensive andsimplistic fractionation method allowed for similar or superior depthof proteomic profiling as well as a substantial increase in the numberof unique identifications of membrane-bound and extracellular matrixrelated proteins. Additionally, the method provides information aboutspatial localization and differential abundance of proteome subsetsin the cell or organelle architectural compartments. These resultssuggest that a very straightforward approach for the preparation ofcellular and organellar specimens prior to proteomic characterizationcan be reproducibly and reliably utilized by biological and medicallaboratories that have no previous analytical biochemistry expertise.232 Detecting Immune System ResponseProteins in a 500 Year-old IncaMummyA. Koller 6 , A. Corthals 1 , L. Davalos 2 ,D.W. Martin 3,4 , R. Rieger 3 , E.I. Chen 3,51City University of New York, John Jay College ofCriminal Justice, Department of Sciences, New York,NY, United States; 2 Department of Ecology andEvolution and Consortium for Inter-DisciplinaryEnvironmental Research, State University of NewYork Stony Brook, Stony Brook, NY, United States;3Stony Brook Proteomics Center, State Universityof New York Stony Brook, Stony Brook, NY, UnitedStates; 4 Department of Medicine, Division ofHematology, State University of New York StonyBrook, Stony Brook, NY, United States; 5 StateUniversity of New York Stony Brook, Department ofPharmacological Sciences, Stony Brook, NY, UnitedStates; 6 State University of New York Stony Brook,Stony Brook, NY, United StatesDisease detection in ancient human samples currently relies ongenomic-based assays, which are error prone due to contamination andcannot distinguish between active and latent pathogenic infection. Onthe other hand, protein-based assays such as global protein profilingoffer complementary alternatives for the pathological diagnosis ofarcheological specimen. The discovery of three Inca mummies in 1998,perfectly preserved in the permafrost of the high Andes, allowed us toanalyze mummy samples by protein-based and genomic-based assay.A buccal swab from one of the 500 year old mummy was analyzed byshotgun proteomics to detect the protein profile. Among the identifiedproteins, we found a signature of proteins indicating an immuneresponse to a bacterial infection at the time of the mummy’s death.Based on the external visible symptoms and the gamut of immuneresponse proteins obtained from the mouth swab, we suspectedthat the pulmonary infection was caused by Mycobacterium. PCRassay followed by direct sequencing of the PCR products confirmedthe presence of Mycobacterium sp. in the mouth swab. Until now,immunoassays have been the only way to detect an active immuneresponse and infer infection in historical samples, but these wereplagued by low specificity and sensitivity. However, we demonstratehere the feasibility of incorporating global protein profiling in thediagnosis of infection from archeological samples. Protein signaturesobtained from these samples could be extremely useful in determiningthe status of infection while genomic-based assays can be used todetect the identity of the pathogen.233 A New Technology for HighlyEfficient and Reproducible AlbuminDepletion from Whole SerumC. Bolcato, H. Anderson, M. Maust, G. Boyce,M.J. PowellProtea Biosciences, Morgantown, WV, United StatesOne of the major problems faced in the analysis of the human serumproteome is the broad dynamic range of its protein constituents.High abundance proteins, such as human serum albumin (HSA) andgamma-immunoglobulin (IgG), which together comprise 75% of totalserum protein content, inhibit the analysis of lower abundant proteinsof interest. Affinity-based depletion strategies are often employed toselectively remove these high abundance proteins in order to enrichthe lower abundant proteins and facilitate their analysis. Traditionally,antibody-based schemes are used to produce affinity-binding ligandsfor HSA and IgG. Variability in the production of these antibodies leadsto a wide variety of specificities and selectivities. Thus, the efficienciesfor antibody-based depletion strategies for the selective removalof high abundance proteins in human serum can vary broadly, from70 to 95%, and can dramatically affect the reproducibility of humanserum proteome studies. Here, a new approach to affinity-based serumalbumin removal is presented. Instead of utilizing a traditional antibodybaseddepletion approach, the ProteaPrep albumin depletion captureligand is a recombinant bacterial protein that is highly purified forrobust, efficient removal of albumin from human serum samples. Therecombinant ligand technology produces binding constants withhuman serum albumin (Kd ~ 1 × 10-11M) that are significantlystronger than antibody-based methods, which have binding constantsthat range from Kd’s of 1 × 10-6 to 1 ×10-8 M. The resultsshow that the functionalized beads bind and remove albumin fromserum in a highly efficient and rapid manner. Depletion efficiencies are>99% for the selective removal of HSA from serum samples in less than20 minutes. The recombinant bacterial protein ligands are shown to beeffective for the depletion of albumin from the sera of other species,including mouse, rat, pig, horse, cow, and monkey.234 Qualitative and QuantitativeCharacterization of Proteomes UsingIon Mobility Mass Spectrometry withData Independent AcquisitionM. Stapels, K. Fadgen, S. Geromanos, J. LangridgeWaters Corporation, Milford, MA, United StatesA central goal in a proteomics study is to fully characterize a sample bothqualitatively and quantitatively. A data-independent analysis yieldsreproducible fragmentation and peak area information for peptides inPoster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 97

Poster Abstractsa complex mixture (1). The addition of ion mobility into this analysisinserts an orthogonal, gas-phase separation, occurring in the millisecondtimescale, which is poised nicely between chromatographic and TOFmass spectrometry timescales. Peptides and their correspondingfragment ions are aligned by chromatographic retention time in dataindependentanalyses. When ion mobility is used, peptides and theirfragment ions also share the same mobility drift time which dramaticallyimproves specificity. In this study, eukaryotic and prokaryotic sampleswere analyzed by one-dimensional (1D) and 2D chromatography usinghigh-low pH RP-RP separations, both with and without ion mobilityseparation. The number of proteins identified in a sample doubled byusing a five fraction 2D separation compared to 1D chromatography.Utilizing the mobility separation yielded an additional increase inthe number of proteins and peptides by at least 20%, without anyadditional instrument time. The amount of information obtained from asample depended on the amount loaded as well as the time dedicatedto analysis of the sample. For one prokaryotic sample, 552 proteinswere reproducibly identified with 1D chromatography without ionmobility from 0.75 ug of material. Loading 5 ug and performing 5fractions, along with ion mobility, yielded 1260 reproducible proteinidentifications. Stoichiometry between proteins was determined bycomparing the average intensity of the top three peptides to everyprotein to that of an internal standard (2). Agreement was foundbetween the experimental stoichiometric ratios and those found inliterature for many protein groups. 1. Geromanos, S.J., et. al. Proteomics.2009 (6):1683-95. 2. Silva, J.C., et. al. Mol Cell Proteomics. 2006 (1):144-56.235 Characterization of UnexpectedBy-Products in a Recombinant TAUProtein by Capillary LC Coupled toMALDI-Top-Down-Sequencing (LC-MALDI-TDS)A. Asperger 2 , B. Kovacech 1 , A. Kovac 1 , D. Suckau 21Institute of Neuroimmunology of Slovak Academyof Sciences, Bratislava, Slovakia; 2 Bruker Daltonics,Bremen, GermanyCompared to Edman sequencing, MALDI-TOFMS based top-downsequencing of proteins (MALDI-TDS) is much faster, cheaper, candeliver significantly longer sequence readout from both, protein N-and C-terminus, and has no limitations in case of terminally modifiedproteins. These features make MALDI-TDS an extremely appealingmethod for the QC analysis of recombinant protein products, forinstance biopharmaceuticals. Direct MALDI-TDS analysis requirespurified, homogeneous protein samples. However, coupling MALDI-TDS to upfront LC separation allows to analyze mixtures of proteins.Since recombinant protein products often contain unexpectedheterogeneities, LC-MALDI-TDS represents a promising methodto characterize such undesired by-products. We describe here thecharacterization by LC-MALDI-TDS of unexpected by-productsoccurring in a recombinant TAU protein. Initial MALDI-TOF analysisof the protein sample revealed at least two minor components beingdifferent from the expected protein mass. To separate these sampleconstituents, capillary LC was used. The LC fractions were depositedon a MALDI target pre-spotted with sDHB matrix. Subsequently,MALDI-ISD spectra of the LC separated compounds were acquiredusing a Bruker Ultraflex III MALDI-TOF/TOF. Analysis of the MALDI-TDS data was performed using Bruker’s BioTools software. By means ofLC-MALDI-TDS, one of the by-products, detected at 16.6kDa, turnedout to represent a truncated version of the expected TAU protein,containing the sequence range [1-161]. C-terminal ISD fragmentsyielded unambiguous evidence for the correct assignment of thetruncation site. Another unexpected by-product, being approx. 130Dalower in mass than the expected protein product, was lacking theN-terminal methionin residue. This was concluded from the assignmentof the respective N-terminal fragments in the ISD spectrum. As shownabove, LC-MALDI-TDS turned out to be very efficient with regard toanalysis time and lab work required, especially when comparing themethod to alternative approaches like Edman sequencing or geLCworkflows, i.e. SDS-PAGE with subsequent LC-MS/MS analysis of thedigested proteins.236 Novel Proteomic Analysis ofEsophageal InflammationK. Jonscher 1 , A. Kendrick 1 , S. Fillon 2 ,Z. Robinson 2 , J. Masterson 2 , S.J. Ackerman 3 ,G.T. Furuta 21Department of Anesthesiology, University ofColorado Denver, Aurora, CO, United States;2Department of Pediatrics, University of ColoradoDenver, United States; 3 Digestive Health Institute,University of Illinois at Chicago, Chicago, IL, UnitedStatesIntroduction: Esophageal inflammation associated with eosinophilicesophagitis (EoE) and gastroesophageal reflux disease (GERD) requireinvasive endoscopy and biopsy for diagnosis and testing efficacy oftreatments. In the 1970’s, the Enterotest, a weighted gelatin capsulefilled with and attached to a 90 cm long nylon string, was introduced as aminimally invasive method to identify intestinal infections. Patients tapethe string end extruding from the capsule to their cheek and swallow thecapsule; the string subsequently deploys into the small intestine and thecapsule released. After incubation for 15 min to overnight the string ispulled back out of the mouth. Here we present pilot data demonstratingthat secretions adhering to the string may be recovered and analyzedusing shotgun proteomics. Methods: Strings from the distal esophagusof patients with GERD and normal controls (n=2/group) were cut into 2cm sections and proteins recovered by incubating strings in lysis buffer.Tandem mass spectrometry analysis using a high-capacity quadrupoleion trap was performed on trypsin-digested samples and data weresearched with X!Tandem through the LABKEY interface. Spectralcounting provided relative quantitation of proteins between samples.Results: After removal of isoforms, 34 proteins common to all stringsamples (2 each from 2 patients/subjects) were identified. Annexin A1,keratins 16, 4 and 13, and MUC5B were most highly upregulated inesophageal secretions from GERD subjects as compared with normalcontrols. In addition, functional clustering revealed that anti-apoptoticproteins and those involved in extracellular signaling were present inonly GERD and not normal controls. Conclusion: The Esophageal StringTest can be used to sample esophageal secretions in health and disease.Host response proteins are present in the esophageal lumen duringinflammation. This work was supported by R21-AI079925, ThrasherFoundation, CURED Foundation, Pappas Foundation and the ColoradoClinical Translational Scientific Institute (CCTSI) NIH/NCRR GrantNumber 5UL1RR026314-02.98 • ABRF 2011 — Technologies to Enable Personalized Medicine

237 Multi-Site Assessment of ProteoRedPlasma Reference Sample forBenchmarking LC-MS PlatformPerformance238 In Depth Characterization of ProteinChanges Induced by GlucoseLimitation Using ComplementaryIonization TechniquesA. Campos 1 , K. Asanov 1 , E. Oliveira 1 , N. Colome 2 ,S. Martínez-Bartolomé 3 , J-P Albar 3 , F. Canals 2 ,ProteoRed ISCIII Consortium 41Proteomics Platform, Barcelona Science Park,Barcelona, Spain; 2 Proteomics Laboratory,Medical Oncology Research Program, Vall dHebron Institute of Oncology VHIO Vall d HebronUniversity Hospital, Barcelona, Spain; 3 CentroNacional de Biotecnologia-CSI, Madrid, Spain;4ProteoRed ISCIII Consortium, Madrid, SpainOne of the missions of the Spanish Proteomics Network (ProteoRedISCIII) is to assist its proteomics core facilities in evaluating theircapabilities to perform qualitative and quantitative proteomicsanalysis. In 2010, the ProteoRed’s Sample Collection and HandlingGroup designed a moderately complex plasma standard referencesample primarily to be used for routine quality assurance monitoringof laboratory instrumentation, as well as inter-laboratory performanceassessment, and development and validation of novel technologies.The ProteoRed Plasma Reference (PPR) sample is a subset of highlyabundant well-characterized human plasma proteins with a numberof isoforms, in addition to 4 spiked-in proteins, altogether distributedover 5 orders of magnitude in concentration. The PPR sample wasrecently stress tested in the latest ProteoRed Multicenter Experiment(PME6) that counted with the participation of 17 proteomics facilitiesusing a wide range of LC-MS platforms. Although each laboratorywas allowed to use its own favorite methodology, we requested thesample be analyzed in a single LC-MS run in experimental triplicate(3 different digestions).Evaluation of the results submitted by thestudy participants revealed moderate discrepancies at the peptideidentification level, and poor overlap at the protein identificationlevel. In an attempt to identify the source of such irreproducibility,raw data of 8 laboratories (24 LC-MS runs) were reanalyzed centrallyusing a standardized data analysis pipeline, which included proteininference using ProteinProphet software. We found that the majorityof protein identification discrepancies across submitted reports ofthese 8 laboratories were due to inconsistencies on how data analystsand computational tools group and/or infer proteins. Immunoglobulinvariable chain identifications were particularly conflicting throughoutidentification lists, even in the centralized analysis. Using a series ofLC-MS performance metrics, we benchmarked the performance of 8LC-MS instruments (Orbitraps) and identified system components thatvary the most across laboratories.B. Müller 1 , W. Jabs 2 , M. Behrens 2 , D. Suckau 2 ,C. Baessmann 2 , K. Niehaus 3 , T. Noll 11Institute of Cell Culture Technology, Facultyof Technology, Bielefeld University, Bielefeld,Germany; 2 Bruker Daltonik GmbH, Bremen,Germany; 3 Proteome and Metabolome Research,Faculty of Biology, Bielefeld University, Bielefeld,GermanyComprehensive quantification of specific changes in biological systemsin response to a certain treatment or perturbation is one of the mostimportant but also among the most challenging tasks in proteomics.Different kinds of separation techniques, mass spectrometer types,identification strategies, and quantification strategies can be appliedfor this purpose. Here, we investigate the benefits of combining theresults obtained by two main ionization techniques used in proteomics,ESI and MALDI. Results are derived with the aid of a bioinformaticsplatform, which integrates different proteomics techniques. Humancell cultures obtained from the periventricular region of the brainwere grown under normal and under glucose limitation conditions.To investigate the changes induced by the glucose limitation, stableisotope labeling with amino acids in cell culture (SILAC) was used.After labeling, samples were mixed and fractionated. 40µg ofthe cytosolic protein fraction was separated by 1D-SDS PAGE, whichwas cut into 10 bands. Each band was digested and then analyzed withnanoscale liquid chromatography (nLC) coupled to an ESI-ion trapand a MALDI-TOF/TOF, respectively. The use of ESI-MS in additionto MALDI-MS resulted in a 46% increase in protein identificationand a 124% increase in peptide identification. Combining data ofboth ionization techniques, 664 proteins consisting of 4658 peptideswere identified. Peptides identified with ESI-MS are significantly morehydrophobic than peptides identified with MALDI-MS. An analysisof the amino acid composition of the identified peptides revealed asignificant bias of ESI-MS for aliphatic and hydroxyl-containing aminoacids while MALDI-MS favors peptides with aromatic, basic and amidegroup-containing amino acids. The results show, that the use of nLC-ESI-MS in addition to nLC-MALDI-MS greatly improves the numberof identified and quantified proteins and peptides. The experimentrevealed little changes in the cytosolic proteome of cells growing underthe applied glucose limitation.239 Evaluation of Two SeparationTechniques, SCX and OFFGEL andof Two Fragmentation Methods,CAD and PQD, to Asses iTRAQQuantitation EfficiencyPoster AbstractsG. Grigorean 1 , A. DiFonzo 2 , P. Soffientini 2 ,T. Manousidou 21European Institute of Oncology, IFOM-IEOCampus, Milan Italy; 2 Cogentech, IFOM-IEOCampus, Milan, ItalyTo evaluate various sample preparation and analysis platforms forisobaric tags for iTRAQ experiments, we compared two separationand two fragmentation techniques: Strong Cation Exchange (SCX) withABRF 2011 — Technologies to Enable Personalized Medicine • 99

Poster Abstractsisoelectric point separation. In addition, we compared two types ofmass analyzers: a quadrupole and a linear ion trap, the former withina quad-TOF hybrid mass spectrometer, and the later within an LTQ-FThybrid MS. This was done by comparing two fragmentation techniques,collisionally activated dissociation (CAD) with pulsed Q dissociation(PQD). Proteins from lysed HeLa cells were trypsinized and peptideswere iTRAQ labeled. Half of the labeled digest was separated via SCXinto20 fractions. The other half of digested peptides was separatedwith an OFFGEL isoelectric point fractionator into 24 fractions. Eachdried peptide fraction was desalted, and submitted to reverse-phaseC-18 HPLC on-line with the mass spectrometer. Quantitative dataanalysis for the LTQ-FT data was performed using Proteome Discoverer(Thermo Electron) and for the QStar data, PEAKS Studio (BioinformaticSolution Inc.). R software was used for the statistical validation ofresults. The LTQ-FT data was also analyzed with the PEAKS softwareto asses the viability of PEAKS as a universally applicable data analysispackage. Comparison between separation and dissociation was basedon a) number of proteins quantified b) reproducibility of technicalreplicates based on protein distribution c) standard deviation for dataobtained from the two dissociation techniques. Fractionation withOFFGEL increased the number of identified and quantified of proteinsby approximately 20%. Number of mass spectra which can be used forquantitation is higher from the QStar instrument implying that the orderof magnitude increased sensitivity of the LTQ-FT relative to the quad-TOF is lost to the inefficiency of the PQD. Reproducibility of technicalreplicates from PQD is lower than that obtained with data from CAD.240 Quantification of Eicosanoid PathwayProteins in Human CerebrospinalFluid Using a Dual Pressure Linear IonTrap Mass SpectrometerR. Biringer 1 , J. Horner 1 , A. Fonteh 2 , S. Kauffman 2 ,A. Huhmer 1 , M. Harrington 21Thermo Fisher Scientific, San Jose, CA, UnitedStates; 2 Molecular Neurology Program, HuntingtonMedical Research Institutes, Pasadena, CA, UnitedStatesObjective: To develop a linear ion trap mass spectrometer-basedprotocol for quantifying eicosanoid pathway enzymes and to apply thisprotocol to monitor enzyme changes in episodic migraine. Methods:Peptides diagnostic for eicosanoid pathway enzymes were determinedfrom trypsin digests using standard LC/MSn methodologies.Corresponding stable isotope peptide analogs (SI-peptides) wereselected and synthesized. For each assay, a cocktail of all SI-peptidesis spiked into delipidated cerebrospinal fluid (CSF) from migrainestudy participants when well and sick. After trypsin digestion, eachof the SI-peptides and the corresponding endogenous peptideswere simultaneously quantified using LC/stable isotope selectedreaction monitoring (SI-SRM) using a dual pressure linear ion trap massspectrometer. Results: Linear dynamic range for this assay spans nearlyfour orders of magnitude (0.5-4800 fmol). The LOQ is in the low to subfmolrange depending on the peptide, and corresponds to eicosanoidproteins at concentrations in the low ng/mL CSF range. Eicosanoidprotein concentrations exhibit both preparation and migraine statedependencies. Conclusions: We have developed a robust, targetedfull scan MS/MS linear ion-trap based quantitative assay (LC/SI-SRM) for eight enzymes of the eicosanoid pathway. Further, we havedemonstrated that this assay can be used to quantify changes in theexpression of eicosanoid proteins during episodic migraine.241 Optimization of Nanospray Voltageand Spray Stability: Impact onChromatographic Peak Area forAnalyte QuantitationG. Valaskovic 1 , M. Lee 2 , A. Berg 11New Objective, Inc., Woburn, MA, United States;2Milestone Development Services, Newton, PA,United StatesState-of-the art liquid chromatography-tandem mass spectrometry(LC-MS/MS) analysis uses a constant electrospray (ESI) voltage fordata acquisition. Modern qualitative and quantitative LC-MS/MSmethods depend on highly efficient gradient elution chromatography.The changing chemical composition of mobile phase during gradientelution results in an inherent disconnect with single point ESIvoltage optimization. A constant ESI voltage limits spray stability andcompromises chromatographic peak area quantification, limiting totalpeak area and increasing peak area relative standard deviation (RSD).We have adapted a previously reported closed-loop “orthogonal”feedback control system for qualitative proteomics† to one suitablefor targeted peptide quantification. A digitally controlled nanospraysource was modified to use the high-voltage output from an external,computer controlled power supply. A software program controllingthe nanospray emitter stage position and the external power supplysupported a programmable spray compensation voltage duringchromatograpghic analysis. One microliter of a standard peptide(MRFA) was autosampler injected (CTC PAL) directly on column (NewObjective PicoFrit, 75 um x 10 cm, C18) to the source equipped linearion trap mass spectrometer (Thermo Scientific, LTQ). Repetitive oncolumninjections at different (fixed) target ESI voltage settings yieldsa apparent compound dependant response curve in which totalchromatographic peak area for selected ion currents reaches a maximumvalue. Spray stability and chromatographic quality was directlycorrelated with the observed spray mode. Altering the ESI voltage from2 kV to 3 kV resulted in a peak area increase of 38%. Qualitative visualcomparison of noise in the molecular ion signal (523.5 m/z) showed amarkedly higher level at 2.0kV. Fourier transform spectrum analysis ofselected ion current, revealed a ten-fold increase in noise at 2 and 4Hz for the 2 kV acquisition. Spray instability results in increased noiseof the reconstructed chromatogram, increasing the uncertainly of peakarea measurement. †Valaskovic, G.A, Murphy, J.P., Lee, M.S. “AutomatedOrthogonal Control System for Electrospray Ionization”, J. Am. Soc.Mass Spectrom., 2004, 15, 1201-1215.242 Development of a High-SensitivityTargeted MRM Assay for Peptide andProtein QuantificationT. McKenna, A. Bartlett, C. Hughes, J. LangridgeWater Corporation, Manchester, United KingdomDiscovery phase proteomics has generated numerous candidate proteinmarkers for a wide variety of biological processes and disease types.To assess the viability of these protein expression changes requires theanalysis of a larger number of samples, preferably in a targeted fashion,and hence the use of MRM has become routine. Specific peptides fromthe proteins of interest are targeted as surrogate markers for that protein,in a screening assay using a triple quadrupole mass spectrometeroperating in the multiple reaction monitoring (MRM) mode. The MRMmethod which is used to detect specific ions from target molecules hasthe capability to simultaneously quantify large numbers of proteins100 • ABRF 2011 — Technologies to Enable Personalized Medicine

with good limits of quantification (LOQ) and linear dynamic range. Inthis mode of analysis the sensitivity and dynamic range are improvedand providing sufficient data points across a chromatographic peak arerecorded then quantitation is accurate (CV 5-10%). This high sensitivitycoupled with the specificity/selectivity afforded by MRM transitionsallows extensive panels of peptide biomarkers to be monitored ina single experiment from complex mixtures. We will describe thedevelopment and implementation of novel high-sensitivity MRM assaysfor large scale peptide quantification.243 High Sensitivity Protein QuantitationUsing a Triple Quadrupole with aDual Ion FunnelC. Miller, Y. Yang, K. WaddellAgilent Technologies, Palo Alto, CA, United StatesAssays that are both specific and quantitative for target proteins arecritical for preclinical validation of putative biomarkers. Such assays aretypically multiplexed, multiple reaction monitoring (MRM) analyseswhich can provide the high-throughput required. Sensitivity is a keyrequirement for such assays as protein biomarker concentrations maybe quite low in commonly used biofluids such as serum and plasma.Improving the sensitivity of LC/MS can be achieved by using nanoflowLC, and by enhancing the sampling and transmission of ions in themass spectrometer. This study demonstrates the 5-10x sensitivity gainachieved for peptides using a triple quadrupole mass spectrometermodified with a dual ion funnel. The sensitivity achieved using amicrofluidic-based nanoflow LC system compared to a standard LCsystem will be shown discussed.244 Development of a Label-FreeQuantitative PhosphoproteomicsPlatform Applicable to Non-CellCulture Biological MatricesE. Soderblom 1 , M.W. Foster 2 , J.W. Thompson 1 ,M.A. Moseley 11Duke Institue of Genome Science and Policy,Durham, NC, United States; 2 Duke UniversitySchool of Medicine, Durham, NC, United StatesThe most commonly employed LC-MS based quantitativephosphoproteomics experiments are performed using cultured celllineswith incorporated stable-isotope labeled amino acids. Althoughthese systems simplify experimental manipulation and providerelatively large amounts of soluble protein, they do not account forthe complexity of signal regulation from other factors or cell typespresent in the cell’s native physiological environment. Here wedescribe the general analytical and informatic methodologies usedfor the global quantitation of specific sites of phosphorylation fromclinically relevant matrices such as human tissue or biological fluidscompatible from a variety of different experimental designs. As anexemplar application of the methodology, a quantitative analysis oflipopolysaccharide challenged and ethyl nitrite protected mouse lungtissue was performed. Phosphopeptide enrichments from 600 ugaliquots of trypsin digested homogenized mouse lung tissue from fourunique treatment groups was accomplished using an optimized TiO2spin-column protocol and were subjected to triplicate 1D-LC-MS/MSanalysis on a Waters NanoAcquity UPLC coupled to a Thermo LTQOrbitrap XL. Following accurate mass and retention time alignmentacross all LC-MS runs performed within Rosetta Elucidator, a robustmean normalization of precursor ion intensities was applied and areaunder the curve quantitative measurements were made for all precursorions. Qualitative identifications were assigned to each precursor ionfollowing Mascot searches against a mus musculus database appendedwith reverse entries and scoring thresholds were adjusted to yield apeptide 1% FDR. Across all four treatment groups, a total of 778phosphopeptides were qualitatively identified. Analytical plus TiO2enrichment variation of pre-digested bovine alpha-casein spiked intoeach sample prior to enrichment yielded a phosphopeptide intensityvariation of < 20% RSD across approximately three orders of magnitude.In summary, these data demonstrate an effective and straight-forwardmethodology to study global changes in phosphorylation directly fromclinically relevant biological samples.245 Studying Whole-Body ProteinTurnover Using Stable-IsotopeLabeled MiceE. Chen 1,2 , A.A. Koller 2 , J. Relucio 1 , S. Nik 1 ,B. Dost 3 , H. Colognato 1 , N. Bandeira 31Department of Pharmacological Sciences, StonyBrook University, Stony Brook, NY, United States;2Stony Brook University, Proteomics Center, SchoolOf Medicine, NY, United States; 3 Department ofComputer Science and Engineering, University ofCalifornia, San Diego, CA, United StatesProtein turnover is a neglected dimension in functional genomicsstudies delineating the dynamic changes of protein regulation and linkstranscriptome, proteome, and potentially metabolome. It is known thatproteins are degraded and synthesized continuously at the cellularlevel. The balance of degradation and biosynthesis is tightly regulatedand has great implications in normal physiology, cellular regulation, andhuman diseases. Recent technological advances in high-resolution massspectrometry open up new opportunities to make proteome-wideprediction of protein turnover. However, studies of proteome turnoverin live animals require high efficiency of labeling using labeled aminoacids and bioinformatics to deal with highly complex mass spectradata. To unravel the dynamics of protein turnover in intact animals,we developed an in vivo pulse-chase strategy and bioinformatics tools(APCIE) to perform analysis of proteome turnover. We pulsed C57BL/6mice with 15N labeled amino acids through diet and chased 15N labeledyoung (3 weeks old) and old (4 months old) mice with unlabeled dietfor 2 weeks. Lungs from these two groups of mice were harvested andanalyzed by mass spectrometry-based shotgun proteomic techniques.Using APCIE, we determined protein degradation (loss of 15N labeledamino acids) and protein synthesis (incorporation of 14N aminoacids) of over 300 proteins commonly found in young and old miceand observed aged-dependent differences in proteome turnover. Asexpected, most proteins are turned over faster in young mice than oldmice reflected by massive loss of 15N amino acids and incorporation of14N amino acids. Our newly developed in vivo pulse-chase techniqueand algorithm can be used for global profiling of protein turnoverin intact animals to study complex mouse models mimicking humandiseases. Combining global profiling of protein turnover and proteinexpression will make a tremendous impact towards mechanism-driventherapeutics based on dynamic regulation of proteins and proteinnetworks.Poster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 101

Poster Abstracts246 Purification of Bacterial APOA-1and Characterization of Novel Anti-Cancer Drug Delivery SystemT. Young 1 , A. Lacko 21North Carolina State University, Raleigh, NC,United States; 2 University of North Texas HealthScience Cente, Fort Worth, TX, United StatesAlthough chemotherapy regimens have proved effective in attackingcancer cells and tumors, side effects and drug resistance remain amajor concern during cancer therapy. The use of reconstituted highdensity lipoprotein (rHDL) nanoparticles has been investigated as adrug delivery system, including the transporting of small interferingribonucleic acid (siRNA). The use of rHDL nanoparticles has greatpotential, in this regard, due to their ability to specifically targetcancer cells via the HDL (SR-B1) receptor. The goal of these studieswas to improve the purification of Apolipoproteins A-1 (ApoA-1), amajor component of rHDL, and the preliminary characterization ofthe siRNA carrying rHDL nanoparticles. E.coli cells transfected withthe apo A-I gene was grown at 37OC, until an optical density of 0.6was reached. The cells were then induced with 0.5mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and centrifuged. Subsequently, thepellets were suspended in the lysate solution and loaded onto a Nickel-Sepharose column. Thereafter, rHDL nanoparticles using siRNA wereprepared. 160 mg per liter of purified ApoA1 was obtained. Particlemeasurements and the chemical composition of the particles are beinginvestigated. Further investigation regarding the efficiency of theincorporation of siRNA, the physical and chemical properties as wellas cytotoxicity of the particles will contribute to the assessment of theefficiency of these particles as a novel anti-cancer drug delivery system.247 Screening for Optimal PurificationConditions for Histidine-TaggedWater-Soluble and MembraneProteins Using Magnetic BeadsMethodsH. Hedlund, E. Mascher, G. RisbergGE Healthcare Life Sciences, Uppsala, SwedenThe development of methods for efficient expression screening ofmultiple numbers of recombinant proteins or optimization of thepurification conditions of proteins is essential to shorten the time fromgene to drug targets. The combination of para- magnetic beads andaffinity purification using IMAC resins (pre-charged with Ni2+-ions)for capture of histidine tagged proteins is an easy-to-use format anda powerful tool allowing for rapid, small-scale purification. We willdescribe two studies for optimization of purification conditions ofhistidine-tagged proteins. All experiments were done using Ni2+chargedHis Mag Sepharose Ni. In the first study optimal purificationconditions for a water-soluble histidine-tagged protein was investigatedat eight different buffer conditions and four different sample loads(varying from 25 % to 100 % of the total binding capacity). The studywas performed using a liquid handling workstation.A good balancebetween yield and purity was obtained at 40 mM imidazole and at asample load between 50 % and 100 % of the total binding capacity. Inthe second study screening for optimal solubilization and purificationconditions for an integral membrane protein was performed, usingseven detergents. The screening protocol involved a rapid purificationstep before analyses, evaluation and final choice of detergent(s).Obtained results from this screening gave reliable results that could alsobe fed into further scale-up experiments. We will present results fromWestern blotting, gel filtration and SDS-PAGE. The para-magnetic beadformat simplified and shortened the handling procedures through-outboth screening workflows.248 Production of Recombinant MouseFlower Protein in E. coli: Applicationof Mistic Fusion to Improving theExpression of Membrane ProteinsJ.L. Martinez-Torrecuadrada 1 , M. Marenchino 2 ,R. González 1 , J. López-Alonso 2 ,R. Campos-Olivas 2 , G. Roncador 3 , E. Moreno 41Proteomics Core Unit, Spanish National CancerCentre (CNIO), Madrid, Spain; 2 Spectroscopyand Nuclear Magnetic Resonance Unit. SpanishNational Cancer Centre (CNIO), Madrid, Spain;3Monoclonal Antibodies Core Unit, SpanishNational Cancer Centre (CNIO), Madrid, Spain;4Cell Competition Group, Spanish National CancerCentre (CNIO), Madrid, SpainThe structural and functional studies of membrane proteins havebeen greatly hindered due to difficulties in their over-expression andproduction. It is also often difficult to generate effective antibodies tomembrane proteins. In our laboratory, we have addressed the problemof producing high level amounts of membrane proteins in Escherichiacoli by use of Mistic, a Bacillus subtilis protein, as a fusion partner. Flower(Fwe) is a membrane protein that is conserved in animals and proposedto be a Ca2+ channel in neurons. It has been recently reported thatin Drosophila Flower is a component of the cell competition responsethat is required and sufficient to label cells as “winners” or “losers”,promoting the elimination of weaker cells from a growing populationin order to optimize tissue fitness. This process may have biomedicalimplications because imbalances in cell fitness appear during aging,cancer formation and metastasis. In this work, we employed themembrane protein Mistic to assist in the production of this protein.The construct 6xHis-Mistic – Flower carrying a TEV cleavage site wasefficiently overexpressed in E. coli. The IMAC-based purification andcleavage of the recombinant protein was achieved in the presence ofthe detergent lauryldimethylamino oxide (LDAO). Circular dichroismshowed a high content in helical structure as predicted from the aminoacid sequence by the program TMHMM. Also, the recombinant Mistic–Flower was used as antigen source to produce monoclonal antibodies inKO mice. The generated monoclonal antibodies were able to recognizeFlower protein by Western blot and immunohistochemistry, indicatingthat this recombinant protein retained the antigenicity of the nativeform. These antibodies will facilitate importantly further functionalstudies on Flower protein.102 • ABRF 2011 — Technologies to Enable Personalized Medicine

249 Rapid Monoclonal Antibody GlycanProfiling Using an IntegratedMicrofluidic-Based mAb-Glyco Chipand Quadrupole Tim-of-Flight MassSpectrometryC. Miller, N. Tang, K. WaddellAgilent Technologies, Palo Alto, CA, United StatesProtein biologics now represent a significant share of pharmaceuticalsales and future growth potential, particularly in an era of increasingpatent expirations. A range of analytical methods is required todetermine the purity, identity and integrity of protein biologics atmultiple points along the manufacturing process, from cell culture todownstream purification, product characterization and lot release.Characterization of glycans from antibodies is fundamentally importantin biotherapeutics design and disease progression and detection. Theability to characterize glycans rapidly has been limited by the samplepreparation steps and structural complexity of the glycoproteins.To address this problem, we have developed a microfluidic chipthat performs rapid on-line cleavage of glycans from monoclonalantibodies, captures the released glycans and then separates themprior to nanospray ionization in the mass spectrometer. The entire runtime is 12 minutes. A glycan accurate mass database was establishedallowing quick assignment and identification of the glycans.250 Protein Cleavage, Disulfide BondsReduction, Metabolite Synthesis andMuch More Using Electrochemistry/MSJ. Powers 1 , J. Purkerson 1 , A. Kraj 2 , M. Eysberg 2 ,J.P. Chervet 21Antec, Palm Bay, FL, United States; 2 Antec,Zoeterwoude, The NetherlandsRecently, the scope of Electrochemistry (EC) upfront MS has beenextended from mimicking drug metabolism towards new applicationssuch as: protein/peptide cleavage, disulfide bonds reduction, covalentdrug-protein binding, etc. In this presentation we will show theapplication of on-line EC/MS as a powerful tool to simulate variousoxidation and reduction processes in life sciences. A specially designedµ-preparative electrochemical flow cell will be presented. The cellallows the synthesis of sufficient amounts of metabolites in a few minutesfor subsequent use as reference material (e.g. NMR or MS). Newscanning method was applied for oxidation of the highly concentratedsamples (mM range) to achieve high yield in the metabolites formation.Stable oxidation conditions were obtained without the need of anycell maintenance for a prolonged period of time. Electrochemistryup front MS can be applied for protein and peptide cleavage (as apromising new approach to enzymatic digestion). Electrochemicalcleavage of proteins and peptides occurs very specifically at C-terminalof the Tyrosine and Tryptophan peptide bonds. Examples of oxidativecleavage will be presented. Disulfide bonds are one of the mostimportant post-translational modifications for proteins. In this posterwe present the structural analysis of biologically active peptidesand proteins containing disulfide bonds (e.g., somatostatin, insulin,etc) using electrochemistry (EC) combined with mass spectrometry.Therefore the sample undergoes electrolytic disulfide cleavage in theelectrochemical flow cell followed by online MS analysis. Based on theintact protein mass and the resulting fragments and the MS/MS dataunambiguous assignment of the disulfide bonds becomes possible.All these applications illustrate the tremendous power and broadapplicability of electrochemistry as a tool to mimic nature’s Redoxreactions within a few seconds or minutes.251 Protein Sequencing Research Group(PSRG): Results of the PSRG 2011Study: Sensitivity Assessment ofEdman and Mass SpectrometricTerminal Sequencing of an UnknownProteinH. Remmer 1 , J.S. Smith 2 , W. Sandoval 3 , B. Xiang 4 ,K. Mawuenyega 5 , D. Suckau 6 , V. Katta 3 ,J.J. Walters 7 , P. Hunziker 81University of Michigan, Ann Arbor, MI, UnitedStates; 2 University of Texas Medical Branch,Galveston, TX, United States, 3 Genentech,Inc., South San Francisco, CA, United States,4Monsanto Company, St. Louis, MO, United States,5Washington University School of Medicine,St. Louis, MO, United States, 6 Bruker Daltonics,Bremen, Germany, 7 Sigma-Aldrich, St. Louis,MO, United States, 8 University of Zurich, Zurich,SwitzerlandEstablishing the N-terminal sequence of intact proteins plays a criticalrole in biochemistry and potential drug development. N-terminalsequence analysis is necessary for quality control of protein biologics,for determining sites of signal peptide cleavage events, as a first stepin elucidating the sequences of genes from uncommon species andfor the characterization of monoclonal antibodies. Automated Edmandegradation has been the method of choice for these analyses. However,alternate methods for N-terminal sequence analysis have emerged. Therecent PSRG studies have established that Edman sequencing and massspectrometry based techniques have varied strengths and weaknessesdepending on several experimental factors and both play an importantrole in terminal sequencing. With this complimentary role realized,the 2011 PSRG study attempts to evaluate the sensitivity limits of thevarious sequencing techniques. The PSRG distributed three samplesets of 3 tubes each, varying by sample format (lyophilized, gel sliceor membrane piece). Each set of three samples contains the samerecombinant protein with increasing amounts of material. The sequenceof this protein is not listed in any database. Participants could requestany one, two, or all three sample sets. Including PSRG committee, a totalof 38 participants requested 74 sample sets. The participants wereasked to determine as many amino acids from both termini by theirmethod of choice, and were encouraged to try multiple methods forsequence elucidation. Study participants were directed to a websiteto anonymously upload sequences and supporting data. Our analysisfocuses on the length and accuracy of the sequence calls reported bythe participants, and how that compares with decreasing amounts ofprotein and the type of sample format analyzed. A comparison of theresults obtained by Edman chemistry and by alternative technologiesas well as information on the type of instruments and protocols isreported.Poster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 103

Author IndexPlease note that only primary authors of poster presentations are listed in this index.AGKAuthor IndexAdams, P. - 125Ahmed, A. - 103Albanese, J. - 207, 216Asperger, A. - 235BBarsch, A. - 222, 223Berg, A. - 215Biringer, R. - 240Bishop, J. - 131Bodi, K. - 156Boland, J. - 146Bolcato, C. - 233Bramlett, K. - 163CCampos, A. - 237Chamberlain, S. - 123Chen, E. - 245Chen, S.M. - 217Chuu, J. - 149Curchoe, C. - 128DDiaz, C. - 178Diaz Acosta, B. - 112Diaz-Candelas, P. - 142Donnelly, R. - 126Duhr, S. - 182, 201EEckart, S.P. - 179FFadgen, K. - 227Fleming, B. - 106Freeman, R. - 171, 208Gadush, M. - 173Ganter, B. - 119Garge, N.R. - 195Gendeh, G. - 183, 184, 191, 198Giuffre, A. - 129Grigorean, G. - 239Grills, G. - 105, 113, 121Gross, V. - 219Grove, D. - 157HHagen, A. - 102Hamilton, J. - 107Hawke, D.H. - 189Hebert, N. - 210Hedlund, H. - 247Hemström, T. - 196Hendrickson, W. - 115Heuermann, K. - 130Hicks, B. - 104Hicks, L. - 177Hinerfeld, D. - 144Holbrook, J. - 122, 165Hughes, C. - 218JJaggi, R. - 164Jen, J. - 161Jennings, S.F. - 114Johnson, D. - 209Jonscher, K. - 236Joshi, S. - 147Jovanuvich, S. - 162Kain, S. - 136Kamberov, E. - 145Khanna, A. - 150Kinross, C. - 152Koller, A. - 232Küster-Schöck, E. - 176LLangmore, J. - 188Lea, K. - 168Liao, A. - 118Lin, S. - 108Liu, C.S. - 134Liu, J. - 135Lopez, P. - 175Lübbert, C. - 214Luna, L. - 200MMa, B. - 194Magpiong, I. - 193Marshall-Waggett, C. - 212Martinez-Torrecuadrada, J.L. - 248Masri, F. - 228McCary, A. - 141McGuigan, J. - 133McKenna, T. - 242McMillan, M. - 124Miller, C. - 221, 243, 249Müller, B. - 238Murage, E. - 172Murray, J. - 185Myers, J. - 153NNarayanan, A. - 197Needleman, D. - 127104 • ABRF 2011 — Technologies to Enable Personalized Medicine

OOng, J. - 117PPatel, S. - 169Peake, D. - 224Person, M. - 109Powers, J. - 160, 225, 250QQu, L. - 170RRavi, H. - 151Remmer, H. - 251Resemann, A. - 192, 204, 230Rezenom, Y. - 220Rosato, C. - 101Ruddat, V. - 202SSalowsky, R. - 229San Jose Hinahon, C. - 132Schweiger-Hufnagel, U. - 203Shan, P. - 190Smith, T. - 139Snyder-Leiby, T. - 205Soderblom, E. - 244Sondej, M. - 181Stapels, M. - 234Stewart, F.J. - 155Supunpong Hernandez, N. - 158TThai, K. - 148Thompson, J.W. - 199Turner, M. - 137Turpen, P. - 110VVacek, G. - 140Valaskovic, G. - 241van Soest, R. - 211Vincent, E. - 116Vogel, C. - 143WWang, X. - 226Whitley, P. - 138Williams, R. - 174Winkvist, M. - 186, 187Wojtkiewicz, M. - 180XXiang, F. - 206YYang, C. - 111Young, T. - 246ZZavadil, J. - 120Zianni, M. - 154, 159Author IndexABRF 2011 — Technologies to Enable Personalized Medicine • 105

ABRF 2011 Exhibits*All ABRF 2011 Meeting attendees are invited to visit the ExhibitHall featuring the leading experts in life sciences research andbiotechnology.Exhibit HoursSunday through TuesdayLevel 2, Grand Oaks BallroomSunday, February 20 ....................................10:00 am – 6:30 pmMonday, February 21 ..................................10:00 am – 6:30 pmTuesday, February 22 ..................................10:00 am – 2:00 pmThe purpose of the exhibition is to further the education ofthe scientists working in resource and research biotechnologylaboratories. The exhibitors will showcase instruments, productsor services for use in teaching, research, books or otherpublications in scientific fields of relevance.*Participation in the Exhibits Program, including attendance ofExhibitor Presentations, does not constitute an endorsement bythe Association of Biomolecular Resource Facilities (ABRF) of theclaims, products, or services offered.Exhibitor Directory in Alphabetical OrderExhibitors confirmed as of January 29, 2011.Exhibitor DirectoryAB SCIEX Booth 501110 Marsh DriveFoster City, CA 94404United StatesP +1 508-383-7640F +1 650-627-2601www.absciex.comAB SCIEX is a global leader in the development of life scienceanalytical technologies that help answer complex scientific challenges.The company provides mass spectrometry instrumentation,software and services for scientific analysis, including coreand applied research.ABRF Booth 3179650 Rockville PikeBethesda, MD 20814United StatesP +1 301-634-7306F +1 301-634-7455abrf@abrf.orgwww.abrf.orgThe Association of Biomolecular Resource Facilities (ABRF) is aunique membership association comprised of approximately800 scientists working in resource and research biotechnologylaboratories. Our members represent over 140 internationalcore laboratories in government, academia, research, industryand commercial settings. ABRF promotes the education andcareer advancement of scientists through conferences, a quarterlyjournal, publication of ABRF Research Group studies, andconference travel awards. The ABRF Research Group studies aresponsored annually to help researchers assess and incorporatenew biotechnologies into their laboratories, and are world-renownedfor their practical and educational benefit.Advanced Analytical Booth 6022711 South Loop DriveAmes, IA 50010United StatesP +1 515-296-6600F +1 515-294-7141webinfo@aati-us.comwww.aati-us.comAdvanced Analytical is a leading innovator of analytical instrumentsthat improve laboratory throughput. The AdvanCE TMFS96 and AdvanCE TM FS12 are 96 and 12-channel parallel capillaryelectrophoresis fluorescent instruments for analyzing dsD-NA fragments and RNA. A specialized reagent kit is available forautomated, sensitive and rapid analysis of Next-Gen Sequencingfragments.Agilent Technologies Booth 3012850 Centerville RoadWilmington, DE 19808United StatesP +1 800-227-9770F +1 302-633-8944agilent_inquiries@agilent.comwww.agilent.com/chemAgilent is a leading supplier of life science instrumentation. LC,GC & mass specs, DNA microarrays and scanners, thermal cyclers,automation instruments, reagents and software productsare used globally. Agilent has developed products and servicesutilized along the entire discovery value chain, from basic biologicalresearch through drug discovery and manufacturing.106 • ABRF 2011 — Technologies to Enable Personalized Medicine

AnaSpec, Eurogentec Group Booth 41734801 Campus DriveFremont, CA 94555United StatesP +1 510-791-9560F +1 510-791-9573admin@anaspec.comwww.anaspec.comAs a subsidiary of Eurogentec, AnaSpec offers expertise inpeptides, detection reagents, antibodies, assay kits, oligonucleotides,and qPCRs. AnaSpec carries a broad product line ofbiochemicals and reagents for basic research, high-throughputscreening and drug discovery.Bioinformatics SolutionsInc. (BSI) Booth 402470 Weber Street North, Suite 204Waterloo, Ontario N2L6J2CanadaP +1 519-885-8288F +1 519-885-9075info@bioinfor.comwww.bioinfor.comBSI produces life science software to serve the needs of pharmaceutical,biotechnological and academic scientists; and tothe progression of drug discovery research. Software solutionsinclude: ZOOM for next-generation sequencing, PEAKS forproteomic mass spectrometry, RAPTOR for protein structureprediction and PatternHunter for general purpose homologysearching.Bioo Scientific Booth 7183913 Todd Lane, Suite 312Austin, TX 78744United StatesP +1 888-208-2246nextgen@biooscientific.comwww.biooscientific.comBioo Scientific provides innovative, custom solutions to corefacilities. These include DNA and RNA AIR TM Next-GenerationSequencing library preparation kits, which offer increased sensitivity,flexibility and speed. We also offer the ExoMir TM kit forthe isolation of RNA from exosomes and MaxDiscovery TM kits fortoxicity and cytotoxicity analysis.Bio-Synthesis, Inc. Booth 104612 East Main StreetLewisville, TX 75057United StatesP +1 972-420-8505F +1 972-420-0442biosyn@biosyn.comwww.biosyn.comBio-Synthesis provides synthetic peptides including libraries andarrays, Antibodies, PNA, Bioconjugates, Custom DNA synthesisand its analogs, Gene synthesis, PCR based detection kits, Cellline authentication, HLA typing and Analytical services. Withover two decades of experience producing custom productsfor both research and pharmaceutical use, BSI has developed areputation for both small and large scale synthesis using optimalprocesses that meet your specifications.Bruker Daltonics Inc. Booth 21440 Manning RoadBillerica, MA 01821United StatesP +1 978-663-3660F +1 978-667-5993www.bdal.comBruker is a leading provider of Separation and Mass Spectrometryinstruments for the Analytical Sciences. Our innovative andeasy to use product families (ESI-TOF, Ion Trap, FTMS, MALDI-TOF, GC, GCMS, ICP-MS) provide the highest performance,highest value systems for a wide range of small molecule andprotein analysis applications.Caliper Life Sciences Booth 30468 Elm StreetHopkinton, MA 01748United StatesP +1 508-435-9500www.caliperLS.comCaliper Life Sciences is a premier provider of cutting-edge technologiesenabling researchers in the life sciences industry tocreate life-saving and enhancing medicines and diagnostic testsmore quickly and efficiently. Caliper is aggressively innovatingnew technology to bridge the gap between in vitro assays andin vivo results and then translating those results into cures forhuman disease. Caliper’s portfolio of offerings includes state-ofthe-artmicrofluidics, lab automation & liquid handling, opticalimaging technologies, and discovery & development outsourcingsolutions.Exhibitor DirectoryABRF 2011 — Technologies to Enable Personalized Medicine • 107

Exhibitor Directory — ContinuedExhibitor DirectoryCEM Corporation Booth 303P.O. Box 200, 3100 Smith Farm RoadMatthews, NC 28106United StatesP +1 704-821-7015F +1 704-821-8710info@cem.comwww.cem.comMake higher purity peptides faster with the Liberty MicrowavePeptide Synthesizer. This system has the fastest cycle times availablefor routine and difficult peptides, including peptides youcan’t access under conventional conditions. The Discover® Systemprovides improved sequence coverage and higher efficienciesfor your enzymatic digests in only 15 minutes.chemagen USA Booth 10067 Millbrook StreetSuite 522Worcester, MA 01606United StatesP +1 508-459-7727F +1 508-459-7548www.chemagen.comchemagic automation and reagents perform fast and reliablemagnetic bead-based DNA and RNA extractions from samplevolumes from 10 ul to 10 ml of blood, tissue, bacteria, food, PCRand sequencing products, etc. Advantages of this unique systemare fast processing, unmatched sample volume range and robustchemistry.Dionex Corporation Booth 2151228 Titan Way/P.O. Box 3603Sunnyvale, CA 94088-3603United StatesP +1 408-737-0700F +1 408-730-9403angie.jinks@dionex.comwww.dionex.comDionex has developed a large portfolio of powerful instrumentsand chemistries for separations of biomolecules, such asproteins, peptides, monoclonal antibodies, nucleic acids, andcarbohydrates that serve in diverse applications ranging frombiomarker discovery to drug R&D and QA/QC. Visit us at booth215 to learn more about ground-breaking life science solutionsfrom Dionex.DNAnexus Booth 404420 Florence StreetSuite 210Palo Alto, CA 94301United StatesP +1 650-204-1938info@dnanexus.comwww.DNAnexus.comDNAnexus provides solutions for both DNA sequencing centersgrowing their next-gen capacity, and the researchers workingwith next-gen sequence data. Our web-based platform solvesthe data management and analysis challenges common to bothwith a single, unified system. We support sequencing operationsand research organizations of any size, with absolutely no upfronthardware investment needed.eagle-i Consortium Booth 221One Kendall Square, Suite B6303Cambridge, MA 02139United StatesP+1 617-384-8787info@eagle-i.orgwww.eagle-i.orgThe eagle-i Consortium, made up of nine member institutionsacross the United States, is building a prototype of a nationalresearch resource discovery network. The eagle-i Network willconnect biomedical researchers with valuable resources, services,and technologies in a faster and more cost-effective way thanpreviously possible.EdgeBio Booth 300201 Perry ParkwaySuite 5Gaithersburg, MD 20877United StatesP +1 800-326-2685F +1 301-990-0881info@edgebio.comwww.edgebio.comEdgeBio offers next-generation sequencing services. From experimentaldesign to bioinformatics, EdgeBio is the perfect solutionfor all your sequencing needs. EdgeBio also sells kits forsequencing reaction cleanup, PCR purification, and plasmid purification,as well as competent cells.108 • ABRF 2011 — Technologies to Enable Personalized Medicine

FASEB MARC Program Booth 4169650 Rockville PikeBethesda, MD 20814United StatesP +1 301-634-7020F +1 301-634-7353marc@faseb.orgwww.faseb.org/marcFASEB MARC Program provides a variety of activities to supportthe training of underrepresented minority students, postdoctorates,faculty and scientists in the biomedical sciences. We offertravel awards for scientific meetings, research conferences, andstudent summer research opportunity programs. We also sponsorCareer Development Programs including grantsmanship andleadership training seminars.Fluidigm Corporation Booth 6147000 Shoreline Court, Suite 100South San Francisco, CA 94080United StatesP +1 866-358-4354F +1 650-871-7152www.fluidigm.comFluidigm develops, manufactures and markets life-science systemsbased on integrated fluidic circuits (IFCs). This technologyfurthers research by minimizing costs and enhancing sensitivityfor applications such as single-cell gene expression profiling,high-throughput SNP genotyping, and next-generation sequencing.Fluidigm products are used for research only.GE Healthcare Booth 714800 Centennial AvenuePiscataway, NJ 08854United StatesP +1 800-526-3593F +1 877-295-8102cs-us@ge.comwww.gelifesciences.comGE Healthcare Life Sciences provides tools for drug discovery,biopharmaceutical manufacturing and cellular technologies, soresearch scientists and specialists around the world can be moreproductive, effective and creative. Our vision is to be the startto-finishbioprocessing solution provider, the partner of choicein cell and protein research, and the leader in life sciences services.Gene Codes Corporation Booth 220775 Technology DriveSuite 100AAnn Arbor, MI 48108United StatesP +1 734-769-7249F +1 734-769-7074gcinfo@genecodes.comwww.genecodes.comGene Codes is an international software firm. Our DNA sequenceanalysis software, Sequencher TM is the industry leader.Customer-driven product development, combined with the latestprogramming techniques, and rigorous quality control to developproducts of the utmost usability and relevance has beenthe key to our success.Genetix Booth 616120 Baytech DriveSan Jose, CA 95134United StatesP +1 408-719-6400F +1 408-719-6401bernette.jones@genetix.comwww.genetix.comGenetix provides unrivalled solutions for imaging and imageanalysis. The company’s imaging platforms offer solutions topick microbial colonies in genomic studies, to screen and selectmammalian cell lines, monitor cell growth, evaluate cellular responseand quantify tissue biomarkers.GENEWIZ, Inc. Booth 316115 Corporate BoulevardSouth Plainfield, NJ 07080United StatesP +1 877-436-3949www.genewiz.comGENEWIZ, Inc. provides award-winning DNA sequencing, molecularbiology, and genomic services. We build codon-optimizedgenes through our powerful Gene Synthesis technologies,and provide high-quality plasmids from mini-to-giga scales usingour Plasmid Preparation services. With over10 years experiencein DNA sequencing, we are well-equipped to handle sequencingprojects of all complexities.Exhibitor DirectoryABRF 2011 — Technologies to Enable Personalized Medicine • 109

Exhibitor Directory — ContinuedGenomatix Software, Inc. Booth 2183025 Boardwalk, Suite 160Ann Arbor, MI 48108United StatesP +1 877-436-6628F +1 734-622-0477grant@genomatix-software.comwww.genomatix-software.comWith a focus on translating NGS data from sequence files tomeaningful biological results, Genomatix leverages it’s 12+ yearhistory of software and data base development to bring youa state-of-the-art, comprehensive, integrated platform for theanalysis of your NGS data.Geospiza Booth 519100 West Harrison, North Tower, Suite 330Seattle, WA 98119United StatesP +1 206-633-4403F +1 206-633-4415info@geospiza.comwww.geospiza.comDesigned by biologists for biologists, Geospiza is the marketleading developer of GeneSifter® software, providing integratedsolutions for Next Generation Sequencing analysis and LIMS.For over 10 years, Geopsiza has set the industry standard forhigh value, low cost, “out of the box” solutions serving the lifesciences market.Hamilton Robotics Booth 2234970 Energy WayReno, NV 89502United StatesP +1 800-548-5950F +1 775-858-3024roboticsales@hamiltoncompany.comwww.hamiltonrobotics.comHamilton Robotics is dedicated to the design and manufactureof automated liquid handling workstations. Key to our productsis our air displacement pipetting and monitoring technologyand software controlling our systems. Our workstations andsoftware serve as a common high precision and flexible baseupon which to provide automated solutions.iLab Solutions, LLC Booth 700124 Lexington AvenueCambridge, MA 02138United StatesP +1 617-297-2805F +1 877-812-6477heather.evans@ilabsolutions.comwww.ilabsolutions.comLab Solutions provides core facility management software tostreamline requests, track usage, and automate billing. The webbasedsolution supports the entire shared service work flow,from scheduling and request approvals through communication,billing and reporting. Integration available with financial systemsuch as SAP, PeopleSoft, Banner, and Lawson.Illumina Booth 5159885 Towne Centre DriveSan Diego, CA 92121United StatesP +1 858-202-4500F +1 858-202-4766info@illumina.comwww.illumina.comIllumina enables discovery by providing comprehensive DNAand RNA analysis solutions, including genotyping and copynumber analysis, gene expression, sequencing, and epigenetics:the cornerstones of genetic research. Our portfolio of tools integratesour sequencing and microarray technologies, as well aspowerful assay protocols to make possible a new scale of biologicalresearch.Intavis Booth 600945 West George StreetSuite 201Chicago, IL 60657United StatesP +1 773-572-5799www.intavis.comIntavis is an industry leader in manufacturing robotic instrumentsfor in situ hybridization, immunohistochemistry, peptidesynthesis, protein digestion, and MALDI spotting.Exhibitor Directory110 • ABRF 2011 — Technologies to Enable Personalized Medicine

IntegenX, Inc. Booth 2015720 Stoneridge DriveSuite 300Pleasanton, CA 94588United StatesP +1 925-701-3400F +1 925-754-7373info@integenx.comwww.integenx.comIntegenX, Inc. produces automated sample processing systemsfor life sciences and applied sciences markets. The company’sproducts: The new Apollo 324 automates Next-Gen librarypreparation and the Apollo 100 automates sample processingfor Sanger cycle sequencing. Our products dramatically reduceprocess time and reagent expenses.Integrated DNA Technologies Booth 3051710 Commercial ParkCoralville, IA 52241United StatesP +1 800-328-2661custcare@idtdna.comwww.idtdna.comServing the areas of academic research, biotechnology, clinicaldiagnostics and pharmaceutical development, IDT is the largestmanufacturer of custom oligo products in the United States.From oligos in plates to gene knockdown products, from fluorescentDNA probes to gene synthesis, IDT offers the highestquality, customer service and turnaround time.Kapa Biosystems, Inc. Booth 619600 West Cummings ParkSuite 2250Woburn, MA 01801United StatesP +1 781-497-2933F +1 781-497-2934sales@kapabiosystems.comwww.kapabiosystems.comKapa Biosystems, Inc. offers a portfolio of best-in-class PCR reagentsengineered using our high-throughput molecular evolutionplatform. The novel DNA polymerases contained within ourkits confer dramatic improvements to performance that enableresults and applications not possible wtih wild-type enzymes.Kapa has engineered high performance reagents for real-timePCR, high fidelity PCR, next-generation DNA sequencing, fastPCR, crude sample PCR, robust PCR, and molecular diagnostics.LC Sciences Booth 3182575 West Bellfort Street, #270Houston, TX 77054United StatesP +1 713-664-7087F +1 713-664-8181info@lcsciences.comwww.lcsciences.comLC Sciences is a genomics and proteomics company offeringinnovative and quality products and services. We provideunique and customizable oligonucleotide and peptide microarrayproducts designed for nucleic acid and protein-profiling,biomarker-screening, drug screening, and development of diagnostic-devices.Leica Microsystems Booth 2172345 Waukegan RoadBannockburn, IL 60015United StatesP +1 800-248-0123F +1 847-236-3009info@leica-microsystems.comwww.leica-microsystems.comLeica Microsystems offers the latest imaging technology. Withthe high sensitivity Leica TCS SP5, you can buy a basic confocalmicroscope today that is easily upgraded to keep pace withyour science. Other innovations: the Leica SCN400 slide scannerwith dynamic focus tracking for fast scanning while revealingmore details; and the Leica LMD6500 laser microdissectionsystem that uses gravity to collect specimens in a contact-freemanner.Life Technologies Booth 6015791 Van Allen WayCarlsbad, California 92008United StatesP +1 760-603-7200rose.mori@lifetech.comwww.lifetechnologies.comLife Technologies is a global biotechnology tools companydedicated to improving the human condition. Our customersdo their work across the biological spectrum, working to advancepersonalized medicine, regenerative science, moleculardiagnostics, agricultural and environmental research, and 21stcentury forensics.Exhibitor DirectoryABRF 2011 — Technologies to Enable Personalized Medicine • 111

Exhibitor Directory — ContinuedExhibitor DirectoryMassTech, Inc. Booth 1036992 Columbia Gateway Drive, #160Columbia, MD 21046United StatesP +1 443-539-1758F +1 443-539-1759www.apmaldi.comMassTech, Inc. manufactures AP/MALDI ion sources and conductson-going research and development in mass spectrometryrelated technology. MassTech’s AP/MALDI ion source is compatiblewith mass spectrometers from manufacturers such as: Agilent,Applied Biosystems/MDS SCIEX, ThermoFinnigan, Waters/Micromass, Bruker and JEOL, and is available for OEMs and directsales around the world.Matrix Science Booth 70464 Baker StreetLondon, W1U 7GBUnited KingdomP +1 800-716-6702F +1 800-716-6704info@matrixscience.comwww.matrixscience.comTake the guesswork out of protein identification with MascotServer. Get closer to your raw data with Mascot Distiller, givingdirect access to all popular file formats for peak picking, de novosequencing, quantitation, and more. Bring it all together withMascot Integra, a relational database system for proteomics.Michrom Bioresources Booth 6151945 Industrial DriveAuburn, California 95603United StatesP +1 530-888-6498F +1 530-888-8295michrommkt@michrom.comwww.michrom.comMichrom Bioresources is a premier supplier of LCMS instrumentation,accessories, and consumables for life science researchersaround the world. Michrom’s new Advance splitless nano-capillaryUHPLC and revolutionary “Plug and Play” CaptiveSprayionization source interface to most MS instruments, providingoptimum LCMS performance for qualitative and quantitativeproteomics applications.Morehouse School ofMedicine/RCMI Program Booth 204720 Westview Drive, SWAtlanta, GA 30310United StatesP +1 404-752-1500www.msm.eduMorehouse School of Medicine (MSM) is a private, historicallyblack institution established in 1975. Major support for the biomedicalresearch infrastructure at MSM is received through theNIH/NCRR funded RCMI Program. The MSM/RCMI supportedcore facilities are: Cell/Tissue Imaging, Gene Profiling, AnalyticalChemistry/Protein Profiling and Biomedical Technology ServiceLaboratories.New England Biolabs, Inc. Booth 205240 County RoadIpswich, MA 01938United StatesP+1 800-632-5227info@neb.comwww.neb.comNew England Biolabs, Inc. leads the industry in the discoveryand production of enzymes for molecular biology applicationsincluding sample preparation for next-generation sequencing.NEB’s global reputation for manufacturing products of the highestquality coupled with best in class technical support makesNEB a first choice for customers demanding optimized reagentsfor advanced technologies.New Objective Booth 2032 Constitution WayWoburn, MA 01801United StatesP +1 781-933-9560F +1 781-933-9564sales@newobjective.comwww.newobjective.comNew Objective brings state-of-the-art systems to nano- ESI-MSand capillary LC/MS. In 2011, New Objective introduces the revolutionaryand rugged PicoFrit Chip. This integrated column/high-voltage system provides high-throughput sample analysiswithout compromising sensitivity or flexibility. Our industryprovenPicoView® sources and PicoFrit columns continue to providehigh-performance and sensitivity in LC-MS.112 • ABRF 2011 — Technologies to Enable Personalized Medicine

Nonlinear Dynamics Booth 1052530 Meridian Parkway, 3rd FloorDurham, NC 27713United StatesP +1 866-GELS-USAinfo@nonlinear.comwww.nonlinear.comNonlinear Dynamics’ range comprises analysis software for label-freeLC-MS, 2D electrophoresis gels, and MALDI data as wellas tools for multivariate statistical analysis. Our unique analysisapproach has transformed proteomics research — it providesfaster more accurate quantitation and has enabled cross-lab reproducibility.Omega Bio-Tek, Inc. Booth 6051850-E Beaver Ridge CircleNorcross, GA 30071United StatesP +1 770-931-8400info@omegabiotek.comwww.omegabiotek.comOmega Bio-Tek manufactures a complete line of DNA|RNA isolationkits utilizing magnetic beads and silica filter technology forboth high throughput facilities and individual labs. Our novel nospin, high-throughput magnetic bead based plasmid DNA purification,PCR cleanup and DTR provides an affordability andselection that is unmatched.OpGen, Inc. Booth 603708 Quince Orchard Road, #160Gaithersburg, MD 20878United StatesP +1 301-869-9683www.opgen.comOpGen, Inc. provides the Argus Optical Mapping System forgenerating high-resolution, ordered, whole-genome restrictionmaps from single DNA molecules. Independent of sequenceinformation, Optical Maps enable a comprehensive view of genomicarchitecture. Use Optical Maps for comparative genomics,strain typing and sequence assembly.OriGene Technologies, Inc. Booth 4009620 Medical Center DriveSuite 200Rockville, MD 20850United StatesP +1 888 267 4436F +1 301-340-8606techsupport@origene.comwww.origene.comOriGene Technologies, Inc., is a gene centric life sciences companydedicated to support academic, pharmaceutical and biotechcompanies in their research of gene functions and drugdiscovery. OriGene develops proteins, antibodies, and othermolecular tools to allow researchers to analyze their data ona multiplex level. OriGene’s novel product line includes theworld’s largest cDNA and shRNA clone collections, over 5,000purified human proteins, high quality monoclonal antibodies(TrueMAB TM ), 100,000 highly validated human tissues, and proteinmicroarray products and services. OriGene also provides abroad range of antibody validation products including genomewidetagged antigen standards and extensive IHC slides derivedfrom our tissue collection.Peak Scientific, Inc. Booth 10119 Sterling RoadBoston, MA 01862United StatesP +1 866-647-1649F +1 978-608-9503infona@peakscientific.comwww.peakscientific.comOver the years our dynamic company has stimulated Peak designengineers into some remarkable developments in the manufactureof Laboratory Gas Generators. Smoother, quieter andmore efficient than anything on the market, our Generators offersuperb technical performance in the lab and our world-class aftersales service comes as standard.Phenix Research Products Booth 51873 Ridgeway RoadCandler, NC 28715United StatesP +1 800-767-0665F +1 828-670-7020sales@phenixresearch.comwww.PhenixResearch.comPHENIX Research was founded in 1990 and is recognized as atechnical leader in providing outstanding laboratory productsat market beating prices. Our core competency and focus is toprovide value added solutions to life science researchers in University,Biotech and Government settings. We offer outstandingcustomer service 8:00 am - 5:30 pm EST and a nationwide directtechnical sales force.Exhibitor DirectoryABRF 2011 — Technologies to Enable Personalized Medicine • 113

Exhibitor Directory — ContinuedExhibitor DirectoryPhotometrics Booth 5163440 East Britannia DriveTucson, AZ 85706United StatesP +1 520-889-9933F +1 520-573-1944info@photometrics.comwww.photometrics.comDeveloper of the new Evolve 128, Photometrics is the world’spremier designer and manufacturer of high-performance CCDand EMCCD cameras for life science research. Photometrics fordecades has led the industry with state-of-the-art imaging instrumentationfor the scientific community. Researchers globallyrely on Photometrics imaging solutions to meet their demandingrequirements.PolyLC Inc. Booth 2229151 Rumsey Road,Suite 175Columbia, MD 21045United StatesP +1 410-992-5400F +1 410-730-8340info@polylc.comwww.polylc.comUnique HPLC columns for life sciences. Featuring ERLIC! 1) Selectiveisolation and separation of tryptic phosphopeptides andglycopeptides; 2) Fractionation of tryptic peptides by isoelectricpoint in volatile solvents. Products for proteomics: PolySULFOE-THYL A for 2-D SCX-RPC separations; mixed-bed ion-exchangeand other columns for predigest fractionation of intact proteins,including histones and membrane proteins, metabolomics, andQC of biotech protein variants.Protea Biosciences, Inc. Booth 503955 Hartman Run RoadMorgantown, WV 26507United StatesP +1 877-776-8321F +1 304-292-7101www.proteabio.comWe are a diversified biotechnology company that applies novelbioanalytical technologies to the development of new pharmaceuticals,products and services. Our products and services empowerResearchers with new capabilities in their own laboratoryto improve the speed, quality and reproducibility of proteinmass spectrometry data obtained from their biological samples.Proteome Software Booth 5141340 SW Bertha BoulevardSuite #10Portland, OR 97219United StatesP +1 800-944-6027F +1 928-244-6024jana.lee@proteomesoftware.comwww.proeomesoftware.comProteome Software produces industry standard products forMS/MS-based proteomics. Scaffold identifies biologically importantresults by comparing data from Mascot, SEQUEST, ProteomeDiscoverer, IdentityE, SpectrumMill, Phenyx, OMSSA andX!Tandem. Scaffold Q+ adds iTRAQ and TMT quantification.MassQC monitors and troubleshoots LC-MS performance. ScaffoldPTM determines sites of phosphorylation and other modifications.Randox Pharma Services Booth 30255 Diamond RoadCrumlin C. Antrim, BT29 4QYUnited KingdomP +44 (0) 2894-422 413F +44 (0) 2894-452 912marketing@randox.comwww.randoxpharmaservices.comRandox Pharma Services offers the most comprehensive rangeof biomarker solutions available to central labs, CRO’s and pharmaceuticalcompanies worldwide. Our award-winning, FDA approved,Biochip Array Technology is recognized as a ‘gold standard’in biomarker multiplexing for the analysis of proteins, genemutations, gene expression and SNP analysis.Research Scientific Services Booth 216P.O. Box 9188Gaithersburg, MD 20898United StatesP +1 301-977-9344F +1 301-977-9572jpeter@resci.orgwww.resci.comResearch Scientific Services (RSS) has provided professionalservice on Waters and AB Sciex mass spectrometers and sellingrefurbished LC/MS/MS instruments for 20 years. Service isprovided by a nationwide network of highly experienced formerOEM engineers. Customer attentiveness and responsive serviceat a competitive price is why laboratories trust RSS service.114 • ABRF 2011 — Technologies to Enable Personalized Medicine

Roche Applied Science Booth 5009115 Hague RoadIndianapolis, IN 46250United StatesP +1 800-262-1640www.roche-applied-science.comRoche Applied Science’s new GS Junior system uses 454’s ultrafastpyrosequencing technology to generate 400 to 500 bp sequencingread lengths for amplicon and transcriptome sequencingprojects. Ask about the newest version of our SequenceCapture sequence-enrichment technology, plus other new NimbleGenmicroarray products. Investigate real-time PCR with theLightCycler® 480 and LightCycler® 1536 Instruments, plus theaddition of rat and mouse targets to our preplated qPCR assays.Sage Science Booth 617500 Cummings CenterSuite 3150Beverly, MA 1915United StatesP +1 978-922-1832F +1 617-812-0540info@sagescience.comwww.sagescience.comSage Science manufactures the Pippin Prep TM DNA size selectionsystem. The Pippin Prep automates size selection of DNAfor next-gen sequencing library preparations, providing a laborsavingalternative to manual gel extraction and purification. Inaddition to time savings, one can achieve higher yields and eliminatelower molecular weight adapter contamination.SeqGen, Inc. Booth 6041725 Del Amo BoulevardTorrance, CA 90501United StatesP +1 877-377-3743F +1 800-790-4830info@seqgen.netwww.seqgen.netSeqGen is a full service provider of high quality, refurbished,DNA Sequencers, PCR Instrumentation, and Service Contracts.We maintain a large inventory of equipment/parts and havemany years of experience supporting Biotech laboratories. Ourexpert SeqGen Engineers conduct installations, warranty calls,service contracts, and paid day service calls.SeqWright, Inc. Booth 7022575 West Bellfort, Suite 2001Houston, TX 77054United StatesP +1 800-720-4363F +1 713-528-6232sales@seqwright.comwww.seqwright.comSeqWright, a CLIA certified and GLP compliant facility, providesFDA, Clinical, and Research-level Genomic services to the Pharmaceutical,Biotechnology and Academic communities. Specializingin Next Generation Genomics, Sequencing, ExpressionAnalysis and Molecular Biology services, SeqWright has earneda reputation for quality, technical expertise and a willingness toprovide client-specific solutions.Sigma Life Science Booth 5053050 Spruce StreetSt. Louis, MO 63103United StatesP +1 314-771-5765custserv@sial.comwww.wherebiobegins.comSIGMA® Life Science offers a wide portfolio of innovative technologies,products and services spanning cell biology, protein assays,genomics, functional genomics, biomolecules, epigenetics,stem cell research and transgenic animal models. Through continuedinvestment in innovation and quality we are the leadingdestination for life science researchers to access deep biologicalinformation and market leading products that improve the qualityof life.SoftGenetics Booth 110100 Oakwood AveSuite 350State College, PA 16803United StatesP +1 814-237-9340F +1 814-237-9343info@softgenetics.comwww.softgenetics.comFeaturing NextGENe® software for analysis of all massively parallelsequencer data with newly included RNA-Seq module; enhancedcapabilities of functionalities added to GeneMarker®Software include carbohydrate analysis, new large sizing algorithms,kinship analysis of wild populations, genotype mergetool and project comparison tool. SoftGenetics is providingdemonstrations and 30-day trials.Exhibitor DirectoryABRF 2011 — Technologies to Enable Personalized Medicine • 115

Exhibitor Directory — ContinuedTecan Booth 4194022 Stirrup Creek DriveSuite 310Durham, NC 27703United StatesP+1 919-361-5200info@tecan.comwww.tecan.comTecan is a leading global provider of laboratory instruments andsolutions. The company specializes in the development, productionand distribution of instruments and automated workflowsolutions for laboratories in the life sciences sector. Its clients includepharmaceutical and biotechnology companies, universityresearch departments, forensic and diagnostic laboratories.Thermo Scientific Booth 200355 River Oaks ParkwaySan Jose, CA 95134United StatesP +1 800-532-4752analyze@thermofisher.comwww.thermoscientific.comThermo Scientific brand solutions leverage the market-leadingsample preparation products and ion trap mass spectrometersfor definitive quantitation of protein mixtures, greatly improvingprotein characterization, accelerating protein database searchesand reducing false identification rates.Vanderbilt UniversityMedical Center Booth 3193401 West End BuildingSuite 300Nashville, TN 37203-8375United StatesP +1 615-875-3363F +1 615-936-6530coresinfo@vanderbilt.eduwww.mc.vanderbilt.edu/CORESVICI Valco Instruments Booth 102P.O. Box 55603Houston, TX 77255United StatesP +1 800-347-8424F +1 713-956-3119sales_usa@vici.comwww.vici.comVICI Valco Instruments is a designer/ manufacturer of standard/custom valves and fittings for precision analytical, biomedical,and biocompatible instrumentation. Product variety includes;pneumatic and electric actuators, tubing, sampling loops, heatedenclosures, valve sequence and temperature controllers, gaspurifiers, GC detectors, and digital interfaces. Devices and instrumentationfor generation of calibration gas standards, containmenttraps and gas specific purifiers.Waters Corporation Booth 10834 Maple StreetMilford, MA 01757United StatesP +1 508-478-2000F +1 508-872-1990info@waters.comwww.waters.comWaters helps laboratory-dependent organizations by providingbreakthrough technologies and solutions. Pioneering a connectedportfolio of separation and analytical science, laboratoryinformatics and mass spectrometry, Waters provides the tools toimprove the quality of today’s science and explore the infinitepossibilities of tomorrow. Waters, The Science of What’s Possible.Vanderbilt University Medical Center (VUMC) is a comprehensivehealthcare facility. Vanderbilt’s mission is to advance healthand wellness through preeminent programs in patient care, education,and research. CORES (Core Ordering & Reporting EnterpriseSystem) is a web-based system developed by VUMC usedto process core billing and produce usage reporting.Exhibitor Directory116 • ABRF 2011 — Technologies to Enable Personalized Medicine

Exhibit Hall FloorplanGrand Oaks Ballroom, Level 2Exhibit Hall FloorplanABRF 2011 — Technologies to Enable Personalized Medicine • 117

Exhibitor List in Alphabetical OrderExhibitors confirmed as of January 29, 2011.M = ABRF Annual Meeting SponsorC = ABRF Corporate SponsorCompanyBoothAB SCIEX .................................................................................................501ABRF .........................................................................................................317Advanced Analytical ...........................................................................602Agilent Technologies M C ............................................................301AnaSpec, Eurogentec Group M C ..............................................417Bioinformatics Solutions Inc. (BSI)...................................................402Bioo S cientific ........................................................................................718Bio-Synthesis, Inc. .................................................................................104Bruker Daltonics Inc. ............................................................................214Caliper Life Sciences C ....................................................................304CEM C orporation .................................................................................303chemagen U SA ......................................................................................100Dionex Corporation ............................................................................215DNAnexus ..............................................................................................404eagle-i Consortium ..............................................................................221EdgeBio C ............................................................................................300FASEB MARC Program ........................................................................416Fluidigm Corporation .........................................................................614GE H ealthcare ........................................................................................714Gene Codes Corporation ..................................................................220Genetix ....................................................................................................616GENEWIZ, I nc. .......................................................................................316Genomatix Software, Inc ....................................................................218Geospiza .................................................................................................519Hamilton Robotics ...............................................................................223iLab Solutions, LLC ...............................................................................700Illumina M C ......................................................................................515Intavis .......................................................................................................600IntegenX, Inc. C ..................................................................................201Integrated DNA Technologies .........................................................305Kapa Biosystems, Inc. ..........................................................................619LC S ciences .............................................................................................318CompanyBoothLeica M icrosystems ..............................................................................217Life Technologies .................................................................................601MassTech, In c. ........................................................................................103Matrix Science M ................................................................................704Michrom B ioresources ........................................................................615Morehouse School of Medicine/RCMI Program ........................204New England Biolabs, Inc. ..................................................................205New O bjective ......................................................................................203Nonlinear D ynamics ............................................................................105Omega Bio-Tek .....................................................................................605OpGen, In c. ............................................................................................603OriGene Technologies, Inc. ..............................................................400Peak Scientific, Inc. ...............................................................................101Phenix Research Products ..................................................................518Photometrics ..........................................................................................516PolyLC Inc. ..............................................................................................222Protea Biosciences, Inc. .......................................................................503Proteome S oftware ..............................................................................514Randox Pharma Services ....................................................................302Research Scientific Services...............................................................216Roche Applied Science M C ........................................................500Sage Science ..........................................................................................617SeqGen, In c. ...........................................................................................604SeqWright, In c. ......................................................................................702Sigma Life Science ................................................................................505SoftGenetics...........................................................................................110Tecan ........................................................................................................419Thermo Scientific .................................................................................200Vanderbilt University Medical Center ..........................................319VICI Valco Instruments .......................................................................102Waters Corporation M C ..............................................................108Exhibitor List – Alpha118 • ABRF 2011 — Technologies to Enable Personalized Medicine

Exhibitor List in Booth OrderExhibitors confirmed as of January 29, 2011.M = ABRF Annual Meeting SponsorC = ABRF Corporate SponsorBooth Company100 .................. chemagen U SA101 .................. Peak Scientific, Inc.102 .................. VICI Valco Instruments103 .................. MassTech, In c.104 .................. Bio-Synthesis, I nc.105 .................. Nonlinear D ynamics108 .................. Waters C orporation M C110 .................. SoftGenetics200 .................. Thermo S cientific201 .................. IntegenX, In c. C203 .................. New O bjective204 .................. Morehouse School of Medicine/RCMI Program205 .................. New England Biolabs, Inc.214 .................. Bruker Daltonics Inc.215 .................. Dionex C orporation216 .................. Research Scientific Services217 .................. Leica M icrosystems218 .................. Genomatix Software, Inc.220 .................. Gene Codes Corporation221 .................. eagle-i C onsortium222 .................. PolyLC Inc.223 .................. Hamilton R obotics300 .................. EdgeBio C301 .................. Agilent Technologies M C302 .................. Randox Pharma Services303 .................. CEM C orporation304 .................. Caliper Life Sciences C305 .................. Integrated DNA Technologies316 .................. GENEWIZ, I nc.317 .................. ABRF318 .................. LC S ciences319 .................. Vanderbilt University Medical CenterBooth Company400 .................. OriGene Technologies, Inc.402 .................. Bioinformatics Solutions Inc. (BSI)404 .................. DNAnexus416 .................. FASEB MARC Program417 .................. AnaSpec, Eurogentec Group M C419 .................. Tecan500 .................. Roche Applied Science M C501 .................. AB S CIEX503 .................. Protea Biosciences, Inc.505 .................. Sigma Life Science514 .................. Proteome S oftware515 .................. Illumina M C516 .................. Photometrics518 .................. Phenix Research Products519 .................. Geospiza600 .................. Intavis601 .................. Life Technologies602 .................. Advanced A nalytical603 .................. OpGen, In c.604 .................. SeqGen, In c.605 .................. Omega B io-Tek614 .................. Fluidigm C orporation615 .................. Michrom B ioresources616 .................. Genetix617 .................. Sage Science619 .................. Kapa Biosystems, Inc.700 .................. iLab Solutions, LLC702 .................. SeqWright, Inc.704 .................. Matrix S cience M714 .................. GE H ealthcare718 .................. Bioo ScientificExhibitor List – BoothABRF 2011 — Technologies to Enable Personalized Medicine • 119

Vendor PresentationsSunday, February 20 — 12:00 pm – 1:30 pmAlyssum Room, Level 3Exhibit Booth: 214Moving Beyond ProteomicsSpeaker: Detlev Suckau, Bruker Daltonik GmbHIn current proteomics studies as well as biopharmaceutical developmentand characterization, post-translational modificationsobtain increasing attention. Amongst these, glycosylation isprominent but the lack of bioinformatics solutions slowed downthe analysis of glycoproteins in both, the biopharmaceutical industryas well as in proteomics studies.In this seminar, we introduce new solutions that include a newglycoprotein and glycoproteomics enabling bioinformatics platformand show examples from selected glycoprotein characterizationsincluding glycan and direct glycopeptide analysis usingBrukers leading ESI-MSn and MALDI platforms.Dedicated solutions for the development and quality control ofBiologicals and Biosimilars will be described that show you howto rapidly characterize therapeutic proteins with the confidencethat is derived from the high specificity of Brukers ultra-highresolution QTOF maXis.As a further extension of classical protein characterization andvalidation workflows, we describe recent advances in Top-Downprotein characterization tools that will make your C- and N-terminaldefinitions specific and rapid. The ESI-ETD and MALDITop-Down Sequencing approaches will also forward your proteomicsresearch as they provide simultaneous access to PTManalysis, protein isoform differentiation and proteolytic regulationevents that are not addressed with current technologies.Grand Oaks Ballroom,Rooms N&O, Level 2Exhibit Booth: 71412:00 pm – 12:30 pmAdvances in Sample Preparation of LowAbundant Proteins using Magnetic BeadsSpeaker: Helena Hedlund, Global Product Manager, GEHealthcare Life SciencesLow abundant proteins are often biologically important butdifficult to detect. Some proteins are present in a few copieswhereas others have a short transient presence. To be able toincrease the detection and identification rate, efficient samplepreparation methods are necessary. We have combined establishedaffinity chromatography methods with magnetic beadtechnology for the enrichment of such challenging proteins. Inthe first study changes in tyrosine phoshorylation in cancer cellsupon drug treatment was investigated. Immmonoprecipitationusing Protein G Mag Sepharose TM was used for enrichment ofphosphorylated proteins prior to a 2-D DIGE experiment. Theeffect of the treatment will be presented as well as protein identificationfrom MS analysis.In the second study screening for optimal purification conditionsfor a number of integral membrane proteins was performed. Vitalparameters as choice of detergent wash and elution bufferconcentrations were evaluated. All experiments were done usingNi2+-charged His Mag Sepharose Ni. Results from screeningfor several detergents gave reliable results that could be fed intofurther scale-up experiments. The magnetic bead format simplifiedand shortened the handling procedures through-out thesetwo different workflows.Vendor Presentations12:30 pm – 1:00 pmQuantitative Western Blotting withAmersham TM ECL TM PrimeSpeaker: Maria Winkvist, Scientist, GE Healthcare LifeSciencesWestern blotting is a well established technique in life scienceresearch. Traditionally the technique has been restricted toqualitative protein analysis. However, development of refineddetection methods and reagents has opened up the possibilityto use the technique in quantitative analysis, for accurate monitoringof small changes in protein abundance and for detectionof posttranslational modifications.120 • ABRF 2011 — Technologies to Enable Personalized Medicine

This workshop will present ways to achieve quantitative Westernblotting by optimizing the imaging system and detection reagent.The impact of normalization, signal-to-noise, backgroundnoise and other experimental factors will also be discussed.1:00 pm – 1:30 pm2-D DIGE Analysis of Multicellular TumorSpheroids in Evaluation of Breast CancerTreatmentSpeaker: Viola Ruddat, Ph.D., Senior Applications Scientist,GE Healthcare Life SciencesMany cancers can be diagnosed using Positron Emission Tomography(PET) to visualize tumors. PET can also be used to monitorhow effective various treatments are in individual patientsby observing the decrease in tumor tissue. Tumor spheroids arecancer cells grown on agar coated dishes forming a 3D structure.They are widely used in preclinical cancer research, where themulticellular tumor spheroid model is considered biologicallyand physiologically similar to in vivo grown tumors.Grand Oaks Ballroom,Rooms P&Q, Level 2Exhibit Booth: 515Illumina’s New 2011 Portfolio andAnnouncementsIllumina’s broad portfolio of sequencing and array solutionsempowers scientists in labs of all sizes to take their researchfurther, faster. Please join us to hear the details about the recentannouncements of the terabase sequencing milestone, ourability to enable our customers to reach 600 Gb/run this spring,and our newest Platform, MiSeq.We’ll also describe our progressive research path for GWAS,our sequencing service without compromise and Eco, our ultraaccessibleqPCR system.In this workshop, we will present how we have used Two-dimensionalDifference Gel Electrophoresis (2-D DIGE) analysis to gainmore insight in changes of protein expression as a result of drugtreatment of multicellular tumor spheroids.Monday, February 21 — 12:00 pm – 1:30 pmGrand Oaks Ballroom,Rooms R&S, Level 2Exhibit Booth: 616Advanced Technologies for AntibodyDiscovery and Cell ScreeningSpeakers: Mark Truesdale, Gabe Longoria and AlisonGlaserThe use of antibodies in investigating and treating diseases isuniversal, however traditional methods for antibody developmentare time consuming and costly. The Genetix ClonePix TM FLsystem improves operational efficiency by reducing timelines,minimizing resource requirement and allows higher throughputof projects.Understanding disease mechanisms through the culture andstudy of relevant cell lines requires multiple technologies. Imagingsystems from Genetix are used to characterize cell growthand quantify the responses of cells to different treatments. Forrapid determination of cell growth in microwell plates, Clone-Select Imager TM provides objective, quantitative and consistentcharacterization of cell growth prior to investigation of cellularresponses to different treatments such as siRNA, small moleculeor antibody. To determine the responses of cells, CellReporterTM is used for fluorescent cell- and bead-based assays. Intuitiveworkflows provide flexibility to optimize and standardize assaysfrom image acquisition through to data analysis. Typical applicationsinclude cell-based assays for cell cycle analysis, apoptosisor monitoring protein translocation and bead-based homogeneousassays for quantifying cellular protein production.This seminar will present the latest technologies and applicationsfor screening cells, identification and isolation of clones,and data-rich cell based assays.Vendor PresentationsABRF 2011 — Technologies to Enable Personalized Medicine • 121

Vendor Presentations — ContinuedMonday, February 21 — 12:00 pm – 1:30 pm (Continued)Vendor PresentationsGrand Oaks Ballroom,Rooms N&O, Level 2Exhibit Booth: 601From Discovery to TranslationalGenomics with Life Technologies’Comprehensive Sequencing SolutionsLife Technologies offers the most comprehensive and complementaryportfolio of research sequencing solutions — fromthe market leading Capillary Electrophoresis solutions, throughthe recently launched Ion Torrent Personal Genome Machine,(PGM) to SOLiD TM systems — with the 5500 series SOLiD TM systemsbecoming available soon. The re-engineered 5500 seriesSOLiD TM system generates up to 30 gigabases of mapped dataper day and can be used in a myriad of research applications— including complete cancer genome re-sequencing, single celltranscriptome analysis, interrogation of methylation status, andde novo sequencing. The increasing throughput of the evolvingSOLiD TM platform and other next generation sequencingtechnologies now makes it possible to sequence a whole humangenome. As additional human genomes are sequenced, newbiological insights are obtained, and we begin to truly appreciatethe critical need for sequencing accuracy. Consensus accuracycan overcome some inherent error rates, however a flooris ultimately reached, thereby limiting identification of low frequencysomatic mutations or SNV’s when using low coveragetechniques for GWAS based projects. A new chemistry incorporatingerror-correction codes has been developed that enablesthe SOLiD TM system to achieve 99.99% accuracy within a singlesequencing read. This, and other technological improvements,will be discussed in the context of the emerging “TranslationalGenomics” era that we have recently entered.PostLight Sequencing with Semiconductor ChipsIon Torrent has invented the first device—a new semiconductorchip—capable of directly translating chemical signals intodigital information. The first application of this technology is sequencingDNA. The device leverages decades of semiconductortechnology advances, and in just a few years has brought theentire design, fabrication and supply chain infrastructure of thatindustry—a trillion dollar investment—to bear on the challengeof sequencing. The result is Ion semiconductor sequencing, thefirst commercial sequencing technologythat does not use light,and as a result delivers unprecedented speed, scalability andlow cost.Next Generation qPCR solutions from Life TechnologiesIn 2010, Life Technologies TM released the Applied Biosystems TMViia TM 7 Real-Time PCR System. This seventh generation systemcombines advances in optical and thermal cycling technologyto provide one the most powerful and flexible Real-Time PCRinstruments available on the market today. After a brief reviewof the Viia TM 7 Real-Time PCR System, we will explore the nextgeneration of qPCR systems.The OpenArray® Real-Time PCR platform employs 3072 wellreaction plates to increase throughput and to lower costs forqPCR. The throughput and cost efficiencies of the OpenArray®Real-Time PCR platform are enabling applications such as Next-Gen Sequencing library quantitation by digital PCR, gene expressionpathway analysis, high-throughput genotyping studies,and miRNA profiling. The OpenArray® Real-Time PCR platformdelivers TaqMan® sensitivity and specificity in miRNA profilingwhile achieving cost savings, comparable to microarray systems.The throughput for miRNA profiling is up to 9 samples completedin a 2.5 hour run, and up to 36 samples per day.The next generation of Life Technologies’ TM qPCR systems alsoincludes a novel technology platform based upon a continuousflow liquid-bridge manifold. This will enable high throughputGenotyping and Gene Expression applications. The Stokes Bionanodroplet qPCR technology will be reviewed during this talk.Alyssum Room, Level 3Exhibit Booth: 500Amplicon Sequencing with GS FLX andGS Junior SystemsAccess Array TM System High-Throughput ResequencingSpeaker: Steven Hoffman, Fluidigm CorporationFlexible Next-Generation Sequencing Approaches Enabledby the GS FLX and GS Junior SystemsSpeaker: Bruce Taillon, Ph.D., 454 Life SciencesFeaturing GS FLX Titanium series chemistry, the Genome SequencerFLX and GS Junior Systems offer a powerful combinationof long sequencing reads (400 to 500 base pairs) and dedicatedGS Amplicon Variant Analyzer (AVA) software, enablingthe sequencing, alignment, and analysis of amplicons against areference sequence.122 • ABRF 2011 — Technologies to Enable Personalized Medicine

Grand Oaks Ballroom,Rooms P&Q, Level 2Exhibit Booth: 505Controlling Experimental VariablesSpeaker: Jim Walters, Ph.D., Principal Investigator, AnalyticalResearch Department, Sigma Life ScienceThe enormous amount of data generated from complex proteomicanalyses with mass spectrometry is often overwhelming;requiring iterative analysis and optimization of a number of variables.To counteract the effects of potentially false data, a numberof defined protein and peptide standards have been developedto allow for universal performance comparisons acrossplatforms and between labs. We will highlight our efforts in thedevelopment, commercialization, and utilization of proteomicsstandards. We will discuss our newly developed standards whichinclude both phosphopeptide standards as well as a syntheticprotein designed to fully assess standard LC-MS platforms.Begonia Room, Level 3Exhibit Booth: 108Recent Developments in WatersProteomics TechnologySpeaker: Martha Stapels, Principle Research Scientist,WatersWe will be discussing advances in ETD, Ion Mobility andseparations. Also featured will be a presentation by J. WillThompson from Duke University: “Absolute Quantitation Using2DLC-MSE and Species-Specific Correction to InvestigateChlamydia trachomatis Developmental Forms.”Monday, February 21 — 7:30 pm – 9:00 pmAlyssum Room, Level 3Exhibit Booth: 500Reviews of SeqCap EZ Exome v2.0Vendor PresentationsABRF 2011 — Technologies to Enable Personalized Medicine • 123

Vendor Presentations — ContinuedTuesday, February 22 — 12:00 pm – 1:30 pmAlyssum Room, Level 3Exhibit Booth: 214The Latest Solutions for Metabolomicsand Small Molecule ResearchSpeaker: Aiko Barsch, Bruker Daltonik GmbHMetabolomics nowadays has several analytical challenges toovercome. The identification of metabolites is often defined asthe major bottleneck in metabolomics applications as well as ingeneral small molecule research.The confident generation of molecular formulae is the first stepin the identification of unknowns. Especially for m/z values above500, the number of ambiguous assignments increases exponentially.maXis TM Ultra high resolution Q-TOF accurate mass andisotopic pattern data from MS and MS/MS spectra significantlyextends the m/z range for reliable formula suggestions. The molecularformula can then be queried against public databasesto quickly match already known compounds. Hyphenating gaschromatography with soft atmospheric pressure ionisation(APCI) to high resolution Q-TOF-MS technology can preservethe molecular ion information which enables the identificationof unknowns in GC-MS data.One crucial advantage of applying maXis UHR-TOF in untargetedmetabolomic studies is the speed of analysis by couplingU-HPLC systems without compromising the performance interms of mass accuracy or resolution even at high acquisitionrates up to 20Hz. Overall, the sample throughput is increased,thus addressing the necessity of handling large sample numbersneeded for statistically relevant data.Grand Oaks Ballroom,Rooms N&O, Level 2Exhibit Booth: 501Breakthrough Solutions for Proteomics,Biomarker, and Lipidomics ResearchSpeakers: Doug Simmons and Jenny Albanese, AB SCIEXJoin us for lunch and learn about powerful new breakthrough innovationsthat can really boost your productivity in proteomics,biomarker, and lipidomics research!Begonia Room, Level 3Exhibit Booth: 108Recent Developments in WatersMetabolomics and Lipidomics BiomarkerTechnologiesSpeaker: Henry Shion, Principal Applications Scientist,WatersWe will be discussing innovations in metabolomic/lipidomicseparations with UPLC, comprehensive data collection with MSEand QuanTOF technology, and advanced data processing anddatabase searching.Vendor Presentations124 • ABRF 2011 — Technologies to Enable Personalized Medicine


Demos (as of January 29, 2011)Monday, February 21 — 12:00 pm – 12:15 pmExhibit Hall Demo StageMonday, February 21 — 12:30 pm – 12:45 pmExhibit Hall Demo StageExhibit Booth: 503DemosInnovations in protein massspectrometry from Protea Biosciences:New bioanalytical tools for proteinanalysis and characterizationSpeakers: Christopher A. Bolcato, Technical ServicesManager, and Haddon E. Goodman, Product MarketingManagerPresentation will include discussion around innovative technologiesand techniques in gel protein separation and recovery, highabundant protein depletion, and new surfactant technologydeveloped by Protea Biosciences. Several new products will bediscussed that aid in the preparation of protein samples for massspectrometry.Monday, February 21 — 12:15 pm – 12:30 pmExhibit Hall Demo StageExhibit Booth: 515Illumina’s Genotyping Portfolio Featuringa New FFPE SolutionThe revolutionary Infinium HD FFPE Restore kit has expandedIllumina’s broad portfolio of array solutions to enable genotypingof formalin-fixed, paraffin-embedded (FFPE) samples on theInfinium platform. Join us to hear more about this exciting optionavailable for the CyotSNP and OmniExpress arrays. We’ll alsodescribe the latest evolution of iSelect, the most flexible customgenotyping option available today, and give a brief update onthe Omni GWAS Roadmap.Comprehensive Genomic Tools fromClinical Research to TranslationalMedicine: Paving the Way forPersonalized MedicineSpeaker: Jessica Parra, Ph.D., Marketing Manager, GlobalField MarketingClinical researchers are making personalized medicine a realityby translating biomarker discoveries into clinical diagnostictests. Although many biomarker signatures have been identifiedin research labs, only a few microarray-based clinical diagnostictests have received clearance for commercialization. Clinical researchersface many challenges from the discovery process tovalidation and routine testing. Using a single platform from clinicalresearch discovery to clinical applications helps researchersto overcome the operational hurdles of commercialization. Affymetrixis paving the path to market with a toolkit that includesinstrumentation, arrays, and reagents for use in developing clinicaldiagnostic tests. The convenience of a cGMP-manufacturedkit helps to reduce rework and risk during the developmentprocess to fast-track commercializationMonday, February 21 — 1:00 pm – 1:15 pmExhibit Hall Demo StageExhibit Booth: 110NextGENe’s RNA-Seq Analysis toolSpeaker: Megan R. ManionNextGENe’s RNA-Seq Analysis tool, which includes a proprietaryalignment algorithm specifically designed for transcriptomedata, will be presented. Project set-up and results will be shown.Results, which include mutation detected, expression analysisand alternative splicing analysis, are displayed in the interactiveNextGENe Viewer which includes detailed annotation. Next-GENe is a standalone Windows-based program for the analysisof Next Generation Sequencing data and is compatible withdata from Illumina Genome Analyzers, Roche/454 GS FLX, FLXTitanium and Junior, the SOLiD System and Ion Torrent’s IonPersonal Genome Machine. In addition to RNA-Seq analysis,NextGENe can also be used for SNP/Indel detection, ChIP-Seq,miRNA analysis, de novo assembly, and metagenomic analyses.126 • ABRF 2011 — Technologies to Enable Personalized Medicine

Tuesday, February 22 — 12:00 pm – 12:15 pmExhibit Hall Demo StageExhibit Booth: 515My samples. My study. MiSeq TM .Introducing Illumina’s personalsequencing system.The MiSeq personal sequencing system delivers the fastest timeto answer, a revolutionary workflow, and the widest breadth ofsequencing applications, all in a compact and economical instrument.This session will provide an introduction to the instrumentand showcase the revolutionary workflow — come experience itfor yourself.DemosABRF 2011 — Technologies to Enable Personalized Medicine • 127

Membership ApplicationAssociation of Biomolecular Resource Facilities9650 Rockville Pike, Bethesda, MD 20814-3998 (USA)Telephone: (301) 634-7306 Fax: (301) 634-7455 Email: abrf@abrf.orgContact InformationYour NameCompany/InstitutionAddressCity State Zip CountryPhoneEmailFaxReferring ABRF Member NameImportant! Your membership confirmation will be sent via e-mail, along with other ABRF communications.Membership OptionsPlease check one: Member, $100.00 Student Member*, $35.00*To qualify for Student membership you must be a full-time undergraduate, graduate or medical student. Your department chair orstudent advisor must complete the following information:I certify that the applicant is a full-time student inEmail address of Department Chair/Major AdvisorSignature of Department Chair or Major AdvisorAdditional InformationAre you part of a resource laboratory? Yes NoIf yes, please list your director/manager’s name:Facility Name:Payment MethodPlease return this form with your full payment to assure proper credit. ABRF Tax ID: 56-1659510. Check enclosed for full amount made payable to ABRF. (Check must be in U.S. dollars and drawn on U.S. Bank.) Credit card (full amount will be charged): VISA MasterCard American ExpressMembershipApplicationCredit Card Number Expiration Date / /Signature of CardholderSend this form with payment to: ABRF, 9650 Rockville Pike, Bethesda, MD 20850-3998 (USA)Fax: (301) 634-7455 (Faxed membership forms must include credit card information.)128 • ABRF 2011 — Technologies to Enable Personalized Medicine

MARCH 16-20, 2012 • DISNEY’S CONTEMPORARY RESORT • ORLANDO, FLORIDAMARK YOURCALENDARS

The Your partner of in choicefor translating genomicdiscoveries intoclinical toolsWith Partnering Affymetrix with solutions Affymetrix for for medical your medical genomics, genomics corelabs research can offer gives clinical you access researchers to a broad a broad range range of tech-oftechnologies, expertise, and and experience to to help help turn turn yourtheir findings into into reliable reliable clinical clinical tools. toolsAffymetrix Take advantage offers: of the expertise thatA platform Affymetrix for offers all applications to help advance – Solutions your research: for everystep in the biomarker discovery, qualification, andvalidation A platform process for all applications – Solutions forevery step in the biomarker discovery, qualification,Extensive and validation scientific process validation – More than22,000 peer-reviewed papers published usingour Extensive technology scientific validation – More than22,000 peer-reviewed papers published usingProven our technology path to market – Multiple diagnosticproducts launched through the Powered byAffymetrix Proven path Program to market – Multiple diagnosticproducts launched through the Powered byThe Affymetrix first FDA-cleared Programmicroarray system –The GeneChip ® System 3000 Dx v2 for moleculardiagnostic Visit www.affymetrix.com/medicalgenomics applicationstolearn now published scientists have advanced theirVisit translational www.affymetrix.com/medicalgenomics research using Affymetrix technology. to learnhow published scientists have advanced their translationalresearch using Affymetrix technology.Unless otherwise noted, Affymetrix products are for research use only and are notfor use in diagnostic procedures.For research use only. Not for use in diagnostic procedures.©2011-12 Affymetrix, Inc. All rights reserved.©2010 Affymetrix, Inc. All rights reserved.

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