Conference Program Book

ABRF 2011 An International Symposium of the Association of Biomolecular Resource Facilities

Technologies to Enable Personalized Medicine February 19-22 • San Antonio, TX • abrf2011.ABRF.org • JW Marriott San Antonio Hill Country

Program & Abstracts Sunday, February 20th, 2011 7:30 pm – 10:30 pm

Take a live band, an old time dance hall, toss in the Texas two-step, old and new friends, blue margaritas and a full barbeque buffet (with all the trimmins’) and we’ve got what’s sure to be a winner of an evening! Leon Springs Dance hall

1515 Rogers Avenue San Antonio, Texas

(Shuttle service provided between the JW Marriott and Leon Springs)

Sponsored by Agilent Technologies

Monday, February 21 Level 1, Exhibit Ballroom C 3:15 pm – 4:15 pm

The ABRF Executive Board is pleased to announce that the recipient of the 2011 ABRF Annual Award for Outstanding Past ABRF Award Winners Contributions to Biomolecular Technologies is Sir Alec John Jeffreys. 1994 Frederick Sanger 1995 Klaus Bieman Biological research is driven by technology. 1996 David Lipman Awards As new instruments and chemistries are 1997 Lloyd Smith conceived and implemented, new frontiers 1998 Bruce Merrifield are created and new possibilities emerge. 1999 Marvin Caruthers Technology, from the invention of the 2000 Leroy Hood light microscope to the deciphering of 2001 Csaba Horváth the Human Genome, opens doors into 2002 John Fenn the unknown that otherwise remain firmly 2003 Franz Hillenkamp and Michael Karas sealed. The ABRF Award recognizes those pioneers responsible 2004 Edwin Southern for developing these powerful new tools that serve as the 2005 Stephen Fodor foundation of the modern biological research enterprise. The 2006 Roger Tsien* ABRF Award is sponsored by Agilent Technologies and is 2007 Don Hunt presented at the annual ABRF meeting. 2008 Ruedi Abersold 2009 Mathias Uhlen About Sir Alec John Jeffreys 2010 Patrick O. Brown Prof. Sir Alec Jeffreys studied biochemistry and genetics at Merton College, Oxford. He then began an EMBO Postdoctoral *also won the 2008 Nobel Prize in Chemistry Fellowship at the University of Amsterdam where, with Dr. Richard Flavell, he was one of the first to discover split genes. He and kinship. His current work concentrates on developing moved in 1977 to the Department of Genetics at the University new approaches to analyzing variation and mutation in human of Leicester where he currently holds the positions of Professor chromosomes. of Genetics and Royal Society Wolfson Research Professor. Sir Alec’s work has received widespread recognition, including Sir Alec’s research at Leicester has focused on exploring human his election to the Royal Society in 1986 and a Knighthood for DNA variation and the mutation processes that create this services to genetics in 1994. Other awards include the Louis- diversity. He was one of the first to discover inherited variation Jeantet Prize for Medicine (2004), the Lasker Award (2005) and in human DNA, then went on to invent DNA fingerprinting, the Heineken Prize (2006). He was also one of the four finalists showing how it could be used to resolve issues of identity for the Millennium Prize in 2008.

12 • ABRF 2011 — Technologies to Enable Personalized Medicine ABRF Outstanding Scientist/Technologist Travel Awards Recipients of the ABRF Travel Awards are those ABRF members who have made outstanding contributions in their institutional core facilities; have developed new biotechnologies with applications in core facilities; and/or have been active in ABRF activities.

The 2011 awardees are:

Pamela “Scottie” Adams Taslimarif Saiyed Trudeau Institute Centre for Cellular and Molecular Platforms, Saranac Lake, NY, United States NCBS-TIFR Bangalore, India

Elke Küster-Schöck Laurence Sandberg Proteomics and Genomics Coordinator, Loma Lind University

Microscopy Associate Loma Linda, CA, United States Awards CIAN (Cell Imaging and Analysis Network), McGill University Montreal, QC, Canada

Anoja Perera Toni Whistler Stowers Institute for Medical Research Chronic Viral Diseases Branch Kansas City, MO, United States Centers for Disease Control and Prevention Atlanta, GA, United States

Michelle Cilia United States Department of Agriculture, Robert W. Holley Center for Agriculture and Health ABRF 2011 Travel Award Selection Committee: Ithaca, NY, United States Michelle M. Detwiler (Chair), Roswell Park Cancer Institute Debbie Adam, University of British Columbia Dr. Richard T. Pon, University of Calgary Dr. Satya P. Yadav, Cleveland Clinic Foundation Research Institute

ABRF 2011 — Technologies to Enable Personalized Medicine • 13 ABRF Robert A. Welch Outstanding Research The Journal of Biomolecular Techniques ( JBT) Group / Committee Member of the Year Award Award The ABRF Robert A. Welch Outstanding This annual award recognizes the best research article published Research Group/Committee Member of each year in the Journal of Biomolecular Techniques, the ABRF the Year Award recognizes the significant society publication. This year’s JBT award recipient is: contributions of members of the ABRF Research Groups and Committees. The The Comparison of Different Pre- and Post- 2011 award will be presented to Jeff Analysis Filters for Determination of Exon-Level Kowalak. Alternative Splicing Events Using Affymetrix Arrays

ABRF Lifetime Membership Award Toni Whistler, Cheng-Feng Chiang, Jin-Mann Lin, William Lonergan, and William C. Reeves This award is given by the Executive Board to members with Chronic Viral Diseases Branch, Division of Viral and longstanding contributions to the success of ABRF and who Rickettsial Diseases, Centers for Disease Control and have been keystones to its missions. The 2011 Award will be Prevention, Atlanta, Georgia presented to Lowell Ericsson. Published in the Journal of Biomolecular Techniques, 2010 April; ABRF Poster Awards

Awards 21(1) Sponsored by Waters Corporation FASEB MARC (Minority Access to Research Waters Corporation sponsors prizes for the four best research Careers) Travel Awards posters presented at ABRF 2011. Eligible posters focus on The FASEB MARC Program sponsors ABRF 2011 Travel Awards the latest scientific research results enabled by advanced life to help support the participation of full-time faculty/mentors, sciences technologies, methods and software tools that facilitate postdoctoral fellows, and students from minority institutions applications, and the latest technology developments in the and historically black colleges and universities. biotechnology field. This year ABRF Education Committee reviewed all poster abstracts submitted to the meeting and The 2011 Faculty/Mentors and Students Travel awardees selected 11 semifinalists for award consideration. are:

This year’s semifinalists are: • Dr. Natalia Vinas (Faculty), Jackson State University • Christina Bernard (Undergraduate Student), Jackson State • , (Poster 137) Mark Turner Proteome Software, Inc. University • , (Poster 143) Christine Vogel New York University • Charnia Hall (Undergraduate Student), Jackson State • , (Poster 149) Jenny Chuu Life Technologies University • Jin Jen, Mayo Clinic (Poster 161) • , (Poster 169) Shripa Patel Stanford University The recipients of the 2011 Poster Presenter Travel Award • , (Poster 193) Ilene Magpiong Mount Holyoke College are: • Anand Narayanan, West Virginia University (Poster 197) • J. Will Thompson, Duke Institute for Genome Sciences and • Ilene Magpiong, Mount Holyoke College Policy (Poster 199) • Thurman Young, Langston University • Stefan Duhr, University Munich, Schellingstrasse (Poster 201) • Keith Fadgen, Waters Corporation (Poster 227) • Emily Freeman, Harvard School of Public Health (Poster 231)

The semifinalists are invited to give a short (5 minute) presentation of their research poster in front of a panel of judges on Sunday, February 20. The authors of the four top-scoring posters will present their work on Tuesday, February 22 during the Waters Corporation-sponsored Poster Award Session, 11:00 am – 12:00 pm, Level 2, Grand Oaks Ballroom, Exhibit Hall, Demo Stage.

Semifinalists are designated by ** next to their poster number within the Poster Session Abstract Listing.

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Visit us at ABRF Booth #515 Illumina Technology Workshop Sunday, February 20, 2011 12:00 p.m. – 1:30 p.m. Grand Oaks Ballroom: Rooms P-Q (Level 2) Complimentary lunch will be provided. Program-at-a-Glance 22 EBRUARY Stratification Biomarker , F TM UESDAY T Plenary Session: Development by Selventa of a Therapeutic Diagnostic for Drug Response in Ulcerative Colitis — David de Graaf , President & CEO, Selventa Concurrent Scientific Sessions on the Horizon S7 - NGS Technologies S9 - Proteomics Standardization Poster Session III Status of the NCRR (10:30 am – 11:00 am) (11:00 am – 12:00 pm) Poster Awards Waters in Exhibit Hall Coffee and Tea Meet the Speaker: David de Graaf Lunch in Exhibit Hall Demo Stage Presentations in Exhibit Hall Presentation in Meeting Rooms Vendor Concurrent Research Group Presentations R6 - Joint Session: PERG & MIRG R7 - Light Microscopy (LMRG) R8 - Joint Session: PRG & MRG in Exhibit Hall Coffee and Tea Concurrent Workshops W13 - Institutional Core Management W14 - NGS Software for Data Management Seminar (Part II) (continued) W15b - ETD Workshop ABRF Members Meeting Closing Reception 7:00 pm – 9:00 Journal of Biomolecular Techniques Award Journal of Biomolecular Techniques Registration Open Continential Breakfast Exhibit Hall Open (10:00 am – 2:00 pm) Concurrent Workshops W11 - Business of Running a Core Facility W12 - Microarrays: Still Alive Seminar (Part II) W15b - ETD Workshop ) 21 EBRUARY , F ONDAY M Program-at-a-Glance Registration Open Plenary Session: En Route to the Era of Genomic National Human Medicine — Eric Green , Director, Genome Research Institute Concurrent Scientific Sessions S4 - Epigenetics S5 - High Throughput Genome Centers S6 - Deep Mining of Complex Protein Mixtures Poster Session II (Even Numbered Posters) in Exhibit Hall Coffee and Tea Meet the Speaker: Eric Green Lunch in Exhibit Hall (sponsored by Continential Breakfast Award Outstanding Scientist/Technologist Demo Stage Presentations in Exhibit Hall Presentation in Meeting Rooms Vendor Concurrent Research Group Presentations R4 - Joint Session: PSRG & gPRG R5 - Joint Session: MARG & NARG W7 - Cellular 3D Imaging in Exhibit Hall Coffee and Tea Concurrent Workshops W8 - Production of Functional Proteins W9 - Therapeutic Antibodies from Network Mining W10 - Drug Targets Interface Meeting CAC Hosted ABRF-Vendor Presentations in Meeting Rooms Vendor (7:30 pm – 9:00 pm) CAN-CC Meeting (7:00 pm – 8:00 pm) Seminar (Part I) W15a - ETD Workshop (8:00 pm – 11:00 pm) Exhibit Hall Open ABRF Award Lecture: Sir Alec John Jeffreys ABRF Award (sponsored by Agilent Technologies) 3:15 pm – 4:15 20 ) EBRUARY , F UNDAY Registration Open Continential Breakfast Opening Remarks Plenary Session: Unlocking Biomarker Discovery: Unbiased Human Proteomics at High Scale, and Accuracy — Larry Gold , Chairman, Sensitivity, CEO of SomaLogic Concurrent Scientific Sessions S1 - Improving Human Health S2 - Integration of Bioinformatics for Genomics and Proteomics Data S3 - Single-cell Environmental Genomics Poster Session I (Odd Numbered Posters) in Exhibit Hall (10:30 am – 11:00 am) Coffee and Tea Career Fair Presentation (11:30 am – 11:45 am) Meet the Speaker: Larry Gold Lunch in Exhibit Hall Presentation in Meeting Rooms Vendor Concurrent Research Group Presentations R1 - Joint Session: iPRG & sPRG (ARG) R2 - Antibody Technology R3 - Joint Session: DSRG & GVRG in Exhibit Hall Coffee and Tea Concurrent Workshops W4 - Protein Expression at Industrial Scale & Tricks W5 - Proteomics Tips W6 - Capillary Electrophoresis ABRF Affiliates and Chapters Open Mic Session (6:00 pm – 6:45 pm) Networking Night at the Ranch (sponsored by 7:30 pm – 10:30 Exhibit Hall Open Concurrent Workshops W1 - The Diagnostics Core Facility W2 - Database Searching in Proteomics W3 - Protein Turnover 19 S EBRUARY , F

ATURDAY Satellite Educational Workshops (8:00 am – 4:30 pm) (SW1) Protein Purification for Mass Spectrometry (SW2) An Introduction to Metabolomics (SW3) Next Generation Sequencing Considerations for Core Professionals (SW4) Lean Management in Core Facilities Opening Reception 7:00 pm – 9:00 Registration Open Opening Plenary Session: Raju Kucherlapati Paul C. Cabot Professor, Harvard Medical School Department of Genetics (6:00 pm – 7:00 pm)

Registration Open Registration – 18th February Friday, (4:00 – 6:00 pm) 6:00 – (4:00 S IME T 7:00 am – 6:00 pm 7:00 am – 7:45 7:45 am – 8:00 8:00 am – 8:50 9:00 am – 10:30 10:00 am – 6:30 pm 10:30 am – 12:00 pm 10:30 am – 11:30 12:00 pm – 1:30 1:30 pm – 3:00 3:00 pm – 4:15 4:15 pm – 4:45 4:45 pm – 6:00 6:00 pm –7:00 7:00 pm – 11:00

16 • ABRF 2011 — Technologies to Enable Personalized Medicine Daily Program

Friday, February 18

4:00 pm – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk

Saturday, February 19

7:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk 7:00 am – 8:00 am Continental Breakfast — Level 2, Grand Oaks Ballroom P/Q 8:00 am – 4:30 pm Satellite Educational Workshops See page 28 for more detailed information.

(SW1) Protein Purification for Mass Spectrometry Level 2, Grand Oaks Ballroom R

(SW2) An Introduction to Metabolomics Level 2, Grand Oaks Ballroom S Daily Program

(SW3) Next Generation Sequencing Considerations for Core Professionals Level 2, Grand Oaks Ballroom N

(SW4) Lean Management in Core Facilities Level 2, Grand Oaks Ballroom O

6:00 pm – 7:00 pm Opening Plenary Session — Level 1, Exhibit Ballroom C Personalized Medicine: Opportunities and Challenges Raju Kucherlapati, Paul C. Cabot Professor, Harvard Medical School Department of Genetics and Professor, Department of Medicine, Brigham and Women’s Hospital

7:00 pm – 9:00 pm Opening Reception — Level 1, Exhibit Ballroom Foyer

Sunday, February 20

7:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk 7:00 am – 7:45 am Continental Breakfast — Level 1, Exhibit Ballroom Foyer 7:45 am – 8:50 am Plenary Session — Level 1, Exhibit Ballroom C Unlocking Biomarker Discovery: Unbiased Human Proteomics at High Scale, Sensitivity, and Accuracy Larry Gold, Chairman, Founder, & CEO of SomaLogic, Inc.

9:00 am – 10:30 am Concurrent Scientific Sessions (S1) Improving Human Health from the Ground Up Level 2, Grand Oaks Ballroom R/S Session Organizer: Theodore W. Thannhauser, United States Department of Agriculture/Agricultural Research Service

(S1-1) Vaccine and Therapeutic Protein Manufacture in Plants Terence E. Ryan, iBio, Inc.

(S1-2) Improving Human Nutrition from the Ground Up: Linking Agriculture to Human Health Ross M. Welch, Department of Food Science, Cornell University

ABRF 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 Developing Economies Robin D. Graham, Flinders University of South Australia

(S2) Integration of Bioinformatics for Genomics and Proteomics Data Level 2, Grand Oaks Ballroom P/Q Session Organizer: Cathy Wu, Center for Bioinformatics & Computational Biology, University of Delaware

(S2-1) Integrative Bioinformatics for Genomics and Proteomics Cathy Wu, Center for Bioinformatics & Computational Biology, University of Delaware

(S2-2) Integrated Bioinformatics for MS-Based Proteomics Eric Deutsch, Institute for Systems Biology

(S2-3) Skate Genome Project: Cyber-Enabled Bioinformatics Collaboration James Vincent, University of Vermont

(S3) Single-Cell Environmental Genomics Level 2, Grand Oaks Ballroom N/O Session Organizer: Rob Knight, University of Colorado

(S3-1) Early Elements of a Pipeline for Single Bacterial Cell Genomics Dave Relman, Departments of Microbiology & Immunology, and of Medicine, Stanford University

(S3-2) Assembling Complete Genomes from Complex Mixtures Folker Meyer, Argonne National Laboratory, Mathematics and Computer Science Division Daily Program

(S3-3) Dining in with Trillions of Fascinating Friends: Exploring our Human Gut Microbiome in Health and Disease Jeff Gordon, Center for Genome Sciences and Systems Biology, Washington University School of Medicine

10:00 am – 6:30 pm Exhibit Hall Open — Level 2, Grand Oaks Ballroom 10:30 am – 11:00 am Morning Refreshment Break — Level 2, Grand Oaks Ballroom, Exhibit Hall 10:30 am – 12:00 pm Poster Session I (Odd Numbered Posters) — Level 2, Grand Oaks Ballroom, Exhibit Hall 10:30 am – 11:30 am Meet the Speaker — Level 2, Grand Oaks Ballroom, Exhibit Hall, Demo Stage Larry Gold, Chairman, Founder, & CEO of SomaLogic, Inc.

11:30 am – 11:45 am Career Fair Presentation — Level 2, Grand Oaks Ballroom, Exhibit Hall, Demo Stage 12:00 pm – 1:30 pm Munch & Mingle — Level 2, Grand Oaks Ballroom, Exhibit Hall 12:00 pm – 1:30 pm Vendor Presentations See 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 Spectra Session Organizer & Presenter: Lennart Martens, Ghent University

(R1b) ABRF-sPRG2011 Study: Development of a Comprehensive Standard for Analysis of Post- Translational Modifications Session Organizer & Presenter: Alexander R. Ivanov, Harvard School of Public Health

18 • ABRF 2011 — Technologies to Enable Personalized Medicine (R2) Antibody Technology Research Group (ARG): Antibodies — Moving Closer to Personalized Therapeutics and Diagnostics Level 2, Grand Oaks Ballroom P/Q Session Organizers: Robert Carnahan, Vanderbilt University Medical Center, and Frances Weis-Garcia, Memorial Sloan- Kettering Cancer Center Presenters: Brian Curtis, Blood Center of Wisconsin, John Harlan, Abbott Laboratories, and Robert Umek, Meso Scale Discovery

(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 Genotyping Session 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. Nimblegen Anoja Perera, Stowers Institute for Medical Research Molecular Biology Facility

(R3b-2) A Methodology Study for Metagenomics Using Next Generation Sequencers Sushmita Singh, BioMedical Genomics Center, University of Minnesota

3:00 pm – 4:15 pm Concurrent Workshop Sessions Daily Program (W1) The Diagnostics Core Facility: Harvesting the Promise of Personalized Medicine Level 2, Grand Oaks Ballroom R/S Session Organizer: Katia Sol-Church, Nemours Biomedical Research, A.I. duPont Hospital for Children

(W1-1) Win on Sunday, Sell on Monday: From the Exome Sequencing of One Boy to the Delivery of Clinical Diagnostics Michael R. Tschannen, Human and Molecular Genetics Center, Department of Physiology, Medical College of Wisconsin

(W1-2) Chromosomal Microarray Analysis in the Clinic Lisa D. White, Microarray Core, Baylor College of Medicine

(W1-3) Whole Genome Sequencing in the Clinical Laboratory Tina Hambuch, Illumina Clinical Services Laboratory, Illumina, Inc.

(W2) Spectral and Sequence Database Searching in Proteomics Level 2, Grand Oaks Ballroom P/Q Session Organizer: Lennart Martens, Ghent University

(W2-1) ETD Performance and Complementarity to Other Fragmentation Methods for Proteomic Analysis Robert Chalkley, University of California San Francisco

(W2-2) Discovery, Identification and Localization of Post-Translational Modifications Nuno Bandeira, Center for Computational Mass Spectrometry, Department of Computer Science and Engineering Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego

(W2-3) Building and Using MS/MS Spectral Libraries for Peptide Identifications in Proteomics Henry Lam, The Hong Kong University of Science and Technology

(W3) Quantifying Protein Turnover by In Vivo Metabolic Labeling Level 2, Grand Oaks Ballroom N/O Session 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 in Muscle: Principles and Applications Robert 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 Medicine

4:15 pm – 4:45 pm Afternoon Refreshment Break — Level 2, Grand Oaks Ballroom, Exhibit Hall 4:45 pm – 6:00 pm Concurrent Workshop Sessions (W4) Insights for Expression of Recombinant Proteins for Drug Target Validation Level 2, Grand Oaks Ballroom R/S Session Organizer: Francis Rajamohan, Pfizer, Inc.

(W4-1) New Developments in BacMam Vectors Frederick M. Boyce, Massachusetts General Hospital, Department of Neurology

(W4-2) Strategies for Optimized High-Throughput Cloning, Expression, and Purification of Recombinant Proteins Rebecca Page, Brown University, Department of Molecular Biology, Cell Biology and Biochemistry, Center for Genetics, Genomics and Proteomics

(W5) Proteomics Tips and Tricks: From Discovery to Protein-Protein Interactions Level 2, Grand Oaks Ballroom P/Q Session Organizer: Michelle Cilia, United States Department of Agriculture/Agricultural Research Service, Robert W. Holley Daily Program Center for Agriculture and Health

(W5-1) Proteomics Tips and Tricks: From Discovery to Protein-Protein Interactions Michelle Cilia, United States Department of Agriculture/Agricultural Research Service, Robert W. Holley Center for Agriculture and Health

(W5-2) Protein Interactions and Topologies in Cells James Bruce, University of Washington, Department of Genome Sciences

(W5-3) The Virus-Host Interface: Exploring Dynamic Protein Interactions via Targeted Proteomics Ileana Cristea, Princeton University, Department of Molecular Biology

(W5-4) Towards the Development of Proteomics Workflows for the Analysis of Samples Derived from Refractory Plant Tissues Theodore W. Thannhauser, United States Department of Agriculture/Agricultural Research Service

(W6) Current State and Future of Capillary Electrophoresis and Sanger Sequencing Level 2, Grand Oaks Ballroom N/O Session 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 Genomics Facility, Ohio State University

6:00 pm – 6:45 pm ABRF Affiliates and Chapters Open Mic Session — Level 2, Grand Oaks Ballroom N/O 7:30 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 pm and 10:30 pm.

20 • ABRF 2011 — Technologies to Enable Personalized Medicine Monday, February 21

7:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk 7:00 am – 7:45 am Continental Breakfast — Level 1, Exhibit Ballroom Foyer 7:45 am – 8:50 am Plenary Session — Level 1, Exhibit Ballroom C En Route to the Era of Genomic Medicine Eric Green, Director, National Human Genome Research Institute

9:00 am – 10:30 am Concurrent Scientific Sessions (S4) Epigenetics Level 2, Grand Oaks Ballroom R/S Session Organizer: Benjamin A. Garcia, Princeton University

(S4-1) Next Generation Quantitative Proteomic Tools for Analyzing Histone Modifications Benjamin A. Garcia, Princeton University

(S4-2) Chromatin Dynamics in Melanoma: A Role for MacroH2A Avnish Kapoor, Department of Oncological Sciences, Mount Sinai School of Medicine

(S4-3) Using Protein Domain Microarrays to Read the Histone Code Mark Bedford, Department of Carcinogenesis, Science Park, Research Division, The University of Texas MD Anderson Cancer Center Daily Program (S5) High-Throughput Genome Centers Level 2, Grand Oaks Ballroom P/Q Session Organizer: George Grills, Cornell University

(S5-1) Overview of the Illumina Sequencing Platform at the Broad Institute Kristen Connolly, Process & Technology Development, Genome Sequencing Platform, The Broad Institute of MIT and Harvard

(S5-2) Science and Technology at a High-Throughput Genome Center Lucinda Fulton, The Genome Center at Washington University School of Medicine

(S5-3) High-Throughput Next Generation Sequencing Methods and Applications Donna Muzny, Baylor College of Medicine, Human Genome Sequencing Center

(S6) Strategies for Deep Mining of Complex Protein Mixtures Level 2, Grand Oaks Ballroom N/O Session Organizer: David Speicher, The Wistar Institute

(S6-1) Coverage and Recovery of Upstream Protein Fractionation Methods in LC-MS/MS Workflows Leonard J. Foster, Centre for High-Throughput Biology, The University of British Columbia

(S6-2) Improving the Comprehensiveness of Large-Scale Proteomics Experiments Using Advanced Computational Tools and Accurate Multiple Hypothesis Testing Statistics Michael 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 Strategies David Speicher, The Wistar Institute

10:00 am – 6:30 pm Exhibit Hall Open — Level 2, Grand Oaks Ballroom 10:30 am – 11:00 am Morning Refreshment Break — Level 2, Grand Oaks Ballroom, Exhibit Hall 10:30 am – 12:00 pm Poster Session II — Level 2, Grand Oaks Ballroom, Exhibit Hall 10:30 am – 11:30 am Meet the Speaker — Level 2, Grand Oaks Ballroom, Exhibit Hall, Demo Stage Eric Green, Director, National Human Genome Research Institute

12:00 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 Presentations

See 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 Unknown Protein Session Organizer: Jim Walters, Sigma Aldrich Presenters: Kwasi Mawuenyega, Washington University School of Medicine, Wendy Sandoval, Genentech, Inc., Steve Smith, University of Texas, and Jim Walters, Sigma Aldrich

(R4b) gPRG: Toward Consensus on Glycan Analysis: Reliable Methods and Reproducibility Session Organizer & Presenter: Joseph Zaia, Boston University

(R5) Joint Session: Nucleic Acids Research Group (NARG) & Microarray Research Group (MARG) Level 2, Grand Oaks Ballroom P/Q

(R5a) Determining miRNA Expression Levels in Degraded RNA Samples Using Real-Time RT-qPCR and Microarray Technologies Session Organizer: Sridar Chittur, State University of New York at Albany Presenters: Sridar Chittur, State University of New York at Albany, and Scott Tighe, University of Vermont

Daily Program (R5b) Microarray Research Group Projects, 2010-11 Session Organizer: Don A. Baldwin, Molecular Profiling Facility, University of Pennsylvania Presenters: Nadereh Jafari, Northwestern University, and Natalia Reyero-Vinas, Jackson State University

(W7) Cellular 3D Imaging* Level 2, Grand Oaks Ballroom N/O Session Organizer: Richard Cole, Wadsworth Center, New York State Department of Health

(W7-1) 3D Cellular Imaging: Beyond the Simple Imaging Paradigm Richard Cole, Wadsworth Center, New York State Department of Health

(W7-2) Basics of Colocalization Analyses Judith 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 facilities The ABRF will host a Leica SP5 II confocal equipped with a tandem scanner. This Confocal covers a broad range of requirements in confocal imaging — with the full array of scan speeds at highest resolution. With its high-efficiency SP detection (five channels simultaneously) and the optional AOBS (Acousto-Optical Beam Splitter), the system delivers bright, noise-free images with minimal photo damage at high speed. Additionally, there will be available the latest Autoquant’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 personnel

3:15 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 Hall 4:45 pm – 6:00 pm Concurrent Workshop Sessions (W8) Successful Production of Functional Proteins Level 2, Grand Oaks Ballroom R/S Session Organizer: Jeff Culp, Pfizer, Inc.

(W8-1) Successful Protein Production Jeff Culp, Pfizer, Inc.

(W8-2) Overcoming Problems in Protein Expression and Purification Bill Gillette, SAIC-Frederick, Inc., National Cancer Institute at Frederick

(W8-3) When Proteins Misbehave, Try Adding a Little Pressure Robert 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/Q Session Organizer: Dan L. Crimmins, Washington University School of Medicine Presenters: Dan L. Crimmins, Washington University School of Medicine, John Harlan, Abbott Laboratories, and Frances Weis-Garcia, Memorial Sloan-Kettering Cancer Center

(W10) Identification of Mechanism-Based Biomarkers and Drug Targets Using Pathway Analysis Level 2, Grand Oaks Ballroom N/O

Session Organizer: Alexander Kel, geneXplain GmbH Daily Program

(W10-1) GeneXplain — Identification of Causal Biomarkers and Drug Targets in Personalized Cancer Pathways Alexander Kel, geneXplain GmbH

(W10-2) Pathway Analysis in Expression Proteomics Roman Zubarev, Chemistry I Division, Department of Medical Biochemistry and Biophysics, Karolinska Institute

(W10-3) Metabolic Biomarkers, Metabolic Networks, and Pathway Analysis Vladimir Tolstikov, University of California Davis Genome Center

6:00 pm – 7:00 pm CAC Hosted ABRF-Vendor Interface Meeting — Level 1, Exhibit Ballroom C 7:00 pm – 8:00 pm Core Administrators Network – Coordinating Committee (CAN-CC) Meeting — Level 1, Exhibit Ballroom C 7:30 pm – 9:00 pm Vendor Presentations See 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/O Presenter: 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 Seminar Form. Please visit the Registration Desk to sign up for the session.

Tuesday, February 22

7:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk 7:00 am – 7:45 am Continental Breakfast — Level 1, Exhibit Ballroom Foyer 7:45 am – 8:50 am Plenary Session — Level 1, Exhibit Ballroom C Development by Selventa of a Therapeutic Diagnostic TM Stratification Biomarker for Drug Response in Ulcerative Colitis David de Graaf, President & CEO, Selventa

ABRF 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 Horizon Level 2, Grand Oaks Ballroom R/S Session Organizer: Katia Sol-Church, Nemours Biomedical Research, A.I. duPont Hospital for Children

(S7-1) Towards Optical DNA Sequencing Using Nanopore Arrays Amit 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 Nanocrystal Peter B. Vander Horn, Genetic Systems, Life Technologies

(S7-3) Sequencing with Semiconductor Chips Glenn Powell, Ion Torrent

(S9) Proteomics Standardization Level 2, Grand Oaks Ballroom N/O Session Organizer: David B. Friedman, Proteomics Laboratory Mass Spectrometry Research Center, Vanderbilt University

(S9-1) A Multi-Laboratory Study Assessing Robustness and Reproducibility of Plasma Reference Sample for Benchmarking LC-MS Platform Performance Juan-Pablo Albar, ProteoRed, National Center for Biotechnology-CSIC

(S9-2) Clinical Proteomic Technologies for Cancer: NIH-Funded Measurement Science Christopher Kinsinger, National Cancer Institute, Center for Strategic Scientific Initiatives, Office of Cancer Clinical Proteomics Research Daily Program (S9-3) Why Reproducible Outcomes are Essential in Proteomic Research and Why Standardization of Processes is Essential for Achieving Reproducibility Andy Borthwick, Nonlinear Dynamics Limited

10:00 am – 2:00 pm Exhibit Hall Open — Level 2, Grand Oaks Ballroom 10:30 am – 11:00 am Morning Refreshment Break — Level 2, Grand Oaks Ballroom, Exhibit Hall 10:30 am – 12:00 pm Poster Session III — Level 2, Grand Oaks Ballroom, Exhibit Hall 10:30 am – 11:00 am Status of the NCRR — Level 2, Grand Oaks Ballroom P/Q Presenter: Mark O. Lively, Wake Forest University School of Medicine

11:00 am – 12:00 pm Poster Awards (Sponsored by Waters Corporation) — Level 2, Grand Oaks Ballroom, Exhibit Hall, Demo Stage 10:30 am – 11:30 am Meet the Speaker — Level 2, Grand Oaks Ballroom, Exhibit Hall David de Graaf, President & CEO, Selventa

12:00 pm – 1:30 pm Munch & Mingle — Level 2, Grand Oaks Ballroom, Exhibit Hall 12:00 pm – 1:30 pm Vendor and Demo Stage Presentations

See 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 Study Session Organizer: Cynthia Kinsland, Cornell University

(R6b) Conclusions from the MIRG 2010 Benchmark Study: Molecular Interactions in a Three Component System and Presentation of 2011 Survey Results on Label-Free Technologies Session Organizer: Aaron Yamiuk, Bristol-Myers Squibb Presenters: Satya Yadav, Cleveland Clinic Foundation, and Aaron Yamiuk, Bristol-Myers Squibb

24 • ABRF 2011 — Technologies to Enable Personalized Medicine (R7) Light Microscopy Research Group (LMRG) Level 2, Grand Oaks Ballroom P/Q Session Organizer: Robert Stack, Wadsworth Center, New York State Department of Health

(R7-1) Point Spread Functions, Spectral Calibration, and Beyond Robert Stack, Wadsworth Center, New York State Department of Health

(R7-2) Deconvolution: Core Concepts, Algorithms, and Advanced Issues Brian 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 Services Session Organizer: David Hawke, The University of Texas MD Anderson Cancer Center

(R8b) Metabolomics Research Group 2011 Study Session Organizer: William R. Wikoff, University of California Davis Presenters: John M. Asara, Beth Israel Deaconess Medical Center, Vladimir Tolstikov and William R. Wikoff, University of California Davis

3:00 pm – 4:15 pm Concurrent Workshop Sessions (W11) The Business of Running a Core Facility Level 2, Grand Oaks Ballroom R/S Daily Program Session Organizer: Nicholas Ambulos, University of Maryland School of Medicine Presenters: 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 Exaggerated Level 2, Grand Oaks Ballroom P/Q Session Organizer: Don Baldwin, University of Pennsylvania, Penn Molecular Profiling Facility

(W12-1) Chip or Seq: Helping Clients Choose Don 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 Harmony between Technologies Scott Tighe, University of Vermont

(W12-3) Microarray Futures: Don’t Decommission Your Scanners Just Yet Seth 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/O Presenter: 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 Seminar Form. 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 Foyer 4:45 pm – 6:00 pm Concurrent Workshop Sessions (W13) Institutional Core Management Level 2, Grand Oaks Ballroom R/S Session Organizers: Susan Meyn, Vanderbilt University Medical Center, and Paula Turpen, University of Nebraska Medical Center Presenter: Pam Alexander, Morehouse School of Medicine, Julie Auger, University of California San Francisco, Gregory Farber, National Center for Research Resources, and Sheenah Mische, New York University Langone Medical Center

ABRF 2011 — Technologies to Enable Personalized Medicine • 25 Tuesday, February 22 (Continued)

(W14) Next Generation Sequencing Software for Data Management, Analysis, and Visualization Level 2, Grand Oaks Ballroom P/Q Session Organizer: Kip Bodi, Tufts University School of Medicine

(W14-1) Tools for Next Generation Sequencing Data Analysis Kip Bodi, Tufts University School of Medicine

(W14-2) GenomeView: Visualizing the Next Generation of Data Thomas 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 Calling Greg 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/O Presenter: 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 Seminar Form. Please visit the Registration Desk to sign up for the session.

6:10 pm – 7:00 pm ABRF Member’s Meeting — Level 1, Exhibit Ballroom C 7:00 pm – 9:00 pm Closing Reception — JW Marriott San Antonio Hill Country Resort, Grand Ballroom Terrace Daily Program

26 • ABRF 2011 — Technologies to Enable Personalized Medicine The Future Is Emerging Take an integrated view of biology, with tools from Agilent. A new frontier is emerging. Advances in technology have given researchers the ability to study complex biological systems beyond individual components—and Agilent is 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 biological processes. Informatics tools provide analysis for each of the omics, and offer integrated data sets across the biological system. Agilent is also collaborating with scientists to overcome the challenges of this emerging discipline, helping ﬁ nd answers to some of our most complex questions. Get the full picture at www.agilent.com/lifesciences/biology.

(SW1) Protein Purification for Mass Spectrometry

(SW2) An Introduction to Metabolomics

(SW3) Next Generation Sequencing Considerations for Core Professionals Workshops Satellite Educational

(SW4) Lean Management in Core Facilities

28 • ABRF 2011 — Technologies to Enable Personalized Medicine Satellite Educational Workshops

All 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 Spectrometry

8:00 am – 4:00 pm Level 2, Grand Oaks Ballroom R

Organizers & Presenters: Robert Carnahan, and W. Hayes McDonald, Vanderbilt University Medical Center

Workshop Description: The workshop is intended as a practical guide for investigators wishing to purify proteins and protein complexes for mass spectrometry based analysis. It should be useful for individuals wishing to perform such purifications as well as for mass spectrometrists who will be analyzing these types of samples. We will explore various considerations and possibilities in designing 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 through designing 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 Agenda

7:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk 7:00 am – 8:00 am Continental Breakfast — Level 2, Grand Oaks Ballroom P/Q 8:00 am – 9:00 am Affinity Purification for Mass Spectrometry Presenter: Robert Carnahan, Vanderbilt University Medical Center Satellite Educational Workshops 9:00 am – 10:00 am Mass Spectrometry Analysis of Protein Complexes Presenter: W. Hayes McDonald, Vanderbilt University Medical Center 10:00 am – 10:30 am Morning Refreshment Break — Level 2, Grand Oaks Ballroom P/Q 10:30 am – 12:00 pm Virtual “Hands-On” Workshop Presenters: W. Hayes McDonald and Robert Carnahan, Vanderbilt University Medical Center Reagent generation and QC Biological Optimization Sample Preparation and Processing Data Analysis 12:00 pm – 1:00 pm Lunch — Level 2, Grand Oaks Ballroom P/Q 1:00 pm – 2:30 pm Exploration of Experimental Design Considerations and their Implications Presenters: W. Hayes McDonald and Robert Carnahan, Vanderbilt University Medical Center 2:30 pm – 3:00 pm Afternoon Refreshment Break — Level 2, Grand Oaks Ballroom P/Q 3:00 pm – 4:00 pm Alternative Approaches Presenters: W. Hayes McDonald and Robert Carnahan, Vanderbilt University Medical Center

ABRF 2011 — Technologies to Enable Personalized Medicine • 29 SW2: An Introduction to Metabolomics

8:00 am – 4:30 pm Level 2, Grand Oaks Ballroom S

Organizer: William R. Wikoff, University of California Davis Presenters: William R. Wikoff, University of California Davis, and Pavel Aronov, Stanford University

Workshop Description: Metabolomics can be defined as the systems-level investigation of small molecules and metabolites in biological 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 study design, 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, triple quad, ion trap, TOF, QTOF, and FT-ICR. Ionization sources. Application of LCMS and GCMS, including a comparison of observable compounds in these techniques. Brief coverage of specialized instruments: GC-QQQ, GC-TOF, chemical ionization. Methods for sample extraction. Targeted versus untargeted methods. Peak integration, data alignment, and software. Data analysis approaches will be discussed, including uni- and multi-variate statistics, with an emphasis on selecting straightforward approaches appropriate for a given problem. Basic statistics for metabolomics: types of t-tests, multiple testing correction, False Discovery Rate, basic multivariate methods. Approaches to biomarker discovery; metabolomics in drug discovery. Review and discussion of selected publications from the literature (case studies). Some familiarity with mass spectrometry and analytical chemistry is preferred, but background will be provided.

Workshop Agenda

7:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk 7:00 am – 8:00 am Continental Breakfast — Level 2, Grand Oaks Ballroom P/Q 8:00 am – 8:30 am Introduction and Overview with Historical Background Presenter: William Wikoff, University of California Davis 8:30 am – 10:00 am Instrumentation for Metabolomics: Mass Spectrometry, Mass Analyzers, Resolution and Mass Accuracy. GC/MS: Separation, Mass Spec, and Libraries Presenter: Pavel Aronov, Stanford University 10:00 am – 10:30 am Morning Refreshment Break — Level 2, Grand Oaks Ballroom P/Q Workshops 10:30 am – 12:00 pm Sample Preparation: Liquid Chromatography, Columns and Methods Satellite Educational Presenter: William Wikoff, University of California Davis 12:00 pm – 1:00 pm Lunch — Level 2, Grand Oaks Ballroom P/Q 1:00 pm – 2:00 pm Data Processing: MZmine, XCMS, Databases. Compound Identification Presenter: Pavel Aronov, Stanford University 2:00 pm – 2:30 pm Statistics Part I Presenter: William Wikoff, University of California Davis 2:30 pm – 3:00 pm Afternoon Refreshment Break — Level 2, Grand Oaks Ballroom P/Q 3:00 pm – 3:30 pm Statistics Part II Presenter: William Wikoff, University of California Davis 3:30 pm – 4:30 pm Metabolomics Case Studies, Summary and Discussion Presenter: William Wikoff, University of California Davis

30 • ABRF 2011 — Technologies to Enable Personalized Medicine SW3: Next Generation Sequencing Considerations For Core Professionals

8:00 am – 4:30 pm Level 2, Grand Oaks Ballroom N

Organizer: Kevin L. Knudtson, University of Iowa Presenters: Deborah Grove, Pennsylvania State University, Anoja G. Perera, Stowers Institute for Medical Research, and Peter Schweitzer, Cornell University

Workshop Description: This workshop is intended for the laboratory professional looking to enhance the quality of their recently established Next Generation Sequencing (NGS) services. It is not intended for the seasoned NGS service provider. The organization of the workshop will be similar to the workflow a core professional expert might take when working with an investigator on their next generation sequencing project. The early sessions will focus on platform choice, experimental design, and sample preparation considerations. The latter sessions will focus on the performance of specific applications.

Workshop Agenda

7:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk 7:00 am – 8:00 am Continental Breakfast — Level 2, Grand Oaks Ballroom P/Q 8:00 am – 8:30 am Overview and First Contact Presenter: Kevin Knudtson, University of Iowa 8:30 am – 9:15 am Providing Next Gen Services Using the GS FLX Presenter: Kevin Knudtson, University of Iowa 9:15 am – 10:00 am Providing Next Gen Services using the GAIIx Presenter: Anoja Perera, Stowers Institute for Medical Research 10:00 am – 10:30 am Morning Refreshment Break — Level 2, Grand Oaks Ballroom P/Q 10:30 am – 11:15 am Providing Next Gen Services using the HiSeq2000 Presenter: Peter Schweitzer, Cornell University Satellite Educational 11:15 am – 12:00 pm Providing Next Gen Services using the SOLiD Workshops Presenter: Deborah Grove, Pennsylvania State University 12:00 pm – 1:00 pm Lunch — Level 2, Grand Oaks Ballroom P/Q 1:00 pm – 1:30 pm Agilent Presentation 1:30 pm – 2:30 pm Specialized Protocols for Use with Next Gen Presenters: Anoja Perera, Stowers Institute for Medical Research, and Peter Schweitzer, Cornell University 2:30 pm – 3:00 pm Afternoon Refreshment Break — Level 2, Grand Oaks Ballroom P/Q 3:00 pm – 3:45 pm Data Management /LIMS Presenters: Deborah Grove, Pennsylvania State University, Kevin Knudtson, University of Iowa, Anoja Perera, Stowers Institute for Medical Research, and Peter Schweitzer, Cornell University 3:45 pm – 4:30 pm Panel Discussion Panelists: Deborah Grove, Pennsylvania State University, Kevin Knudtson, University of Iowa, Anoja Perera, Stowers Institute for Medical Research, and Peter Schweitzer, Cornell University

ABRF 2011 — Technologies to Enable Personalized Medicine • 31 SW4: Lean Management in Core Facilities

8:00 am – 4:30 pm Level 2, Grand Oaks Ballroom O

Organizer: Tim C. Hunter, Vermont Cancer Center Presenters: Belynda Hicks, SAIC-Frederick, National Cancer Institute at Frederick, Nicholas Ambulos, University of Maryland School of Medicine, Val Scott, The Jackson Laboratory, Rand Haley, Huron Consulting Group, Wayne Collins, Agilent Technologies, and Tim C. Hunter, University of Vermont

Workshop Description: This workshop is intended for core directors, managers, and facility personnel that are interested in learning new and creative approaches to core management during lean budget times. Sessions will focus on ways to reduce or maintain costs while sustaining the activity necessary to continue meeting the demands of the scientific user base. Topics include: balancing your core budget, non-compensatory approaches to acknowledge high performing staff and foster professional development, negotiating service contracts, money saving tips, and decision making strategies for in-house or outsourcing analyses.

Workshop Agenda

7:00 am – 6:00 pm Registration Open — Level 2, Ballroom Level Registration Desk 7:00 am – 8:00 am Continental Breakfast — Level 2, Grand Oaks Ballroom P/Q 8:00 am – 8:05 am Introductions and Workshop Overview 8:05 am – 9:00 am Cost Savings via Cost Avoidance: Quality Management for the Core Laboratory Presenter: Belynda Hicks, SAIC-Frederick, National Cancer Institute at Frederick 9:00 am – 10:00 am Novel Institutional Approaches to Managing Core Facilities during Difficult Economic Times Presenter: Rand Haley, Huron Consulting Group 10:00 am – 10:30 am Morning Refreshment Break — Level 2, Grand Oaks Ballroom P/Q 10:30 am – 11:30 am Building Synergistic Relationships with Institutional Leadership that Pays Off Presenter: Nicholas Ambulos, University of Maryland School of Medicine 11:30 am – 12:00 pm Non-compensatory Approaches to Acknowledging High Performing Staff and Fostering Professional Development Presenter: Tim C. Hunter, Workshops University of Vermont

Satellite Educational 12:00 pm – 1:00 pm Lunch — Level 2, Grand Oaks Ballroom P/Q 1:00 pm – 2:00 pm How to Develop and Balance your Core Budget Presenter: Val Scott, The Jackson Laboratory 2:00 pm – 2:30 pm Can I Afford that Service Contract? Getting the Most for the Least Presenter: Tim C. Hunter, University of Vermont 2:30 pm – 3:00 pm Afternoon Refreshment Break — Level 2, Grand Oaks Ballroom P/Q 3:00 pm – 3:30 pm Association of Laboratory Managers: Promoting Excellence in Laboratory Management Presenter: Wayne Collins, Agilent Technologies 3:30 pm – 4:30 pm Interactive Roundtable Discussion on Lean Management in Core Facilities Panelists: Belynda Hicks, SAIC-Frederick, National Cancer Institute at Frederick, Nicholas Ambulos, University of Maryland School of Medicine, Val Scott, The Jackson Laboratory, Rand Haley, Huron Consulting Group, Wayne Collins, Agilent Technologies, and Tim C. Hunter, University of Vermont

32 • ABRF 2011 — Technologies to Enable Personalized Medicine Notes Notes

ABRF 2011 — Technologies to Enable Personalized Medicine • 33 Plenary Session Abstracts

(PS1) Personalized Medicine: Opportunities panel, offer hopes for accurate diagnostic information for patients and and Challenges physicians. The first diagnostic products will enter the market soon. (PS3) En Route to the Era of Genomic Medicine R. Kucherlapati Harvard Medical School Department of Genetics and E.D. Green Professor, Department of Medicine, Brigham and Women’s Hospital, Boston, MA, United States National Human Genome Research Institute, Bethesda, MD, United States The completion of the sequencing of the human genome has augured a new era in genetics. The genome program not only provided us The Human Genome Project s completion of the human genome with the complete sequence but also lead to a revolution in many sequence in 2003 was a landmark scientific achievement of historic technologies that are critical to biology and medicine. The cost of significance. It also signified a critical transition for the field of genomics, DNA sequencing has gone down significantly and is expected to go as the new foundation of genomic knowledge started to be used in down further. This technology is enabling us to examine genetic and powerful ways by researchers and clinicians to tackle increasingly genomic differences among and between individuals at a depth that complex problems in biomedicine. To exploit the opportunities has not been possible before. The application of genetic and genomic provided by the human genome sequence and to ensure the productive knowledge to assess disease risk, to accurately diagnose disease and to growth of genomics as one of the most vital biomedical disciplines of help with determining the appropriate therapy is called personalized the 21st century, the National Human Genome Research Institute medicine. Our understanding about human genetic variation and how (NHGRI) is pursuing a broad vision for genomics research beyond the this influences risk of many complex disorders is increasing at a rapid Human Genome Project. This vision includes facilitating and supporting pace. Assessing individual risk may help prevent or postpone the onset the highest-priority research areas that interconnect genomics to of human diseases. The genetics of human disease is complex and the biology, to health, and to society. Current efforts in genomics research use of genetic information may help us better diagnose disease that in are focused on using genomic data, technologies, and insights to turn may alter management of the disease. In the case of cancer, somatic acquire a deeper understanding of biology and to uncover the genetic genetic changes are helping stratify patients and helping choose the basis of human disease. Some of the most profound advances are most appropriate drug or therapy to which the patients is most likely being catalyzed by revolutionary new DNA sequencing technologies; to respond. All of these changes are bringing in a new era where these methods are already producing prodigious amounts of DNA implementation of the principles of personalized medicine would sequence data, including from large numbers of individual patients. result in better health for our population at an affordable cost. Such a capability, coupled with better associations between genetic diseases and specific regions of the human genome, are accelerating our understanding of the genetic basis for complex genetic disorders (PS2) Unlocking Biomarker Discovery: Unbiased and for drug response. Together, these developments will usher in the Human Proteomics at High Scale, Sensitivity, era of genomic medicine. and Accuracy (PS4) Selventa Development of a Therapeutic Abstracts DiagnosticTM Stratification Biomarker for Drug

Plenary Session L. Gold Response in Ulcerative Colitis SomaLogic, Inc., Boulder, CO, United States

Unbiased human proteomics should be used for biomarker discovery, D. de Graaf given that one’s capacity to deduce (from the literature) sensible biomarkers for disease is severely constrained by our limited Selventa, Cambridge, MA, United States knowledge of human biology. Empiricism will rule the field for years to come. We have worked for more than a decade on building a Using a gene expression data set from Ulcerative Colitis patients proteomics platform that allows such empiricism. The platform utilizes treated with Infliximab with objective response criteria as input data a special class of aptamers, called SOMAmers that have enhanced and our proprietary Selventa analytics, we have developed a disease affinities and specificities toward their target proteins. SOMAmer classifier which identifies alternative molecular mechanisms driving the performance is sufficient to allow our platform to operate without pairs disease beyond TNF-alpha, the target of Infliximab. This Therapeutic of affinity reagents for each target protein — in other words, we do not DiagnosticTM classifier identifies at least 3 more strong, independent use “sandwiches.” Today we measure more than 1,000 human proteins disease drivers, including IL-6, and provides the opportunity to stratify from human samples (plasma, serum, tissue extracts, or other matrices), patient a priori by molecular disease mechanism and associated using only a small volume of sample. The measurements have limits treatment(s). In the case of IL6 we provide supporting evidence in a of detection similar to those of good ELISA’s, and low CV’s (around related disease that indicates the complementary nature of the TNF- 5%). We anticipate additional content in the near future — as new alpha and IL-6 disease driving mechanisms, as a partial validation of the content is added to the platform, performance appears to get better. approach. This approach does not rely on response data to develop To date we have analyzed more than 10,000 samples from individuals, classifiers and is easily generalized to deliver patient stratification for and studied a number of diseases. In most cases we have found novel any specific disease population in Early Discovery with an optimized (and unexpected) biomarkers, which, when used together in a small project portfolio as a result.

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(S1) Improving Human Health from the Ground (S1-2) Improving Human Nutrition from the Ground Up Up: Linking Agriculture to Human Health

R.M. Welch T.W. Thannhauser Department of Food Science, Cornell University, Ithaca, NY, United States Department of Agriculture/Agricultural United States Research Service, Ithaca, NY, United States Malnutrition is the leading cause of death globally. Both overt nutrient The focus of this scientific session will be the essential link between deficiencies and diet-related chronic diseases account for over 20 agriculture and human health that is grounded in biotechnology. million deaths a year. The causes of malnutrition are complex but are Advances in medicine, in particular personalized medicine, will rooted in dysfunctional food systems dependent on agricultural systems ultimately demand equal and outstanding advances in agricultural that have never had an explicit goal of improving human health. These biotechnologies to occur in parallel. Such advances promise to deaths are preventable. Linking agricultural systems to human health make highly nutritious food accessible and affordable to most of the could provide sustainable solutions to malnutrition. Various agricultural world’s population. Speakers will describe the ways in which humans tools can be used to improve the health and felicity of people afflicted rely on agriculture, and increasingly, on agricultural technologies, to with malnutrition. Biofortification is one tool that is currently being meet their basic nutritional needs. Direct applications of agricultural employed to address micronutrient malnutrition among resource- biotechnologies for crop improvement, agricultural diversification and poor families in the developing world. Fertilizers provide another tool the production of vaccines and other protein therapeutics will also that has been used successfully to address selenium, iodine and zinc be presented. Members of the ABRF can help to meet the growing deficiencies in several nations. There are numerous other “off the shelf” demand of agricultural research scientists for analytical advances and agricultural tools that could be used to improve the nutrient output throughput via the application of state-of-the-art biotechnologies to of farming systems and improve the health all people dependent on agricultural research. agricultural systems for their sustenance. These include: designing cropping systems to maximize nutrient output, using agronomic (S1-1) Vaccine and Therapeutic Protein Manufacture practices to improve the nutritional and health promoting quality of in Plants food crops, re-diversifying cropping systems, and genetically modifying crops to be more nutritious and healthy. This can only be accomplished T.E. Ryan if explicit links are made between the agriculture, nutrition and health communities. Further, government policies should be reoriented to iBio, Inc., Newark, DE, United States reflect the important roles that agriculture plays in the health of all people. We need to closely link agriculture to health if we want to find The need to contain costs, as well as the desire to expand patient access sustainable solutions to malnutrition globally. to biotherapeutics has increased interest in non-traditional methods of recombinant protein expression. The production of vaccines and (S1-3) A Food Systems Approach to Address biotherapeutic proteins in whole plants holds the promise of dramatically lowering the capital and operating costs for the manufacture of life- Poor Nutritional Health in Both Advanced and saving drugs, and can also be used in developing nations that lack a Developing Economies Abstracts sophisticated drug manufacturing infrastructure. A transient expression Scientific Session system in whole plants using a combination Agrobacterium/viral gene R.D. Graham vector (iBio LaunchTM) has been developed to manufacture vaccines Flinders University of South Australia, Adelaide, South and biotherapeutics in a variety of plant species (including Nicotiana Australia benthamiana), and has been successfully scaled to pilot plant levels of plant biomass (50kg). The speed of protein production in this Since the retreat of the oceans, the land surface of planet Earth has for system makes it highly effective for personalized vaccines or vaccines plant growth become widely deficient in nitrogen and phosphorus and that counter pandemic threats. Influenza vaccines for H N and H N 5 1 1 1 to a smaller extent, potassium and sulphur. A century ago, agriculture isolates generated in whole plants using this system have been shown was focused on adding these nutrients to solve production problems to raise neutralizing antibodies in animal challenge models, and are now of otherwise good soils exhausted from many hundreds of years in human clinical trials. of cultivation and of new, more marginal lands being brought into production. The use of mineral fertilisers such as Chilean saltpetre, superphosphate and potash, together with the other then-known essential minerals, calcium and magnesium, brought production up to expectations, but to experienced eyes, anomalous results hinted at limitations to production as yet unknown. The micronutrients, as they became to be known, contributed greatly to the 20th century crop productivity. 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. The post war explosion in the human population put great stress on our

36 • ABRF 2011 — Technologies to Enable Personalized Medicine food systems in the 1950s but the Green Revolution (1960-1980) more of uniformly processed MS/MS experiments, along with tools to than doubled world food production and mass starvation was avoided. use the information in a variety of ways. SRMAtlas is a resource that Instead, there was in the 1980s and 90s a massive rise in micronutrient enables the design of selected reaction monitoring (SRM) experiments deficiencies, especially in subsistence farming systems, which are still based on information from several different sources. In addition, the unresolved today. It appears that the Green Revolution emphasis interface of these resources with community standardization and on cereal production at the expense of pulses and other nutrient- cooperation efforts such as the Proteomics Standards Initiative and the rich foods is the cause, and new food systems capable of addressing ProteomeXchange Consortium will be presented. all human nutritional needs are being developed, a multifactorial challenge. Dealing with the complex nutritional requirements of new (S2-3) Skate Genome Project: Cyber-Enabled and effective food systems for the future is demanding new analytical Bioinformatics Collaboration capability to support plant breeders and agronomists in the field. J. Vincent (S2) Integration of Bioinformatics for Genomics University of Vermont, Burlington, VT, United States and Proteomics Data The Skate Genome Project, a pilot project of the North East (S2-1) Integrative Bioinformatics for Genomics and Cyberinfrastructure Consortium, aims to produce a draft genome sequence of Leucoraja erinacea, the Little Skate. The pilot project Proteomics was designed to also develop expertise in large scale collaborations across the NECC region. An overview of the bioinformatics and C.H. Wu infrastructure challenges faced during the first year of the project will Center for Bioinformatics and Computational Biology, be presented. Results to date and lessons learned from the perspective University of Delaware, Newark, DE, United States of a bioinformatics core will be highlighted. Systems integration is becoming the driving force for 21st century (S3) Single-Cell Environmental Genomics biology. Researchers are systematically tackling gene functions and complex regulatory processes by studying organisms at different levels of organization, from genomes and transcriptomes to proteomes (S3-1) Early Elements of a Pipeline for Single and interactomes. To fully realize the value of such high-throughput Bacterial Cell Genomics data requires advanced bioinformatics for integration, mining, comparative analysis, and functional interpretation. We are developing D.A. Relman a bioinformatics research infrastructure that links data mining with text mining and network analysis in the systems biology context Departments of Microbiology & Immunology, and of for biological network discovery. The system features include: (i) Medicine, Stanford University, Stanford, CA, United States integration of over 100 molecular and omics databases, along with gene/ A critical aspect of microbial community analysis is to understand protein ID mapping from disparate data sources; (ii) data mining and the contributions of individual cells to overall community structure text mining capabilities for literature-based knowledge extraction; and and function. Together with Steve Quake and Paul Blainey from the (iii) interoperability with ontologies to capture functional properties Department of Bioengineering, we have built a microfluidics device and of proteins and complexes. The system further connects with a data Scientific Session platform that permits sequence-based identification of single bacterial analysis pipeline for next-generation sequencing, linking genomics data Abstracts cells using fluorescence in situ hybridization, single cell sorting using to functional annotation. The integrative approach will reveal hidden laser tweezers, and subsequent on-chip whole genome amplification interrelationships among the various components of the biological (WGA). Current trapping performance enables the sorting of up to 60 systems, allowing researchers to ask complex biological questions and cells per hour, and complete processing of 30 - 46 cells, 0 – 16 negative gain better understanding of biological and disease processes, thereby controls, and two positive controls per day on a routine basis. Each facilitating target discovery. device enables 48 simultaneous single-cell WGA reactions, which can be monitored in real-time with SYBR green fluorescence. In conjunction (S2-2) Integrated Bioinformatics for MS-Based with other developments described in this Session, we envision a near- Proteomics future capability for microbial community genomic analysis at a level of resolution not previously imagined. E. Deutsch Institute for Systems Biology, Seattle, WA, United States (S3-2) Assembling Complete Genomes from Complex Mixtures A typical tandem mass spectrometry (MS/MS) proteomics workflow involves a series of steps including format conversion, spectrum F. Meyer identification, peptide validation, protein inference, quantification, interpretation, and public repository deposition. This talk will provide Argonne National Laboratory, Mathematics and Computer an overview of the proteomic bioinformatics resources developed at Science Division, Argonne, IL, United States the Institute for Systems Biology, covering the Trans-Proteomic Pipeline (TPP) and related tools, the PeptideAtlas public repository, and the Characterizing genomes of un-culturable microbes (most species emerging SRMAtlas resource. The TPP provides an easily-installable on earth) requires new approaches for genome assembly from suite of tools to enable users to perform nearly all steps in an MS/MS environmental or biomedical samples that often contain many analysis workflow. PeptideAtlas is a multi-species public compendium hundreds or thousands of species. Here we show we can reconstruct of peptide and protein identifications assembled from a large number the complete genome (Candidatus Sulfuricurvum sp) via short-read metagenomics and novel approaches for assembly based on simple

ABRF 2011 — Technologies to Enable Personalized Medicine • 37 statistical principles. While previous examples of complete genome (S4) Epigenetics sequences from metagenomes stem from samples of very limited complexity (>10 OTUs), this sequence was obtained from a complex mix of over 300 OTUs. The reason traditional genome assembly (S4-1) Next Generation Quantitative Proteomic approaches fail are varying abundance levels and strain variation and Tools for Analyzing Histone Modifications we show a simple approach to overcome those hurdles. This novel approach will allow the assembly of genomes and via metabolic models B.A. Garcia derived from the genomic sequences, hopefully the cultivation of key species from diverse environments/enrichment cultures. Princeton University, Princeton, NJ, United States (S3-3) Dining in with Trillions of Fascinating Friends: Histones are small proteins that package DNA into chromosomes, Exploring Our Human Gut Microbiome in Health and a large number of studies have showed that several single post-translational modification sites on the histones are associated and Disease with both gene activation and silencing. Nevertheless, what type of effect distinct combinations of simultaneously occuring histone J.I. Gordon modifications (Histone Codes or patterns) have upon cellular events Center for Genome Sciences and Systems Biology, is poorly understood. The main reason for this lack of knowledge is Washington University School of Medicine, St. Louis, MO, that robust high-throughput methods for quantitative characterization United States or even qualitative identification of combinatorial Histone Codes by any standard biological, immunological or physical technique Our genetic landscape is a summation of the genes embedded in do not exist. We plan to specifically address this deficiency by our human genome and in the genomes of our microbial symbionts developing novel mass spectrometry based proteomic methods and (the microbiome). Similarly, our metabolic features (metabotypes) accompanying bioinformatics to quantitatively characterize molecular are an amalgamation of human and microbial traits. Therefore, level descriptions of combinatorial Histone Codes, and apply these understanding of the range of human genetic and metabolic diversity methods to study how these dynamic Histone Codes influence gene means that we must characterize our microbiomes, which contain expression under different biological conditions. Here we present at least several hundred-fold more genes than our human genome, initial proteomics data that describes: (i) high-throughput comparison as well as the factors that influence the properties of our microbial of histone modifications from multiple cellular states (ii) developing communities (microbiota). The results should provide an additional mass spectrometry methods for quantitative tracking of combinatorial perspective about contemporary human biology as we assess Histone Codes (iii) monitoring in vivo Histone Code dynamics, and how our changing lifestyles, cultural norms, socioeconomic status, (iv) investigating the role of Histone Code interpreting proteins in and biosphere are influencing our microbial ecology and health recognizing distinct Histone Codes. Ultimately, we will work towards status. I will discuss the results of our group’s ongoing metagenomic the goal of taking any defined part of the genome and accurately studies of the interrelationships between diet and the structure and quantifying the Histone Codes, detecting all the non-histone proteins dynamic operations of the human gut microbiome. We believe that that reside on these distinct pieces of chromatin, and then mapping understanding these interrelationships is important for advancing our this proteomic data back to specific genomic locations, therefore taking appreciation of the nutritional value of food ingredients, for creating a proteomic snapshot of what that chromosome landscape looks like new nutritional guidelines for humans at various stages of their lifespan, during any nuclear event. These studies in combination with biological and for developing new ways to deliberately manipulate the properties experiments will help provide a systems biology outlook on gene of the gut microbiota to prevent or treat various diseases. We have expression that will lay down the basic scientific foundation to advance developed a translational medicine pipeline that involves metagenomic several applications, such as stem cell reprogramming and cancer analyses of the gut microbial communities of adult mono- and dizygotic progression. Abstracts twins living in the USA who are lean, or concordant or discordant for

Scientific Session obesity, and twins aged 0-3 years living in developing countries who develop normally, or who become malnourished and are treated with a ready-to-use therapeutic food (RUTF). Intact fecal communities from these individuals, or ‘personal’ culture collections that capture the majority of bacterial diversity in their microbiota, are then transplanted into germ-free mice, which are fed the diets of the human donors, or systematically manipulated derivatives of these diets. The impact of diet and microbiota on these humanized mice, including the degree to which the human donor’s physiologic/metabolic phenotypes can be transmitted to gnotobiotic animals via microbiota transplants, are then studied using a variety of methods.

38 • ABRF 2011 — Technologies to Enable Personalized Medicine (S4-2) Chromatin Dynamics in Melanoma: A Role for (S4-3) Using Protein Domain Microarrays to Read the MacroH2A Histone Code

A. Kapoor1,2, M. Goldberg1,2, L. Cumberland1,2*, M.T. Bedford K. Ratnakumar1,2*, M. Segura4,6, P. Emanuel2,3, MD Anderson Cancer Center, Department of Molecular S. Menendez4,6, C. Vardabasso1,2, G. LeRoy7, C. Vidal2,3†, Carcinogenesis, Smithville, TX, United States D. Polsky4,5,6, I. Osman5,6, B. Garcia7, E. Hernando4,6, E. Bernstein1,2 For cells to survive, differentiate, and grow, information has to be 1Department of Oncological Sciences, Mount Sinai School transferred from the cell surface to the nucleus. This process is referred to as signal transduction. A hallmark of cancer is the deregulation of of Medicine, New York, NY, United States; 2Department signal transduction pathways. Signaling events in eukaryotic cells involve of Dermatology, Mount Sinai School of Medicine, New the assembly and disassembly of large protein-protein complexes. 3 York, NY, United States; Department of Pathology, Mount These diverse associations are mediated through interactions of Sinai School of Medicine, New York, NY, United States; a limited number of modular signaling units or protein-domains. 4Department of Pathology, New York University Langone Protein interactions involving domains are often regulated by post- Medical Center, New York, NY, United States; 5Department translational modification (PTM – like phosphorylation, methylation of Dermatology, New York University Langone Medical and acetylation) of the smaller protein motif within the ligand. We Center, New York, NY, United States; 6Interdisciplinary have developed a chip-size protein microarray that harbors a display Melanoma Cooperative Group, New York University of over 300 modular protein-interacting domains including SH2, SH3, Langone Medical Center, New York, NY, United States; PDZ, FHA, 14-3-3, WW, Chromo, Tudor, PHD and MBT domains. In 7Department of Molecular Biology, Princeton University, the emerging proteomic era, it is becoming easier to identify proteins using tryptic digestion followed by mass spectrometric approaches. Schultz Laboratory, Princeton, NJ, United States; †Present These same methods also detect sites of posttranslational modification Address: Department of Dermatology, Saint Louis University on proteins. Many of these posttranslational modifications likely School of Medicine, St. Louis, MO, United States; *These generate docking sites for protein modules. We have developed authors contributed equally to this work. protein-domain microarray technology to help identify proteins that can interact with motifs that are either methylated or phosphorylated. Cancer is a disease consisting of both genetic and epigenetic changes. This high-throughput approach facilitates the rapid identification Although increasing evidence demonstrates that tumour progression of protein-protein interactions in vitro. Further in vivo studies are entails chromatin-mediated changes such as DNA methylation, the needed to confirm that these interactions do indeed occur in biological role of histone variants in cancer initiation and progression currently systems. Protein domains are cloned into a GST expression vector, and remains unclear. Histone variants replace conventional histones within recombinant protein is produced in bacteria. These fusion proteins the nucleosome and confer unique biological functions to chromatin. are then arrayed onto nitrocellulose coated glass slides using a robot. Using well characterized, paired series of murine and human melanoma These slides are probed with biotinylated peptides that are pre- cells lines, we probed the epigenetic profile of melanoma. Analysis conjugated to streptavidin-Cy3. The peptides used in this experiment of histones from both series using multiplexed quantitative mass are synthesized as 15 mers, and both the modified and unmodified spectrometryrevealed changes in several histone posttranslational forms of the peptides are tested on the array. In this manner, we can modifications and histone variants. The loss of mH2A isoforms, identify novel methyl- and phospho-dependent interactions. We have Scientific Session histone variants generally associated with condensed chromatin and built three types of arrays: (1) A phospho-tyrosine reader harbors 70 Abstracts fine-tuning of developmental gene expression programs is positively SH2 domains and 5 PTB domains (total = 75 domains). (2) A phospho- correlated with increasing malignant phenotype of melanoma cells threonine/serine reader that harbors 7 14-3-3 domains, 5 FHA in culture and human tissue samples. Knockdown of mH2A isoforms domains, 15 BRCT domains and a WW domain (total = 28 domains). in melanoma cells of low malignancy results in significantly increased (3) An epigenetic reading array that harbors methyl and acetyl readers. proliferation and migration in vitro and growth and metastasis in This array is composed of 50 tudor domains, 22 bromo domains, 36 vivo. Restored expression of mH2A isoforms rescues these malignant PHD domains, 17 MBT domains, 11 WD40 domains, 9 SANT domains, phenotypes in vitro and in vivo. We demonstrate that the tumour- 28 chromo domains, 15 PWWP domains, 5 BRK domains, 5 CW promoting function of mH2A loss is mediated, at least in part, through domains, and 9 Ank repeats (total = 207 domains). More and more direct transcriptional upregulation of CDK8. Suppression of CDK8, a posttranslational modifications are being discovered on proteins. The colorectal cancer oncogene inhibits proliferation of melanoma cells, roles of many of these methylation and phosphorylation events often and knockdown of CDK8 in cells depleted of mH2A suppresses the remain obscure. This approach provides an easy way for a researcher proliferative advantage induced by mH2A loss. Moreover, a significant to identify potential binding partners for their favorite proteins. inverse correlation between mH2A and CDK8 expression levels exists These arrays thus offer researchers tools to get at “mechanism”. Once in melanoma patient samples. Taken together, our results demonstrate investigators know that they are working with a clearly functional PTM, that mH2A is a critical component of chromatin that suppresses the they can proceed with confidence to generate modification specific development of malignant melanoma, a highly intractable cutaneous antibodies and interrogate the signaling pathway that is engaged by neoplasm. the identified PTM-driven protein-protein interaction.

ABRF 2011 — Technologies to Enable Personalized Medicine • 39 (S5) High-Throughput Genome Centers (S5-3) High-Throughput Next Generation Sequencing Methods and Applications

(S5-1) Overview of the Illumina Sequencing Platform D. Muzny1, M. Wang1, I. Newsham1, Y.Q. Wu1, H. Dinh1, at the Broad Institute C. Kovar1, J. Santibanez1, A. Sabo1, J. Reid1, M. Bainbridge1, E. Boerwinkle2, T. Albert3, R. Gibbs1 K. Connolly 1Baylor College of Medicine, Human Genome Sequencing Process & Technology Development, Genome Sequencing Center, Houston, TX, United States; 2University of Texas Platform, The Broad Institute, Cambridge, MA, United States Health Science Center at Houston, School of Public Health, 3 The constant increase in quality and quantity of Next-Generation Houston, TX, United States; Roche NimbleGen, Inc., sequencing data necessitates a parallel growth in sample preparation Madison, WI, United States and a scalable tracking system. The Broad Institute’s Illumina Sequencing Second Generation high-throughput sequencing technologies have Platform handles a variety of applications and comprises Illumina’s latest revolutionized the genome sequencing applications and will ultimately hardware, software and kit releases, and a high-throughput sample have great impact on personalized medicine. The increase in capacity preparation process. With our automated sample preparation and QC of both the AB/Life Technologies SOLiD 4.0 and Illumina HiSeq processes, we have been able to meet our increased capacity goals of instrumentation and the ability of the platforms to multiplex samples up to 3,840 libraries per week, and have reduced our rework rate to has led to process innovations impacting many ongoing projects 5% through attaining target cluster densities with high reproducibility. at the HGSC. Applications have ranged from regional and whole We continue to work closely with Illumina to develop the sequencing exome capture sequencing to the use of whole genome shotgun for technology, using data to drive process improvements and exploring deep coverage and determining structural rearrangements. Internal methods to improve GC bias. Maximizing platform-wide efficiency is advancements have complemented the higher capacity instrumentation possible through the implementation and continuous development of through the implementation of library automation, low DNA input tools for process quality control and centralized communication, such samples, capture hybridization multiplexing and application of read as our real-time run monitoring dashboard and JIRA tracking system. mapping tools such as BFAST and BWA. Development of sample intake We were able to convert rapidly from GAIIxs to HiSeq2000s by procedures, LIMS tracking and defined reporting metrics has enabled establishing and using an enterprise Knowledge Management system, NexGen sequencing pipelines that can effectively deliver targeted with which we can efficiently accumulate and disseminate a changing and whole genome shotgun data for thousands of samples. These knowledge base. All of these improvements are applicable to both the technical advancements to the pipeline have allowed us to achieve GA and HiSeq platforms. a rate of ~1500 libraries/captures per month. To date the center has (S5-2) Science and Technology at a High completed over 5000 exome and regional capture libraries for The Cancer Genome Atlas (TCGA), NIMH Autism, Cohorts for Heart and Throughput Genome Center Aging Research in Genomic Epidemiology (CHARGE-S) and 1000 Genomes Project. Development of these applications and methods will , R. Wilson, The Genome Center Production L. Fulton be discussed along with key data metrics, process management and Group pipeline organization. The Genome Center at Washington University School of Medicine, St. Louis, MO, United States (S6) Strategies for Deep Mining of Complex Protein Mixtures The Genome Center (GC) at Washington University School of Medicine

Abstracts has developed a state of the art genomics facility. Our scientists work (S6-1) Coverage and Recovery of Upstream Protein

Scientific Session on a variety of cutting edge projects with researchers from around the world. These collaborative research projects lead to cutting edge Fractionation Methods in LC-MS/MS Workflows advances in the field of genomics. The structural organization at the GC reflects these efforts and is centered around six major scientific L.J. Foster areas: Transcriptome Sequencing, Genome Assembly, Whole Genome Centre for High-Throughput Biology, The University of British Sequencing, Human Microbiome, Human Genetics, and Targeted Resequencing. These specific scientific areas are supported by one Columbia, Vancouver, BC, Canada central data production pipeline. Attributes of this pipeline include The proteome of any cell or even any subcellular fraction remains detailed sample screening protocols, sample barcoding capabilities too complex for complete analysis by one dimension of liquid that allow for a broad range of sample cohorts, multiplatform data chromatography-tandem mass spectrometry (LC-MS/MS). Hence, to production, and the ability to select from more than one method of achieve greater depth of coverage for a proteome of interest, most sequencing strategies. All of this is supported by one centralized LIMS groups routinely subfractionate the sample prior to LC-MS/MS so group dedicated to maintaining and developing the data production that the material entering LC-MS/MS is less complex than the original capabilities. The technology development group investigates new sample. Protein and/or peptide fractionation methods that biochemists techniques and instrumentation prior to any changes in the main data have used for decades, such as strong cation exchange chromatography production pipeline. Only robust protocols and instrumentation are (SCX), isoelectric focusing (IEF) and SDS-PAGE, are the most common allowed into the data production pipeline. This strategy allows The prefractionation methods used currently. There has, as yet, been no Genome Center to run a base data production pipeline while constantly comprehensive, controlled evaluation of the relative merits of the infusing high quality advances. Sequence data for each project is sent various methods, although some binary comparisons have been made. into an advanced analysis pipeline built to conduct a multitude of We will discuss the most popular methods for fractionating samples at assessments. When needed, validation (a second sequence event) can both the protein and peptide level, demonstrating quantitatively which be used to confirm variants detected by the analysis software.

40 • ABRF 2011 — Technologies to Enable Personalized Medicine are the best methods for optimal recovery and proteome coverage. strengths and weaknesses, label-free quantitative comparison of LC-MS A novel approach for fractionating samples at the level of protein signals is increasing in popularity and seems adequately reproducible complexes will also be discussed. for most studies. Our laboratory commonly uses two alternative plasma proteome analysis strategies. One powerful approach utilizes a 3-D (S6-2) Improving the Comprehensiveness of Large- protein/peptide profiling method consisting of depleting 20 abundant Scale Proteomics Experiments Using Advanced proteins followed by 1-D SDS PAGE, fractionation of the gel lane into Computational Tools and Accurate Multiple 20 to 60 fractions and LC-MS/MS analysis. Proteins can be quantitatively Hypothesis Testing Statistics compared using label-free analysis of ion current patterns from the MS full scans. An even greater depth of analysis can be achieved using a M.J. MacCoss, J. Egertson, B. Frewen, L. Käll, W. Noble 4-D protein/peptide profiling strategy utilizing microscale solution isoelectrofocusing of proteins prior the SDS gel in the 3-D scheme, Department of Genome Sciences, University of Washington, although throughput is substantially reduced. Seattle, WA, United States (S7) Next Generation Sequencing Technologies Mass spectrometry based technology for the analysis of complex on the Horizon protein mixtures has improved at an amazing rate. With each new instrument release, mass spectrometers have become more sensitive and have faster MS/MS data acquisition speeds. Furthermore, (S7-1) Towards Optical DNA Sequencing Using instruments are continuously improving the dynamic range, mass Nanopore Arrays accuracy, and resolution of the resulting mass spectrometry data. All of these developments have increased the number of peptides that A. Meller can be identified and quantified without extending the overall analysis time. While the technological hardware advances that are required to Department of Biomedical Engineering, Boston University, increase the number of peptide identifications by 50% with a constant Boston, MA, United States analysis time is monumental, we have been able to demonstrate that increase in performance without increasing the analysis time at all. To Next generation DNA sequencing methods that utilize nanometer- accomplish this, we have made use of improved database searching size pores have been subject of numerous studies in past years. One algorithms, spectrum library searching, use of chromatographic of the compelling features of the nanopore technique lies in its ability retention time, powerful machine learning tools, accurate multiple to electrophoretically focus long DNA strands towards the pore area hypothesis testing statistics, and many more. Strategies will be and thread the molecules inside the pore in a highly efficient manner. discussed on how to increase the comprehensiveness of any dataset Thus extremely small copy numbers of the target DNA are required using improved data analysis strategies. for analyses†, circumventing the need for costly and time-consuming target amplification. One of the major bottlenecks for the realization (S6-3) In-Depth Analysis of Human and Mouse of a viable nanopore-based DNA sequencing has been the ability to Plasma Using 3-D And 4-D Fractionation Strategies simultaneously read the electrical signals from hundreds to thousands of nanopores densely fabricated on sub-millimeter size silicon chip. To D.W. Speicher address this issue we develop an extremely high throughput single- molecule DNA sequencing technique, which employs optical, wide-

The Wistar Institute, Philadelphia, PA, United States field readout from DNA molecules electrically mobilized through Scientific Session

the nanopores. Our method consists of two steps: First, target DNA Abstracts In-depth profiling of plasma proteomes can potentially identify novel molecules are converted according to pre-determined code, which is disease biomarkers. But few biomarkers identified by proteomic recognized by molecular beacons with four types of fluorophores (each approaches have advanced to early-stage clinical testing because they uniquely corresponding to one of the four DNA bases). Solid-state often are not sufficiently disease specific. Major challenges in plasma nanopores are then used to sequentially strip off the beacons, leading proteome analysis include the very wide dynamic range of protein to a series of photon bursts that can be detected with a custom made concentrations, the high protein complexity, and the substantial microscope. Notably the method circumvents the use of enzymes in the heterogeneity of most protein concentrations in the normal human readout stage, and is thus not affected by their limited processivity and population. Because most disease-specific biomarkers are present in lifetime. Here we demonstrate the feasibility of this method using a two blood at very low concentrations, extensive fractionation is required color model system, and show for the first time, individual nucleotide prior to LC-MS/MS analysis. In general, more fractionation will result in recognition from multiple nanopores simultaneously‡, allowing greater depth of analysis, but there is a point of diminishing return for straightforward parallelization of our system to nanopore arrays. †. each fractionation method and throughput decreases as the number Wanunu, M., W. Morrison, Y. Rabin, A. Y. Grosberg, and A. Meller. 2010. of LC-MS/MS runs per proteome increases. A common feature of most Electrostatic Focusing of Unlabeled DNA into Nanoscale Pores using a current plasma profiling methods is to first immunodeplete as many Salt Gradient. Nature Nanotechnology 5:160-165. ‡. McNally, B., A. high abundance plasma proteins as possible, followed by extensive Singer, Z. Yu, Y. Sun, Z. Weng, and A. Meller. 2010. Optical Recognition protein fractionation of the depleted plasma prior to trypsin digestion of Converted DNA Nucleotides for Single-Molecule DNA Sequencing and LC-MS/MS. In addition, reliable quantitative comparisons are Using Nanopore Arrays. Nano Letters 10:2237-2244. needed for most types of studies. While all quantitative methods have

ABRF 2011 — Technologies to Enable Personalized Medicine • 41 (S7-2) Single Molecule Real-Time DNA Sequencing (S9) Proteomics Standardization on the Surface of a Quantum-Dot Nanocrystal

P.B. Vander Horn D.B. Friedman Genetic Systems, Life Technologies, Carlsbad, CA, United Proteomics Laboratory Mass Spectrometry Research Center, States Vanderbilt University, Nashville, TN, United States

A single molecule, long read-length, real-time sequencing- The needs and issues related to establishing inter-laboratory by-synthesis technology has been developed by building standardization in quantitative proteomics will be highlighted by a sequencer directly on the surface of a ~ 10 nm quantum- presentations from three international initiatives. By establishing the need for standardization, each group will also highlight the tremendous dot nanocrystal. Fluorescence resonance energy-transfer amount of instrument and experimental variation that is also measured technology (FRET) is utilized for DNA sequence detection, in when trying to determine real biological changes. The Spanish-based which signals from the quantum-dot labeled DNA polymerase ProteoRed consortium addresses multiple proteomics platforms, plus 4 DNA-base-specific acceptor dyes are simultaneously ranging from MS-based to gel-based. The NIH CPTAC network is detected. Precisely engineered sequencing-grade QdotTM focused on MS-based studies, and the UK-based FixingProteomics nanocrystals are smaller than current commercially available initiative covers mostly gel-based methods. Results from these materials (to increase FRET signals), and have an exctinction studies will demonstrate complement and overlap in technology and coefficient ~100X greater than organic-dyes, allowing for very approaches used between these groups, all motivated by the universal low levels of excitation power to be used while sequencing, goal of standardizing these complex technologies across laboratories. Acting as the FRET donor, the QdotTM-polymerase generates a correlated “photon-dip” for every inserted based (termed the (S9-1) A Multi-Laboratory Study Assessing “quantum-correlation-signal”), allowing for more accurate base- Robustness and Reproducibility of Plasma calling. Because the sequencer is not physically bound to any Reference Sample for Benchmarking LC-MS Platform solid substrate, it can be exchanged (like a reagent) during mid- Performance sequence runs, effectively replacing damaged non-functioning 5, A. Campos1,4, E. Oliveira1,4, polymerases mid-reaction. Each exchange cycle lengthens the J.P. Albar S. Martínez-Bartolomé3,4, F. Canals2,4 effective read-length of the sequencer. In this manner, the read-length can be continuously extended without “gaps”. 1Barcelona Science Park, Barcelona, Spain; 2Vall d’Hebron Expanding upon this flexibility, after sequencing a particular University Hospital Research Institute, Barcelona, Spain; length of DNA, the newly synthesized strand can be selectively 3Centro Nacional de Biotecnologia-CSIC, Madrid, Spain; removed. The original genomic DNA strand is then re-primed, 4ProteoRed Consortium, Spanish National Institute of QdotTM-polymerase sequencers are rebound, and the identical Proteomics, Madrid, Spain; 5ProteoRed, National Center for genomic DNA strand can be sequenced again, greatly increasing Biotechnology-CSIC, Madrid, Spain the net accuracy and not requiring circularization of genomic templates. In combining these features, the desired accuracy An increasingly common request for proteomics core facilities is determining qualitative and quantitative differences among clinical and read-length can be “tuned” by adjusting the number of samples such as plasma, CSF, or urine. One of the missions of the reagent exchange cycles. Because each sequencing reaction can Spanish Network of Proteomics Facilities (ProteoRed-ISCIII) is to assist be completed in minutes, multiple exchange experiments can its proteomics core facilities in evaluating their capabilities to perform Abstracts be performed per sequencing hour. These QdotTM-polymerase qualitative and quantitative proteomics analysis. This year, in an attempt Scientific Session sequencers can also bind to ultra-long DNA segments (>10kb) to represent a realistic experiment scenario that might be requested to at multiple positions along the length of the DNA and a proteomics core facility, we provided a moderately complex plasma sequence while moving “horizontally” (parallel to TIRF field), standard reference sample to be used for routine QC monitoring of enabling the possibility of “ordered-reads” for long-phased laboratory instrumentation. The ProteoRed Plasma Reference (PPR) haplotype sequencing. Examples of real-time sequencing of sample is a subset of highly abundant well-characterized human plasma homopolymeric, patterned, and complex templates will be proteins with a number of isoforms, in addition to 4 spiked-in proteins, shown. altogether distributed over 5 orders of magnitude in concentration. The PPR sample was recently stress tested in the latest ProteoRed Multicenter Experiment (PME6) that counted with the participation of 17 proteomics facilities using a wide range of LC-MS platforms. We requested the sample be analyzed in a single LC-MS run in experimental triplicate (3 different digestions). Evaluation of the results submitted by the study participants revealed moderate discrepancies at the peptide identification level, and poor overlap at the protein identification level. In an attempt to identify the source of such irreproducibility, raw data of 8 laboratories (24 LC-MS runs) were reanalyzed centrally using a standardized data analysis pipeline, which included protein inference using ProteinProphet software. We found that the majority of protein identification discrepancies across submitted reports of these 8 laboratories were due to inconsistencies on how data analysis and computational tools group and/or infer proteins. Immunoglobulin

42 • ABRF 2011 — Technologies to Enable Personalized Medicine variable chain identifications were particularly conflicting throughout (S9-3) Why Reproducible Outcomes are Essential identification lists, even in the centralized analysis. Using a series of in Proteomic Research and Why Standardisation of LC-MS performance metrics, we benchmarked the performance of 8 Processes is Essential for Achieving Reproducibility LC-MS instruments (Orbitraps) and identified system components that vary the most across laboratories. A. Borthwick, W. Dracup (S9-2) Clinical Proteomic Technologies for Cancer: Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom NIH-Funded Measurement Science In both 2D electrophoresis and LC-MS proteomic analysis we are dealing with highly complex samples, and there are many complex processes C.R. Kinsinger, E. Boja, T. Hiltke, M. Mesri, A. Rahbar, R. Rivers, H. Rodriguez involved which in turn can be affected by a host of parameters and issues such as reagent batches, column performance, even the temperature of National Cancer Institute, Center for Strategic Scientific the lab. This complexity means that it can be very difficult to generate Initiatives, Office of Cancer Clinical Proteomic Research, the same results from the same samples in different labs and even in Bethesda, MD, United States the same lab at different times. This in turn makes it very difficult for labs to build upon published results, a fundamental principle of the In 2006, the National Cancer Institute (NCI) launched the Clinical scientific method. Quality Control (QC), based on the use of standards Proteomic Technologies for Cancer initiative (CPTC). The overall to monitor levels of technical variation in industrial processes is mission of this initiative was to foster the building of an integrated fundamental in the output of a reproducible product. We argue that foundation of proteomic technologies, data, analysis systems, and because proteomic analysis is significantly more challenging than most reagents and reference materials to systematically advance the industrial processes, employing standards and the standardisation of application of protein science to accelerate discovery and clinical processes in proteomics experiments is key to arriving at reproducible research in cancer. Specifically, the CPTC was charged to address outcomes. Focusing mainly on 2D electrophoresis and to some extent issues of variability and irreproducibility in proteomic measurements. on LC-MS we examine the importance of standardisation and how During the past five years, CPTC investigators have focused on such standards may be applied to Proteomic research in order to assessing proteomic platforms involving mass spectrometry. Inter- facilitate reproducible discoveries. Using studies carried out with single laboratory studies have addressed variability in both unbiased and and multi-users from within and between different laboratories, we targeted mass spectrometric methods. These studies have produced describe our experiences of achieving standardisation using Standard reference materials and data, performance metrics, standard operating samples to provide feedback on the reproducibility of each stage and procedures, and guidance for the community on the current ability as well as the complete proteomic workflow. of mass spectrometry for proteomics. Outputs from the technology assessment aspects of CPTC have leveraged additional developments. First, CPTC inter-laboratory studies provided a basis for engaging the FDA on the metrological requirements for approval in vitro diagnostic- multivariate index assays. Second, the NCI developed a follow-on funding opportunity that applies the technology pipeline developed in the first phase of CPTC. Scientific Session Abstracts

ABRF 2011 — Technologies to Enable Personalized Medicine • 43 Workshop Session Abstracts

(W1) The Diagnostics Core Facility: Harvesting disorders with exon by exon coverage of over 1,700 genes. We have the Promise of Personalized Medicine performed approximately 35,000 postnatal CMA tests. Additionally, inclusive of our reported experience with 300 prenatal cases (PMID 19012303), we now have CMA results on approximately 800 clinical (W1-1) Win on Sunday, Sell on Monday: From the prenatal samples. This talk will cover the utility CMA for chromosomal Exome Sequencing of One Boy to the Delivery of abnormality detection in the clinical lab as well as development and Clinical Diagnostics deployment of clinical tests from the core lab perspective.

M.R. Tschannen (W1-3) Whole Genome Sequencing in the Clinical Laboratory Human and Molecular Genetics Center, Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, T. Hambuch, M. Laurent, B. Sickler, A. Liao, P. Cotter, United States S. Jain, Y. Lyan, J. Bernd, J.O. Daniel, P. Poggio, M. Ross, D. Bentley For several years, there have been discussions about using both Sanger and whole genome sequencing in clinical practice. In late Illumina Clinical Services Laboratory, San Diego, CA, United 2009, the Medical College of Wisconsin initiated the infrastructure States to streamline the delivery of current and emerging DNA technologies into state-of-the-art molecular diagnostics. The online publication of The advent of routine whole genome sequencing creates an opportunity our initial case in Genetics of Medicine in late 2010 further intensified to provide an accurate, comprehensive and cost-effective catalogue of our efforts in this endeavor. However, being relatively new to the germline variation for an individual. The process of sequencing and field of NextGen sequencing, we began with the addition of Sanger delivering genomes for individual use must be driven by clinical and diagnostic sequencing to our already successful research core, which educational opportunities balanced by addressing ethical concerns. at that point had been in operation for almost ten years. This was a Using guidelines issued from professional and accrediting agencies great undertaking, as typically, independent research laboratories as well as an independent ethics board, we developed and launched performing cutting-edge science lack the financial resources and individual genome sequencing (IGS) as a physician-led service. A breadth of experience to launch their custom product or application physician orders the sequence and obtains informed consent from the to the diagnostic industry. An independent research laboratory is able individual; the sample is sequenced within a CLIA certified laboratory to resolve these shortages by partnering with a core laboratory staffed and after a series of quality checks the sequence is returned to the with diagnostic expertise. Due to our lack of diagnostic experience, we physician for communication back to the individual. The sequencing quickly aligned the research core to a consortium of individuals with platform and process were validated for accuracy and precision, and clinical experience to allow us to benefit from established diagnostic accredited following review by a College of American Pathologist facilities on campus. Difficulties faced at the onset of diagnostic startup (CAP) inspection team. Each individual genome is sequenced at >30 were many, including large issues such as accreditation program (CAP fold coverage using paired-end reads of 100 base pairs. Resulting vs. CLIA), SOP generation and validation, competency and proficiency sequence information is provided for >93% of the NCBI36 genome, the testing, and reimbursement, as well as smaller problems like semi- remainder being mostly recently duplicated repeats where ambiguous annual pipette calibration, temperature monitoring, and inventory read alignment is not permitted in our ELAND analysis. On average, we Abstracts control. The purpose of this talk is to give insight into efficient ways to detect over 3 million SNPs most of which are previously documented

Workshop Session Workshop resolve these problems, both large and small, and transform a decade in dbSNP129. The overall accuracy of our base calling is measured as or more of research expertise into a viable diagnostic laboratory. >99.99% and the accuracy for SNP calling is >99.7% based on multiple methodlogical assessments. The aim of the Illumina Clinical Services (W1-2) Chromosomal Microarray Analysis in the Laboratory is to make Individual Genome Sequencing accurate, Clinic accessible and clinically relevant for physicians and patients through a fully accredited process. Here we have established baseline processes, L.D. White tools, and policies to maximize the benefit to patients and minimize potential misuse. Additionally, our ongoing efforts to develop clinically Microarray Core, Baylor College of Medicine, Houston, TX, relevant interpretation tools for physicians are described in a separate United States abstract (see M. Ross). Individual Genome Sequencing has the capacity to replace current genetic testing with a near-complete description of Current Array Comparative Genomic Hybridization (aCGH) or the sequence of an individual. Considering this potential, it is essential Chromosomal Microarray Analysis (CMA) performed at Baylor to engage policy makers and ethicists so that appropriate policies College of Medicine utilizes the latest microarray technology to detect are developed around information access and use of whole genome unbalanced chromosome abnormalities associated with over 210 clinical information.

44 • ABRF 2011 — Technologies to Enable Personalized Medicine (W2) Spectral and Sequence Database and actually disagreed on over 20% of all cases where at least two Searching in Proteomics participants called a modification site. In this talk we will cover current and novel methods for identification of post-translationally modified peptides and automated determination of site localization confidence L. Martens scores and false discovery rates. Ghent University, Ghent, Belgium (W2-3) Building and Using MS/MS Spectral Libraries for Peptide Identifications in Proteomics This session will present an overview of more advanced methods to match acquired MS/MS spectra to peptides, including strategies to detect (unexpected) protein modifications, and the use of spectral H. Lam libraries for peptide identification by spectrum-to-spectrum matching. The Hong Kong University of Science and Technology, Clear Introduced by some of the world’s foremost experts in these proteomics Water Bay, Hong Kong, China informatics challenges, this session is meant to provide a pragmatic introduction to the topics for interested researchers, thus making these Spectral library searching is an emerging approach in peptide methods directly adoptable in the lab. identifications from tandem mass spectra, a critical step in proteomic data analysis. In this approach, a spectral library is first meticulously (W2-1) ETD Performance and Complementarity compiled from a large collection of previously observed and identified to Other Fragmentation Methods for Proteomic peptide MS/MS spectra. An unknown spectrum is then identified by Analysis comparing it to all the candidates in the spectral library for the most similar match. Thanks to the reduction of search space to only the R. Chalkley previously discovered peptides, and the use of real, experimentally observed reference spectra for more precise spectral matching, this University of California San Francisco, San Francisco, CA, approach is considerably faster and more sensitive than the popular United States alternative of sequence searching. This talk will explain the basic principles of spectral library building and searching, describe its Radical-driven fragmentation approaches present an alternative to the advantages and limitations, and provide a starting point for researchers well-established collisional cleavage approaches that have dominated interested in adopting this new approach in their data analysis. It will proteomic research up until now. With the recent availability of also discuss the future outlook on the evolution and utility of spectral electron transfer dissociation (ETD) in commercial quadrupole ion libraries in the field of proteomics. trap and hybrid instruments, this technology is now accessible to many researchers. It has been described as a complementary approach (W3) Quantifying Protein Turnover by In Vivo to CID, and decision tree approaches have been employed where a choice between CID or ETD is made depending on the precursor m/z Metabolic Labeling and charge. In this presentation I will discuss the performance of ETD for peptide identification, drawing heavily on data acquired as part of the (W3-1) Stable Isotope Tracers Applied to Measuring 2011 iPRG study ‘Identification of Electron Transfer Dissocation (ETD) Rates of Protein Synthesis and Breakdown in Muscle: Mass Spectra’. Results will be compared to analyses of the same sample Principles and Applications using CID and HCD, decision tree approaches and the difference Workshop Session in measuring data in the ion trap versus in the orbitrap detector. A Abstracts comparison of search engine performance will be presented and R.R. Wolfe methods for improving database search engine analysis of ETD data University of Arkansas for Medical Sciences, Little Rock, AR, will also be discussed. United States

(W2-2) Discovery, Identification and Localization of Muscle is in a constant state of turnover, meaning that it is continuously Post-Translational Modifications synthesized and broken down. The balance between the rates of synthesis and breakdown determines if an individual is gaining or N. Bandeira loosing muscle mass. It is therefore of interest in a variety of physiological circumstances to quantify the rates of muscle protein synthesis and Center for Computational Mass Spectrometry, Department breakdown. Tracer methodology using both radioactive and stable of Computer Science and Engineering Skaggs School of isotopes has been used in a wide variety of kinetic studies, including Pharmacy and Pharmaceutical Sciences University of measurement of synthetic and breakdown rates of various compounds. California, San Diego, CA, United States Stable isotopes are particularly suited for the study of muscle protein metabolism, as multiple amino acid tracers can be used simultaneously, Mass spectrometry based analysis of post-translational modifications and multiple labels can be used with any individual amino acid, commonly report thousands of modified-peptide identifications including labeling the nitrogen with 15N. Two basic approaches can accompanied by both precisely and ambiguously localized be used to measure muscle protein synthesis. The direct incorporation modification sites. Since these identifications often motivate extensive of a labeled amino acid is the most conventional. This technique follow up studies, the confident identification of the peptide and involves infusion or injection of tracer and measurement of subsequent accurate localization of the modification site(s) remains one of the incorporation into muscle protein over time. The measurement of the major challenges in computational proteomics. As revealed by the precursor enrichment, which is usually taken to be the free intracellular 2010 iPRG study on identification of phosphopeptides and localization pool of the tracer amino acid, is necessary to calculate the actual rate of phosphorylation sites, participants only attempted to call the of synthesis. Alternatively, the rate of muscle protein synthesis can be modification sites for less than 2 out of every 3 identified spectra derived from the rate of uptake of an amino acid from blood. In this

ABRF 2011 — Technologies to Enable Personalized Medicine • 45 case incorporation of the amino acid into protein must be its only the best solubility enhancing tags, and methods for subsequent tag metabolic fate within the muscle. The basic principle of measuring removal, will be presented. Finally, I will also describe two case studies muscle protein breakdown is to determine the rate at which intracellular which required highly tailored expression protocols for the production amino acid tracer is diluted by the appearance of unlabeled amino acid of a bacterial toxin and eukaryotic phosphatase. In summary, the focus that is not coming from the plasma. There are different approaches of this lecture will be to provide practical information that researchers to accomplishing this measurement, and the choice of the optimal actively involved in protein purification can readily implement into depends of the method used to measure protein synthesis. Ideally, their own workflows. This work was supported in part by a medical methods to measure synthesis and breakdown are compatible (i.e., the research grant from the American Cancer Society (RSG-08-067-01-LIB) same units) to enable calculation of the balance between synthesis and and an NSF-CAREER award (MCB 0952550). breakdown to determine if there is a net gain or loss in muscle protein. (W5) Proteomics Tips and Tricks: From (W3-2) In Vivo Stable Isotope Labeling for Discovery to Protein-Protein Interactions Quantifying Amyloid-beta Kinetics in Alzheimer’s Disease: Is it All in our Head? (W5-1) Proteomics Tips and Tricks: From Discovery K.E. Yarasheski to Protein-Protein Interactions

Biomedical Mass Spectrometry Research Laboratory, M. Cilia Washington University School of Medicine, St. Louis, MO, United States United States Department of Agriculture/Agricultural Research Service, Robert W. Holley Center for Agriculture Mass spectrometry has revolutionized the manner in which we identify, and Health, Cornell University, Ithaca, NY, United States characterize, and quantify proteins. In combination with in vivo stable isotope labeling strategies, mass spectrometry-based analyses can Proteins are the functional constituents of cells. The diverse physical provide valuable information about human amino acid and protein properties of proteins enable them to be multifunctional and also pose kinetics, protein production (synthesis), and clearance (proteolysis) challenges to analyzing their abundance, subcellular localization, and rates. Dysregulated or imbalanced protein synthesis and degradation potential interacting partners using proteomics. Some samples are rates is the basis for many clinical disorders. These protein kinetic especially recalcitrant and pose additional challenges in proteomic rates can be quantified in vivo and serve to identify potential targets analysis due to the unanticipated modification of proteins. These for novel drug therapies. Our group uses an intravenous infusion of challenges extend from initial protein extraction to analysis using 13C6-Leu, cerebral spinal fluid (CSF) sampling, affinity isolation of mass spectrometry. Broadly, the focus of workshop will be to discuss relevant proteins, and tandem mass spectrometry to quantify 13C6- the benefits and limitations of using proteomics to study protein Leu incorporation and removal rates from CSF amyloid-beta and identification, protein quantification, protein structure, and protein- apolipoproteins in Alzheimer’s disease patients. This approach has protein interactions. Examples will be drawn from different biological identified slower amyloid-beta clearance rates as a primary lesion that disciplines, including cell biology, host-pathogen biology, and may explain an accumulation of amyloid plaques in Alzheimer’s disease. developmental biology to demonstrate how scientists are solving the problems posed by their discovery methods and biological systems. (W4) Insights for Expression of Recombinant The workshop will include presentations from each panelist followed Proteins for Drug Target Validation by an open discussion and question and answer session. (W5-2) Protein Interactions and Topologies in Cells (W4-2) Strategies for Optimized High-Throughput Abstracts Cloning, Expression and Purification of Recombinant J. Bruce Workshop Session Workshop Proteins in E. coli University of Washington, Department of Genome Sciences, Seattle, WA, United States R. Page Brown University, Department of Molecular Biology, Cell Life on earth has evolved to utilize proteins as functional molecules due Biology and Biochemistry, Center for Genetics, Genomics to the wide diversity of structures and physical properties this class of molecules can exhibit. However, the divergent properties that critically and Proteomics, Providence, RI, United States support life also pose fundamental challenges in all efforts to measure The arrival of structural genomics, in addition to large-scale efforts the proteome. As a result, most proteomics experiments only sample initiated in pharmaceutical companies, have resulted in the development a small subset of expressed molecules and typically appear biased of numerous new methods and strategies to minimize the time required towards cytoplasmic proteins. Furthermore, detection of protein- to optimize the cloning, expression and purification of novel protein protein interactions is even more challenging. For example, most large- drug targets. Here, I will present both best practices and unusual (‘last scale methods such as the yeast two-hybrid, tandem affinity purification, ditch’) methods that are used to successfully express and purify both and co-IP methods are predicated on the production/maintenance prokaryotic and eukaryotic proteins in E. coli. Recent developments in of native protein structures and co-localization of native interacting protein expression, including co-expression with protein partners and partners. These two requirements can result in failure to identify many bacterial chaperones will be presented. In addition, a comparison of bone fide interactions and in false discovery because many interactions

46 • ABRF 2011 — Technologies to Enable Personalized Medicine may not survive cell lysis steps and non-relevant interactions can form (W5-4) Towards the Development of Proteomics in cell lysates during sample preparation. Significant progress on Workflows for the Analysis of Samples Derived from many of these issues has been achieved in recent years, including the Refractory Plant Tissues use of cryogenic cell lysis techniques. Alternatively, covalent linkage of interacting proteins within cells as accomplished with chemical T.W. Thannhauser cross-linking has long held potential for protein interaction studies. If successful, chemical cross-linking approaches mitigate the need United States Department of Agriculture/Agricultural to maintain native interactions and structures during subsequent Research Service, Ithaca, NY, United States proteome sample preparation and may offer unique insight on protein interactions and structures present within cells. However, the analysis of Carrying out proteomic analyses in plant tissues involves dealing with cross-linked peptides presents a different set of challenges related to a number of specialized challenges that can make protein extraction dynamic range and detection specificity. This presentation will highlight and quantification significantly more difficult than in other organisms. advancements in technology, informatics and sample preparation steps In addition to having relatively low protein concentrations, plant tissues we have pursued to enable cross-linked peptide measurements from in are often rich in proteases, protease inhibitors and other materials that vivo cross-linking experiments and will illustrate the unique information impede protein analysis. These compounds include lipids, tannins, that can be derived from this approach. polysaccharides, and a large variety of secondary metabolites. The extent of the problems encountered is dependent on tissue type (W5-3) The Virus-Host Interface: Exploring Dynamic studied. Two major research thrusts in our lab involve detailed Protein Interactions via Targeted Proteomics developmental and time-course studies on staple crops and fruits. An understanding of the proteins involved in the development of fruits, I. Cristea seeds, tubers and other plant organs will enhance our ability to control the agronomically-important traits of these crops including stability Princeton University, Department of Molecular Biology, in storage, disease resistance, and vitamin and mineral content. Two Princeton, NJ, United States recent studies that have posed unique challenges for proteome analysis include 1) comparing protein expression in red ripe tomato fruit to that Dynamic protein interactions carry out the majority of the processes in mature green tomato fruit and 2) studying the effects of storage on within a cell, including cellular responses to environmental stimuli potato tubers. This talk will focus on the problems encountered in these and pathogens. Isolation and characterization of protein complexes recalcitrant tissue types and our efforts to provide plant scientists with can provide invaluable insights into their biological functions. The a broader array of extraction and chemical modification protocols than development of approaches that can access stable and transient is represented by those that constitute the existing paradigm. Efforts interactions is invaluable for numerous fields of study, including that to move away from narrowly defined prescriptive methods and move of temporal and spatial virus-host protein interactions. Viruses have towards results-based methods will also be discussed. co-evolved with their hosts, developing remarkable mechanisms for subverting cellular processes for their own benefit. The study of virus- (W6) Current State and Future of Capillary host interactions has therefore emerged as a driving force in infectious disease research. Despite these efforts, the protein interactome Electrophoresis and Sanger Sequencing remains in large part uncharted, and our knowledge of mechanisms controlling the outcome of an infection is limited. Modern proteomics 1 2 3 4 J. Kieleczawa , D. Adam , P. Schweitzer , E. Vennemeyer , techniques are currently emerging as powerful tools, bringing a new M. Zianni5 Workshop Session perspective to the field of virology. This presentation will describe the Abstracts integration of targeted proteomics with genetic, molecular biology, 1Pfizer, Inc., Cambridge, MA, United States; 2NAPS Unit, and bioinformatics techniques for studying dynamic virus-host protein Michael Smith Laboratories, University of British Columbia, associations. Strategies for isolating protein complexes, quantifying Vancouver, BC, Canada; 3 DNA Sequencing and Genotyping infection-triggered changes in interactions, and assessing interaction Lab, Cornell University, Ithaca, NY, United States; specificity will be presented. In studies of human cytomegalovirus 4LifeTechnologies, Carlsbad, CA, United States; 5Plant- (HCMV) infection, we discovered parallel processes occurring at distinct Microbe Genomics Facility, Ohio State University, Columbus, cellular sites during the assembly of infectious virions. Additionally, we have observed that certain viral proteins recruit chromatin-remodeling OH, United States enzymes, such as histone deacetylases, indicating a possible mean of Capillary Electrophoresis remains a widely used, often irreplaceable, controlling virus or host gene expression. In addition to revealing their sequencing technology continuing to experience increasing demands. functional roles during infection, our studies provided insights into We believe that the speed, simplicity and high quality of reads for low- the regulation of these enzymes outside the context of infection. A volume sequencing needs will maintain the viability of this technology combinatorial proteomics approach, incorporating CID, HCD and ETD for many years to come. Although the main thrust of development peptide fragmentation using a nLC LTQ Orbitrap Velos-ETD, identified in the sequencing space is now in NGS area, there are still steps 17 in vivo phosphorylation sites on HDAC5. Functional phosphomutant which can be improved or adjusted as other methods mature. In this screening and live cell imaging allowed the characterization of novel sites session we propose to cover: The effect of long-term storage of DNA within functional domains, and identified a previously unrecognized under different conditions (temp/buffers) on sequencing, spectral regulatory point of its nuclear import. characteristics, integrity and transformation efficiency; A Description of the Apollo system; and the Future of Capillary Electrophoresis and Sanger sequencing. In addition to the featured talks, there will be a question & answer component in which participants are encouraged to bring up any issues relevant to this topic.

ABRF 2011 — Technologies to Enable Personalized Medicine • 47 (W7) Cellular 3D Imaging (W8) Successful Production of Functional Proteins (W7-1) 3D Cellular Imaging: Beyond the Simple Imaging Paradigm (W8-1) Successful Protein Production

R. Cole J. Culp Wadsworth Center, New York State Department of Health, Pfizer, Inc., Groton, CT, United States Albany, NY, United States Successful production of functional proteins is more than an The modern microscope has advanced so far beyond what Leeuwenhoek immunoreactive band on a Western blot. Availability of multiple invented, it’s doubtful he would even recognize it as a microscope. expression vectors make accessible a variety of expression systems and Modern microscopes, or more acutely, imaging workstations, are parallel expression approaches can speed results and increase chance an integrated compilation of optoelectronic components. It is now of success. The next hurdle is isolation of the protein target in sufficient possible to produce much more than simple images using these amounts and with sufficient purity to support subsequent experimental systems. Quantitation of various metrics are now possible with these work. Occasionally, protein refolding is the only method available to advanced instruments. With the advent of fluorescently-tagged achieve the desired protein. Finally, sufficient characterization of proteins/organelles, it is possible to actually quantify sub-cellular the purified protein is required to verify that the protein is in the amounts of these transgenically produced or exogenously labeled biologically relevant state. Often, the protein is heterogeneous with macromolecules/complexes. Spectral un-mixing of overlapping signals post-translational modifications and may be aggregated or in multiple from within a single image (fluorescent and absorbent) has blurred oligomeric complexes. distinction between imaging and spectroscopy. It is now possible to get chemical data from techniques such as unmixing in addition to (W8-2) Overcoming Problems in Protein Expression more conventional modes such as polarized light microscopy. The and Purification ability to determine if two macromolecules are precisely co-localized (< 100 Å apart) is now routinely accomplished in both living and fixed B. Gillette preparations with Fluorescence Resonance Energy Transfer (FRET). The latest technological advances has been achieving resolution beyond SAIC-Frederick, Inc., National Cancer Institute at Frederick, Abbe’s predicted limits. This has been accomplished both through Frederick, MD, United States hardware and software improvements, such as Stimulated Emission Depletion (STED), and Structured Illumination Microscopy (SIM) to We use a parallel, micro-scale protein purification approach to screen name a couple. purification outcomes for multiple expression constructs from multiple expression systems. Method development and optimization for lead (W7-2) Basics of Colocalization Analyses candidates are then performed on the same platform. Scale-up from the micro-scale format (10-160 microliter column volumes available) to bench-top scale (5-100 milliliter column volume) has proven J. Lacoste predictive in terms of qualitative and quantitative results. The increase Cell Imaging and Analysis Network, Department of Biology, in throughput allows us to follow a “purify first” approach that, while McGill University, Montreal, QU, Canada still providing data on expression levels and solubility, also produces valuable purification information within the same time-frame as a For many fluorescence microscopy-based projects, colocalization traditional solubility assessment approach. analyses are often the main objectives of the study, and detecting Abstracts “yellow” is considered a proof of colocalization. However, the “yellow” (W8-3) When Proteins Misbehave, Try Adding a Workshop Session Workshop statement is essentially a qualitative one which opens the door to Little Pressure more quantitative approaches. This presentation will provide a quick overview of colocalization analysis methods. First the required R.M. Petrovich preliminary steps and words of caution will be discussed. Secondly, intensity correlation coefficient -based methods (both the traditional National Institute of Environmental Health Sciences and more modern ones) will be presented, and object-based analysis Laboratory of Structural Biology, Research Triangle Park, methods will next be introduced. Lastly, it is important to remember NC, United States that colocalization studies are dependent on quantitative microscopy and as such requires careful considerations. The most economical way to produce the mg quantities of purified protein required by many studies still requires expression in E. coli. Unfortunately, between 50% and 70% the time, a given protein will not express properly in bacteria. Of those that are correctly folded, a percentage of them will either lose activity during purification or cold storage once purified. To overcome these obstacles we have begun using high hydrostatic pressure refolding (Barofold) which allows us to refold protein at high concentration (1 mg/ml or higher) without adding and removing chaotropic agents.

48 • ABRF 2011 — Technologies to Enable Personalized Medicine (W9) Therapeutic Antibodies: Over-Hyped study of a breast cancer samples treated with antineoplastic agents “Magic Bullet”, or Under-Explored Technology? including the novel drug compounds — RITA and Nutlin, targeting p53 and Mdm2. We analyzed promoters of downregulated pro-survival genes and identified combinations of transcription factors involved D.L. Crimmins1, J.E. Harlan2, F. Weis-Garcia3 in their regulation. Topological modeling of the signal transduction network upstream of these transcription factors revealed key-nodes 1Washington University School of Medicine, Saint Louis, MO, — potent master-regulators of the cell survival program that prevent United States; 2Abbott Laboratories, Abbott Park, IL, United efficient apoptosis of cancer cells. We considered these key-node States, 3Memorial Sloan-Kettering Cancer Center, New York proteins (e.g. PI3K subunits) as causal biomarkers as well as prospective City, NY, United States targets for novel anticancer drug combinations. We applied a cheminformatics computer tool PASS to these targets and identified Bringing the vision that monoclonal antibodies (mAbs) could be two novel prospective antineoplastic chemical compounds which were medicinal “magic bullets” into practice has been a long and challenging experimentally validated in a cellular assay confirming their synergistic journey which began in 1975 when Kohler and Milstein made it possible potential in highly selective triggering of apoptosis of cancer cells. to generate these homogenous, highly specific homing devices. In the last 35 years, many hurdles have slowed the manifestation of this dream, (W10-2) Pathway Analysis in Expression Proteomics including: target location; host species, specificity, and subclass of the mAb; immunogenicity of murine mAbs; mAb pharmacokinetics (i.e. R.A. Zubarev hepatic and renal clearance); and mAb form (i.e. natural, conjugated to a toxin or radionuclide, humanized murine mAbs, scFvs). The work Chemistry I Division, Department of Medical Biochemistry involved in overcoming these factors explains why few mAb’s have been and Biophysics, Karolinska Institutet, Stockholm, Sweden made it to the clinic in more than 3 decades later. Nonetheless, over 20 mAbs have been FDA-approved for human use since 1994. Since Proteomics studies have revealed unexpected plasticity and dynamic the total global therapeutic monoclonal antibody market increased nature of the human proteome. The paradigm that the time evolution from $40 billion in 2009 to $45 billion in 2010, pursuit of these “magic of a biological system can be described by abundance variation of bullets” is unlikely to wane anytime soon. This workshop is a roundtable relatively few “regulated” proteins has been shuttered, being replaced format what where we will collectively discuss what we have learned on by the growing understanding that the whole proteome is regulated, this long road of bringing mAbs to the clinic and try and look forward and no protein remains unaffected when the system undergoes to where we are going. transition from one state to another. This finding underlines the importance of systems biology analysis of expression proteomics data. Systems biology shifts the analytical focus from thousands of proteins to (W10) Identification of Mechanism-Based hundreds of signaling pathways, thus reducing the number of entities to Biomarkers and Drug Targets Using Pathway be analyzed. Application of these methods required the development Analysis of 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. (W10-1) GeneXplain — Identification of Causal predict not only the activated pathway, but also the relative degree of its Biomarkers and Drug Targets in Personalized Cancer activation. Introducing the quantitative aspect in systems biology is one of the greatest challenges this field is facing today, since the final goal

Pathways of pathway analysis, which is the creation of a quantitative predicting Workshop Session

model of the biological process under investigation. 1. Zubarev, R. A.; Abstracts 1, F. Kolpakov2, V. Poroikov3, G. Selivanova4 A. Kel Nielsen, M. L.; Savitski, M. M.; Kel-Margoulis, O.; Wingender, E.; Kel, 1geneXplain GmbH, Wolfenbuettel, Germany; 2Institute A. Identification of dominant signaling pathways from proteomics of Systems Biology Ltd., Novosibirsk, Russia; 3Institute of expression data, J. Proteomics, 2008, 1, 89-96. 2. Stahl, S.; Fung, Y.M.E.; Biomedical Chemistry of Russian Academy of Medical Adams, C. M.; Lengqvist, J.; Mörk, B.; Stenerlöw, B.; Lewensohn, R.; Lehtiö, J.; Zubarev, R. A.; Viktorsson, K. Proteomics and Pathway Analysis Sciences, Moscow, Russia; 4Microbiology and Tumor Biology Identifies JNK-signaling as Critical for High-LET Radiation-induced Center (MTC), Karolinska Institutet, Stockholm, Sweden Apoptosis in Non-Small Lung Cancer Cells, Mol. Cell Proteomics, 2009, 8, 1117-1129. 3. Marin-Vicente, C.; Zubarev, R. A. Search engine for We have developed a geneXplainTM platform (www.genexplain.com) proteomics, Fact or Fiction? G.I.T. Lab J, 2009, 11-12, 10-11. for causal interpretation of data coming from microarray, proteomics, miRNA and ChIP-chip/seq experiments. GeneXplainTM applies a unique upstream analysis approach based on implementation of (W10-3) Metabolic Biomarkers, Metabolic machine learning and graph topological analysis algorithms in order Networks, and Pathway Analysis to identify causality keynodes in the network of gene regulation and signal transduction and combines it with full genome sequence analysis V. Tolstikov and chemoinformatics methods for drug discovery. The power of this University of California Davis Genome Center, Davis, CA, approach is that we are trying to identify causal biomarkers — those United States which are more than just correlating with disease or treatment outcome but which are parts of the disease mechanism, which may differ in We apply well established MS-based Metabolomics platform towards patient cohorts. Such personalized networks are analyzed in order to the detection, characterization and identification of small molecule find key nodes — most important nodes triggering the disease. These biomarkers. Data acquisition is performed with the assistance of GC/ keynodes and genes directly influenced by them are considered as TOF/MS line and LC/MS line. Sophisticated data mining approach using promising biomarkers which can discriminate patients into cohorts different algorithms deals with electron impact (EI) generated data as well from the disease mechanism point of view. In the current study, we as data generated with the unit resolution, high resolution and ultrahigh analyzed a large scale gene expression and ChIP-seq data from a

ABRF 2011 — Technologies to Enable Personalized Medicine • 49 resolution hybrid instruments having API sources. These competitive (W12-2) Microarray Analysis of Fluorescence approaches deliver exciting results on selection of the predictors’ panel Activated Cell Sorter-Derived Cells: Creating and potential metabolic biomarkers discovery. We here report on Harmony Between Technologies efficiency of Metabolic Biomarkers Discovery Project as a platform for cases study: Kidney diseases (RCC and PKD); Pancreatic Cancer (PDAC); S. Tighe Human Embryonic Stem Cells functional characterization. However, Metabolic Networks simulation and Metabolic Pathway Analysis, University of Vermont, Advanced Genome Technology Core, which could be essential tools for Metabolic Biomarkers validation and Burlington, VT, United States connections with underlying biochemical processes are still difficult and approached not systematically, but rather case wise. Challenges Although microarray technology is well-established in both the and developments in this field will be further presented and discussed. research and clinical fields, it continues to evolve into new areas that require new methods for the successful isolations of nucleic acid from (W11) The Business of Running a Core Facility non-traditional sources. Because RNA specifically is a labile molecule, special procedures and considerations must be implemented to avoid degradation from methods such as fluorescence activated cell sorting N.P. Ambulos, Jr.1, S.A. Bobin2 (FACS) and laser capture microdissection (LCM) to name a few. This presentation will discuss specific methodologies to maximize the success 1 University of Maryland School of Medicine, Baltimore, MD, of nucleic acid recovery from these approaches including instrument United States; 2Dartmouth School of Medicine, Hanover, NH, preparation, extraction methods, and the use of special reagents to United States deal with problematic samples. The success and sustainability of a Core Facility has been dependent (W12-3) Microarray Futures: Don’t Decommission on factors which include (1) the ability to maintain state-of-the- Your Scanners Just Yet art technology, (2) recruiting and retaining talented technical staff, (3) a strong financial management plan, and (4) strong Institutional S.D. Crosby support from both end users and the Administration. Historically, most core facilities enjoyed generous subsidies from their Institutional Department of Genetics, Washington University School of Administration, which have helped defray costs for end users, purchase Medicine, St. Louis, MO, United States new instrumentation, or to hire new staff. In light of the current economy, it is likely that many core facilities are now seeing these subsidies The first attempt to wind down the Washington University Microarray drastically cut, or even eliminated. In essence, core facilities are faced Facility was made by Genome Sequencing Center in 2008. It was with operating as a self-supporting ‘business’, and at the same time, clear to most of us involved in the attempt that, with the advent of ensure compliance with federal guidelines. This workshop will present NGS, microarrays were headed the way of differential display PCR ideas, both conventional and creative, that might provide core directors (remember that!?). The attempt failed, and 2010 was the busiest year with the tools necessary to continue to sustain a successful core facility. yet for our microarray facility. Sequence remains a bit too expensive This will set the stage for an open discussion with the audience to and complex for many to make the leap. In addition, because of the encourage a free-flow of additional ideas that could benefit the future rarity of variants and the modest number of bases required to identify success of our cores. a locus, most sequence data is superfluous. Beyond that, as the actual cost of sequence continues to fall, so do the price of arrays, while the (W12) Microarrays: The Reports of My Death density of elements of the latter rises. It seems that until the advent of Have Been Greatly Exaggerated reagentless sequencing, it is unlikely that the actual cost of sequencing will be less than the cost of an array that covers the same loci. While

Abstracts sequencing technology will remain an important tool for discovery,

Workshop Session Workshop (W12-1) Chip or Seq: Helping Clients Choose over time most exons and biologically relevant variants will be captured in toto on low cost arrays. Hospital or contract labs will (indeed are D. Baldwin already) use small, cheap arrays that capture disease relevant genes and variants. University of Pennsylvania, Penn Molecular Profiling Facility, Philadelphia, PA, United States

The Penn Molecular Profiling Facility provides services using microarray and deep sequencing platforms for a variety of applications. Drawing from experiences helping prospective clients to choose one approach or the other, and sometimes both, this portion of the workshop will address some of the factors to consider when thinking about a new project. The budgeting process will be discussed, with particular focus on the use of interactive cost calculators.

50 • ABRF 2011 — Technologies to Enable Personalized Medicine (W13) Institutional Core Management in a Unix / Linux environment. Researchers unfamiliar with the Unix command line may be unable to use these tools, or face a steep learning curve in trying to do so. Commercial packages exist, such as the CLC S. Meyn1, P. Turpen2, G.K. Farber3, S. Mische4, Genomics Workbench, DNANexus, and GenomeQuest. However, P. Alexander5, J. Auger6 these commercial packages often incorporate proprietary algorithms to perform data analysis and may be costly. Galaxy provides a solution 1Vanderbilt University Medical Center, Nashville, TN, United to this problem by incorporating popular open-source and community States; 2University of Nebraska Medical Center, Omaha, linux command line tools into an easy to use web-based environment. NE, United States; 3National Center for Research Resources, After sequence data has been uploaded and mapped, there are a Bethesda, MD, United States; 4New York University Langone variety of workflows for NGS analyses that use open-source tools. This Medical Center, New York, NY, United States; 5Morehouse includes peak-calling analyses for ChIP-Seq (MACS, GeneTrack indexer, School of Medicine, Atlanta, GA, United States; 6University of Peak predictor), RNA-Seq (Tophat, Cufflinks), and finding small California San Francisco, San Francisco, CA, United States insertions, deletions, and SNPs using SAMtools. Any researcher can apply a workflow to his NGS data and retrieve results, without having to This workshop session will focus on issues related to Institutional interact with a command line. Additionally, since Galaxy is cloud-based, Core Management, in response to the national conversation evolving expensive computing hardware for performing analyses is not needed. around research core facility issues and management. The workshop In this presentation we will provide an overview of two popular open- will be formatted as an experts’ panel; each participant currently source RNA-Seq analysis tools, Tophat and Cufflinks, and demonstrate plays an important role in supporting and developing research core how they can be used in Galaxy. resources at an institutional level. Some of the topics to be discussed include: (1) Core Consolidation — one size fits all? (2) Bottom-up vs. (W14-2) GenomeView: Visualizing the Next- top-down management, advantages and disadvantages of centrally Generation of Data managed cores. (3) Performance metrics and impacts on professional development, core infrastructure support and improved operations. T. Abeel (4) Impacts of NIH-NCRR programs on improving access to research Broad Institute of MIT and Harvard, Cambridge, MA, United resources, including core facilities. We also plan to highlight the new Core Administrators Network Coordinating (CAN). In response States, and VIB Department of Plant Systems Biology, Ghent to an emerging trend to centralize the oversight of research core University, Ghent, Belgium facilities, ABRF has fostered development of this network and a Due to recent advances in sequencing technologies, billions of nucleotide new committee: the Core Administrators Network-Coordinating sequences are now produced on a daily basis. A major challenge is Committee (CAN-CC). The committee seeks input and participation to visualize these data, including both whole genome sequence and from scientists, administrators and others with an interest in issues transcriptome data, for further downstream analyses. Visualization is related to the administration of research core facilities which, by the often overlooked and undervalued, but it is an extremely valuable to nature of their service role, must interface with multiple constituencies explore your data on several levels. A first area where visualization shines within a research enterprise. Today many institutions have established is at the early stages of data analysis to perform sanity checks on your administrative positions designed to assist core facilities with data. Eye-balling your data in a visually pleasing way is the best way to management of economic, regulatory and performance issues. In order get a good feel on what came out of your experiments. Once you have to facilitate greater interaction between and among core scientists a good idea of what is in your data a good visual representation can be Workshop Session and administrators, the mission of the CAN-CC is to contribute to the used to generate new hypotheses and to fine-tune analysis parameters. Abstracts common interests of core administrators, and promote interactions with The appropriate image often makes the solution obvious and as such, it core scientists in a collegial and productive manner. The specific goals really makes it easier to develop algorithms. The ability to interactively of the Core Administrators Network Coordinating Committee (CAN- explore gives you insights in large-scale data sets and definitely CC) are: to identify and reach out to our target community; provide augments our ability to reason about complex data. To this end, we opportunities for networking; and assess goals for program focus and present GenomeView, a stand-alone sequence browser specifically development. designed to visualize and manipulate a multitude of genomics data. GenomeView enables users to dynamically browse high volumes of (W14) Next Generation Sequencing Software aligned short read data, with dynamic navigation and semantic zooming, for Data Management, Analysis, and from the whole genome level to the single nucleotide. At the same time, Visualization the tool enables visualization of whole genome alignments of dozens of genomes relative to a reference sequence. GenomeView is unique in its capability to interactively handle huge data sets consisting of dozens (W14-1) Tools for Next Generation Sequencing Data of aligned genomes, thousands of annotation features and millions of Analysis mapped short reads both as viewer and editor. GenomeView is freely available for academic use as an open source software package at K. Bodi http://genomeview.org. Tufts University School of Medicine, Tufts University Core Facility, Boston, MA, United States

As NGS technology continues to improve, the amount of data generated per run grows exponentially. Unfortunately, the primary bottleneck in NGS studies is still bioinformatics analysis. Not all researchers have access to a bioinformatics core or dedicated bioinformatician. Additionally, much of the software for NGS analyses is written to run

ABRF 2011 — Technologies to Enable Personalized Medicine • 51 (W14-3) Galaxy Next Generation Sequencing (W15) ETD Workshop Seminar Series: Manual Functionality from Sample Tracking to SNP Calling Interpretation of Electron Transfer Dissociation (ETD) Mass Spectra of Peptides G. Von Kuster1, The Galaxy Team1,2 1Center for Comparative Genomics and Bioinformatics, D.F. Hunt Pennsylvania State University, University Park, PA, United States; 2Departments of Biology and Mathematics & Departments of Chemistry and Pathology, University of Computer Science, Emory University, Atlanta, GA, United Virginia, Charlottesville, VA, United States States Electron transfer dissociation mass spectrometry is a break-though A new generation of DNA sequencing technologies has enabled a variety technology for sequencing post-translationally modified peptides. of novel genome-scale experimental techniques. What is perhaps In this technique, radical anions of polyaromatic hydrocarbons most unique about this recent data explosion is that it is distributed (fluoranthene and azulene) are employed to transfer electrons to — relatively inexpensive instruments allow any lab or institution to the carbonyl group in the polyamide backbone of multiply charged produce enormous amounts of data. Yet the infrastructure upstream peptides generated by electrospray ionization. Capture of an electron and downstream of sequencing instruments is largely undeveloped. into the peptide backbone reduces the positive charge on the ion by In addition to the instrument cost labs, core facilities and sequencing one, and forms a carbonyl radical anion that then abstracts a proton service providers are forced to earspend thousands on commercial from a nearby protonated amino group. The resulting carbonyl radical LIMS systems and sequence analysis packages, which are in-turn based triggers cleavage of the adjacent nitrogen-carbon bond to produce on tools from the public domain. Galaxy provides a robust open-source fragments of type c’ and z•. The purpose of the workshop is to alternative. It’s lightweight sample tracking system is aimed at helping provide instruction on how to manually interpret peptide ETD mass small labs and core facilities managing requests for sequencing runs. It spectra. Following a lengthy tutorial about ion structures, fragmentation allows one to track the entire “life-cycle” of sequencing request from pathways, predictable changes in fragment ion isotope patterns, etc., the initial sample to the resulting dataset. Once the run is complete we will outline a general approach for the manual interpretation of the user can apply a variety of NGS tools including format converters, peptide ETD spectra, solve the sequence of several post-translationally mappers, ChIP-seq and transcriptome utilities. Results of these analyses modified peptides, assign homework spectra, and reconvene on the can be visualized, shared, and published. In this presentation we will second day of the workshop to go over the homework problems. A demonstrate sample tracking functionality from the moment of sample packet of lecture notes and handouts will be provided. Attendees submission to the sequencing facility, through the sequencing run, until should bring an inexpensive calculator, pad of paper, and a small ruler. the sample becomes a dataset and can be analyzed with a variety of NGS tools. Abstracts Workshop Session Workshop

52 • ABRF 2011 — Technologies to Enable Personalized Medicine Notes Notes

ABRF 2011 — Technologies to Enable Personalized Medicine • 53 Research Group Presentation Abstracts

(R1) Joint Session: Proteome Informatics (R1b) ABRF-sPRG2011 Study: Development of Research Group (iPRG) & Proteomics Standards a Comprehensive Standard for Analysis of Post- Research Group (sPRG) Translational Modifications

A. Ivanov1, C. Colangelo2, C.P. Dufresne3, J.G. Farmar4, (R1a) iPRG 2011: A Study on the Identification of D.B. Friedman5, C. Kinsinger6, K.S. Lilley7, K. Mechtler8, Electron Transfer Dissociation (ETD) Mass Spectra B.S. Phinney9, S.A. Shaffer10, S.T. Weintraub11

1 L. Martens10, M. Askenazi1, N. Bandeira2, R.J. Chalkley3, Harvard University School of Public Health, Cambridge, K.R. Clauser4, E. Deutsch5, H.H.N. Lam6, W.H. McDonald7, MA, United States; 2Yale University, New Haven, CT, United T. Neubert8, P.A. Rudnick9 States; 3Thermo Fisher Scientific, West Palm Beach, FL, United States; 4University of Virginia, Charlottesville, VA, 1 Dana-Farber Cancer Institute, Boston, MA, United States; United States; 5Vanderbilt University, Nashville, TN, United 2 University of California San Diego, La Jolla, CA, United States; 6National Cancer Institute, Bethesda, MD, United 3 States; University of California San Francisco, San Francisco, States; 7University of Cambridge, Cambridge, United 4 CA, United States; The Broad Institute of MIT and Harvard, Kingdom; 8Research Institute of Molecular Pathology, 5 Cambridge, MA, United States; Institute for Systems Vienna, Austria; 9Proteomics Core, University of California 6 Biology, Seattle, WA, United States; University of Science Davis Genome Center, Davis, CA, United States; 10University 7 and Technology, Hong Kong, China; Vanderbilt University of Massachusetts Medical School, Worcester, MA, United 8 School of Medicine, Nashville, TN, United States; New States; 11University of Texas Health Science Center at San York University School of Medicine, New York, NY, United Antonio, San Antonio, TX, United States States; 9National Institute of Standards and Technology, Gaithersburg, MD, United States; 10Ghent University and VIB, The Proteomics Standards Research Group (sPRG) has initiated a study Ghent, Belgium that focuses on development of a standard that can be used in both assessment of a laboratory’s ability to detect an array of post-translational The field of mass spectrometry based proteomics has seen several key modifications in a complex proteomic sample and development of innovations over the last several years, including novel experimental new approaches for characterization of post-translationally modified methods, new instruments, and unique fragmentation strategies. The proteins. The sample that has been generated for the first stage of this latter, in the form of electron capture dissociation (ECD) and electron study contains a mixture of more than seventy synthetic peptides with a transfer dissociation (ETD) have captured the imaginations of many variety of modifications, in a tryptic digest of six proteins. Modifications researchers, expanding their ability to identify and analyze peptides represented in the sample include acetylation, methylation, nitration, and proteins. However, since ECD/ETD spectra differ substantial from phosphorylation, and sulfation. The individual proteins were purified, more traditional collision induced dissociation (CID) spectra in both digested and analyzed prior to formulation of the sample. The their prominent ion series as well as their preferred bond-breaking synthetic peptides were each analyzed and then mixed with the digests; characteristics, the (automatic) interpretation of ECD/ETD spectra the mixture was aliquoted and lyophilized in sufficient quantities that requires novel algorithm optimizations. Efficient identification of ECD/ would permit evaluation by several analytical approaches. The sample

Research Group Group Research ETD spectra thus remains an active and exciting field of proteomics was fully characterized by members of the sPRG. It is planned that in

Presentation Abstracts Presentation informatics research. In this work, the ABRF Proteome Informatics 2011, the sample will be distributed to requesting laboratories for a Research Group (iPRG) presents the results of a collaborative study larger study focusing on PTM characterization. The sPRG will present focusing on the analysis of an LC-MS/MS dataset from a yeast lysate results obtained at different stages of preparation and characterization digested with Lys-C and enriched for highly charged peptides using of the sample. Approaches for analysis of complex proteomic samples strong cation exchange fractionation. The data derived from one fraction containing various post-translationally modified proteins will also be analyzed exclusively by ETD was distributed to participants for analysis discussed. The sPRG is planning to open the study for sample requests in several equivalent formats, along with a standardized sequence at the ABRF 2011 conference. From the responses returned by the database derived from the UniProtKB/Swiss-Prot yeast complement, participating laboratories, the sPRG will subsequently report on the a decoy version of this database, and an applicable spectral library. effectiveness of the approaches used for characterization of post- Participants were free to use any and all methods available to them to translational modifications in protein digests. identify this fraction, and results were to be submitted using an Excel template. All participant identities were subsequently anonymized, and a survey was used to collect information about participant experience and software tools used to produce the submitted analysis. This uniform collection of data has allowed a thorough comparison of participant results. A summary, including a comparison of results submitted by members of the iPRG, will be presented.

54 • ABRF 2011 — Technologies to Enable Personalized Medicine (R2) Antibodies: Moving Closer to Personalized (R3) Joint Session: Genomic Variation Research Therapeutics and Diagnostics Group (GVRG) & DNA Sequencing Research Group (DSRG) B.R. Curtis1, R. Umeck2, J.E. Harlan3

1 (R3a) Evaluation of DNA Whole Genome Blood Center of Whisonsin, Milwaukee, WI, United States; Amplification Technologies for Genotyping 2Meso Scale Discovery, Gaithersburg, MD, United States; 3 Abbott Laboratories, Abbott Park, IL, United States A. Hutchinson1, C. Dagnall1, C. Nicolet2, H. Escobar3, S. Blake4, B. Sanderson5, B. Kingham6, K. Jonscher7 The possibilities of personalized medicine are largely rooted in successfully exploiting the ever-growing genome and proteome-wide 1Core Genotyping Facility, SAIC-Frederick, Inc., NCI- understanding of human biology. Translating this knowledge base into Frederick, Frederick, MD, United States; 2Data Production the clinic requires, among other things, being able measure the patient’s Facility, USC Epigenome Center, Los Angeles, CA, United genomic and proteomic disease signature in a routine manner. Thus, States; 3DNA Sequencing Operations, Eurofins MWG expanded focus on assays and platforms capable of measuring these Operon, Huntsville, AL, United States; 4DNA Core Facility, specific signatures within tens, hundreds or even thousands of complex 5 patient samples simultaneously is critical. Antibody-based assays and University of Missouri, Columbia, MO, United States; Life 6 platforms have become a centerpiece of many technologies pushing Technologies, Austin, TX, United States; Director, DNA these boundaries because of the antibody’s intrinsic specificity, diverse Sequencing & Genotyping Center, University of Delaware, recognition capacity and ease with which they can be produced. Newark, DE, United States; 7Director, SBCF and TATC Furthermore, these same characteristics have made them an equally Proteomics, University of Colorado Denver, Denver, CO, promising prospect for therapeutic approaches. This seminar trifecta United States will highlight the pivotal role antibodies have in multiplex biomarker assay development and therapeutic venues. Specifically: 1. Brian The evolution of genomic technologies is occurring rapidly and often Curtis will discuss a robust multiplex assay platform to detect human requires large amounts of source DNA. There is also an expanded platelet auto-antibodies and platelet antigen polymorphisms by desire to analyze smaller numbers of cells for higher resolution studies combining bead array technologies with flow cytometry. Such a as well as to take advantage of large numbers of archived samples (e.g. simple, yet high-throughput platform would facilitate routine testing FFPE, serum, etc.). To provide enough material for the newest genomic for platelet disorders like neonatal alloimmune thrombocytopenia, technologies, whole genome amplification (WGA) has reemerged as an multi-platelet transfusion refractoriness, and post-transfusion purpura; important and necessary technique. With some new WGA products 2. Robert Umeck will summarize the challenges in developing on the market, we have evaluated the quantity and quality of WGA antibody-based multiplex assays and how Meso Scale Discovery products generated as well their performance on some of the current utilizes an electrochemiluminescence detection system combined genomic applications. The GVRG has completed a benchmarking with a patterned multi-array format to generate multiplex assays for study evaluation of 6 commercially available WGA kits using several nearly any application. Several actual examples will be presented to genotyping assays. Utilizing 6 samples, the different WGA kits were demonstrate how this approach could support personalized medicine; tested following the manufacturer’s protocols. The samples included 3. John Harlan will introduce and discuss the evolution of therapeutic Coriell DNA from a trio of CEPH individuals, 2 FFPE DNAs, both Presentation Abstracts antibodies. To illustrate where the field is venturing, he will highlight from the same individual (1 newly extracted and the other extracted Research Group Abbott Laboratory’s new bi-specific antibody format. 2 years ago), and 1 sample from the Coriell trio that was fragmented prior to amplification for a total of 6 samples tested. The WGA kits included those from Sigma, NuGen, Qiagen and GE Life Sciences. The 6 samples were amplified as blind duplicates at several GVRG member lab sites resulting in a total of 78 amplified products. Quality metrics were performed on the 78 amplified products using several different techniques in order to provide measurements on yield, fragment size, and concentration. Several widely used genotyping methods were used to evaluate the amplification products, including Illumina Human Omni1 Quad Beadchip, TaqMan copy number and SNP genotyping assays, and STR genotyping. Data will be presented on genotyping concordance and loss of heterozygosity checks across the different platforms where possible.

ABRF 2011 — Technologies to Enable Personalized Medicine • 55 (R3b) DNA Sequencing Research Group (DSRG) (R4) Joint Session: Protein Sequencing Research Group (PSRG) & Glycoprotein Research Group (R3b-1) Comparison of Custom Target Enrichment (gPRG) Methods; Agilent vs. Nimblegen

A. Perera4, K. Bodi1, P.S. Adams2, D. Bintzler3, K. Dewar5, (R4a) PSRG 2011 Study Results: Sensitivity D.S. Grove6, J. Kieleczawa7, R.H. Lyons8, T. Neubert9, Assessment of Terminal Sequencing Techniques A.C. Noll1, S. Singh10, R. Steen11, M. Zianni12 Using an Unknown Protein

1 2 Tufts University, Boston, MA, United States; Trudeau 1, 2, 3, 3 J.J. Walters W. Sandoval K. Mawuenyega Institute, Saranac Lake, NY, United States; DNA Analysis, 4, H. Remmer5, B. Xiang6, D. Suckau7, V. Katta2, 4 J.S. Smith Inc., Cincinnati, OH, United States; Stowers Institute, Kansas P. Hunziker8 City, MO, United States; 5McGill University, Montreal, QC, Canada; 6Pennsylvania State University, University Park, 1Sigma-Aldrich, St. Louis, MO, United States; 2Genentech, PA, United States; 7Pfizer Research, Cambridge, MA, United Inc., South San Francisco, CA, United States, 3Washington States; 8University of Michigan, Ann Arbor, MI, United University School of Medicine, St. Louis, MO, United States, States; 9New York University, New York, NY, United States; 4University of Texas Medical Branch, Galveston, TX, United 10University of Minnesota, Minneapolis, MN, United States; States, 5University of Michigan, Ann Arbor, MI, United 11Harvard Medical School, Cambridge, MA, United States; Stated, 6Monsanto Company, St. Louis, MO, United States, 12Ohio State University, Columbus, OH, United States 7Bruker Daltonics, Bremen, Germany, 8University of Zurich, Zurich, Switzerland Over the last four years, we witnessed the tremendous advances in Next Generation Sequencing (NGS) that have dramatically decreased Establishing the N-terminal sequence of intact proteins plays a critical the cost of whole genome sequencing. However, the cost of sequencing role in biochemistry and potential drug development. N-terminal larger genomes is still significant. In addition and depending on the sequence analysis is necessary for quality control of protein biologics, goal of study, whole genome sequencing creates a large amount of for determining sites of signal peptide cleavage events, as a first step additional/auxiliary data that complicates data analysis. There are in elucidating the sequences of genes from uncommon species and several commercial methods available for isolating subsets of genomes for the characterization of monoclonal antibodies. Automated Edman that greatly enhance the efficiency of NGS by allowing researchers degradation has been the method of choice for these analyses. However, to focus on their regions of interest. For the 2009-11 DSRG study, alternate methods for N-terminal sequence analysis have emerged. The we compared products from two leading companies; Agilent and recent PSRG studies have established that Edman sequencing and mass Nimblegen that offer custom enrichment methods. Both companies spectrometry based techniques have varied strengths and weaknesses obtained the same genomic DNA stock and performed DNA capture depending on several experimental factors and both play an important on the same specified regions. Following capture, the Illumina Genome role in terminal sequencing. With this complimentary role realized, Analyzer IIx system was used, in two different laboratories, to generate the 2011 PSRG study attempts to evaluate the sensitivity limits of the the sequence data. We present our data comparing in terms of cost, various sequencing techniques. The PSRG distributed three sample quality, reproducibility and most importantly completeness and depth sets of 3 tubes each, varying by sample format (lyophilized, gel slice of coverage. Acknowledgements: We would like to thank Agilent, or membrane piece). Each set of three samples contains the same Illumina and Nimblegen for all their support in making this study recombinant protein with increasing amounts of material. The sequence possible. of this protein is not listed in any database. Participants could request any one, two, or all three sample sets. Including PSRG committee, a total (R3b-2) A Methodology Study for Metagenomics of 38 participants requested 74 sample sets. The participants were Research Group Group Research asked to determine as many amino acids from both termini by their

Presentation Abstracts Presentation Using Next Generation Sequencers method of choice, and were encouraged to try multiple methods for S. Singh1, D. Grove2 sequence elucidation. Study participants were directed to a website to anonymously upload sequences and supporting data. Our analysis 1 Biomedical Genomics Center, University of Minnesota, focuses on the length and accuracy of the sequence calls reported by Saint Paul, MN, United States; 2Genomics Core Facility, Huck the participants, and how that compares with decreasing amounts of Institutes for Life Sciences, Pennsylvania State University, protein and the type of sample format analyzed. A comparison of the University Park, PA, United States results obtained by Edman chemistry and by alternative technologies as well as information on the type of instruments and protocols is Metagenomics is one of several genomics applications, which has reported. benefited immensely from the high throughput and cost efficacy of Next generation sequencers. And although hundreds of studies on metagenome 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 sample preparation methods, specifically DNA extraction and amplification approaches, on data output along with a comparative analysis of Next generation sequencing platforms. We will study the effect of these different experimental and technical strategies on determination of sample biodiversity.

56 • ABRF 2011 — Technologies to Enable Personalized Medicine (R4b) gPRG: Toward Consensus on Glycan Analysis: Choice human brain reference RNA sample. Two popular RT priming Reliable Methods and Reproducibility strategies tested in this study include the Megaplex miRNA TaqMan assay (ABI) and the RT2 miRNA qPCR assay (Qiagen/SA Biosciences). J. Zaia1, D. Kolarich2, R. Orlando3 The basis for the ABI assay design is a target-specific stem-loop structure and reverse-transcription primer, while the Qiagen design 1Boston University, Boston, MA, United States; 2Max Planck combines poly(A) tailing and a universal reverse transcription in one Institute of Colloids and Interfaces, Berlin, Germany; 3The cDNA synthesis reaction. For this study, the human brain reference University of Georgia Carbohydrate Research Center, RNA was subject to controlled degradation using RNase A to RIN (RNA Atlanta, GA, United States Integrity Number) values of 7 (good), 4 (moderately degraded), and 2 (severely degraded).These templates were then used to assess both The field has undertaken over the past few years a series of studies to RT methods. In addition to this real-time RT-qPCR data, the same RNA evaluate methods for glycan analysis. There are several methods that templates were further analyzed using universal poly(A) tailing and are widely used at this time for characterization of glycoprotein glycans hybridization to Affymetrix miRNA GeneChips. This talk will provide that appear to be generally reliable. These include (1) reductive insights into RT priming strategies for miRNA and contrast the qPCR amination followed by reversed phase chromatography, (2) reductive results obtained using different technologies. amination with matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS), (3) reductive amination with electrospray (R5b) Microarray Research Group Projects, 2010-11 liquid chromatography-MS, and (4) permethylation with tandem MS. The ABRF glycoprotein research group (gPRG) is planning a study in D. Baldwin1, N.G. Reyero-Vinas2, N. Jafari3 which participant laboratories carry out glycan analysis according to 1 protocols representing best practice in the field. The results will be Penn Molecular Profiling Facility, University of Pennsylvania, 2 presented at the 2012 ABRF meeting. The results will be useful for Philadelphia, PA, United States; Jackson State University, demonstrating the reproducibility of glycan analysis using comparable Jackson, MS, United States; 3Northwestern University, protocols. They will also highlight the extent to which different methods Evanston, IL, United States bias the glycan analysis results obtained. This workshop will summarize the history and use of the glycan analysis methods proposed for the Members of the MARG will discuss our research projects: Comparison 2012 study. There will be opportunity for discussion and comment on of microarray and deep sequencing platforms for microRNA profiling, these methods. Performance of a synthetic human microRNA reference panel, Participation in the SEQC Sequencing Quality Control consortium, and (R5) Joint Session: Nucleic Acids Research Group RNA-Seq profiling of environmental samples exposed to the Gulf oil (NARG) & MicroArray Research Group (MARG) spill. (R6) Joint Session: Protein Expression Research (R5a) Determining miRNA Expression Levels in Group (PERG) & Molecular Interactions Degraded RNA Samples Using Real-Time RT-qPCR Research Group (MIRG) and Microarray Technologies

1 2 3 4 (R6a) PERG Research Group Presentation: Refolding S. Chittur , S. Tighe , J. Holbrook , V. Nadella , Presentation Abstracts 5 3 6 R. Carmical , K. Sol-Church , A.T. Yueng Study Research Group

1State University of New York at Albany, Albany, NY, United C. Kinsland States; 2University of Vermont, Burlington, VT, United States; Cornell University, Ithaca, NY, United States 3Nemours/A.I. duPont Hospital for Children, Wilmington, DE, 4 United States; Ohio University, Athens, OH, United States; Currently, the overwhelming majority of protein purification projects 5The University of Texas Medical Branch, Galveston, TX , start with a recombinant protein expressed in a suitable host. For a United States; 6Fox Chase Cancer Center, Philadelphia, PA, range of reasons, E. coli is the predominant expression host yet a large United States percentage of proteins expressed therein are found in an insoluble form called inclusion bodies. Inclusion bodies have the advantage of The Nucleic Acid Research Group (NARG) has previously conducted consisting of relatively homogeneous protein, which can simplify the studies evaluating the impact of RNA integrity and priming strategies purification process. However, this leaves the challenge of solubilizing on cDNA synthesis and real-time RT-qPCR. The results of last year’s field and refolding the protein into its native and biologically active structure. study as it relates to degraded RNA will be presented. In continuation The conditions for efficient refolding are particular for each protein and of the RNA integrity theme, this year’s study was designed to evaluate there are a wide range of methods to choose from. For this and other the impact of RNA integrity on the analysis of miRNA expression reasons, many researchers are hesitant to pursue a refolding strategy using real-time RT-qPCR. Target section was based on data obtained to obtain a target protein. An overview of refolding methods and by the Microarray Research Group (MARG) and other published data strategies will be presented along with a description of the upcoming from next gen sequencing. These 9 miRNAs represent three groups Protein Expression Research Group (PERG) refolding study. of miRNA that are expressed at low, medium or high levels in the First

ABRF 2011 — Technologies to Enable Personalized Medicine • 57 (R6b) Conclusions from the MIRG 2010 Benchmark (R7) Light Microscopy Research Group (LMRG) Study: Molecular Interactions in a Three Component System and Presentation of 2011 Survey Results on Label-Free Technologies (R7-1) Point Spread Functions, Spectral Calibration, and Beyond A.P. Yamniuk1, S.P. Yadav5, S. Bergqvist2, M.L. Doyle1, E. Eisenstein3, M.K. Robinson4, T. Neubert6 R. Stack, R. Cole 1Bristol-Myers Squibb, Princeton, NJ, United States; 2Pfizer, La Wadsworth Center, New York State Department of Health, Jolla, CA, United States; 3University Maryland Biotechnology Albany, NY, United States 4 Institute, Baltimore, MA, United States; Fox Chase Cancer Modern light microscopes are highly evolved opto-electronic- 5 Center, Philadelphia, PA, United States; Cleveland Clinic mechanical devices. Establishing instrument performance is crucial in Foundation, Cleveland, OH, United States; 6New York ensuring that reliable and accurate images can be acquired. Imagers, as University School of Medicine, New York, NY, United States well as granting agencies, need to be confident that data collected will be uniform and quantifiable both temporally and from instrument to Characterizing the assembly of multi-protein complexes and the instrument. Last year, in phase-one of our world-wide research study competition between multiple protein ligands for a given target are on instrument performance, we successfully concentrated our efforts common challenges faced by core facilities. The MIRG2010 Benchmark on three image-based tests: long and short term stability of illumination study was designed to assess participants’ ability to correctly describe sources, uniformity of field illumination, and co-registration across the interactions between two protein ligands and their target protein various wavelengths. A manuscript summarizing the phase-one study using primarily biosensor technologies such as surface plasmon has been submitted to and accepted by Microscopy & Microanalysis, resonance. Participants were provided with microgram quantities of one of the highest rated imaging journals. In the coming year, phase- three proteins (A, B and C) and asked to determine if a ternary A-B-C two of our instrument performance study will focus on determining the complex can form, or if ligands B and C bind competitively to protein following: the point spread function of an imaging system, the system’s A. This presentation will summarize the conclusions from the 2010 spectral separation ability and the spectral calibration and resolution Benchmark Study, and provide perspective on the potential for future of the detection system. As with the phase-one study, the goal of this application of this system as a reference standard for quantitative study will be neither to compare the performance of different brands characterization of protein-protein interactions using biosensor of instruments, nor to ascertain which brand had better performance technologies. The field of label-free biophysical technologies like in a given area. Instead, the goal of our proposed phase-two study is surface plasmon resonance (SPR) and isothermal calorimetry (ITC) are continue to focus on determining the current state of modern imaging becoming indispensable in translational research and in the discovery systems through straightforward, efficient and robust tests. These tests phase of biotherapeutics. Investigators are much more aware about the will aid imagers in the early detection of system problems. Moreover, developments in biomolecular interaction analysis using SPR and ITC these tests will continue to help define relative standards that will assist and usefulness of these technologies in designing better drugs based both core personnel and imagers in maintaining their instruments in on biomolecules and vaccines. The Molecular Interaction Research optimal operating conditions. Group (MIRG) of ABRF has conducted an on-line survey to capture the recent explosive developments in these technologies. The survey was (R7-2) Deconvolution: Core Concepts, Algorithms targeted to both academia and pharmaceutical industry and the survey and Advanced Issues data will be presented during the meeting. B. Northan, N. Beavers Media Cybernetics, Guilderland, NY, United States Research Group Group Research

Presentation Abstracts Presentation Deconvolution is a computational technique used to remove blur from images. In this presentation image formation in the microscope will be reviewed and it will be explained what deconvolution does and why deconvolution is needed as a post acquisition processing step. The concept of Point Spread Function (PSF) will be introduced. Several approaches to deconvolution and deblurring exist from simple subtractive methods to complex iterative approaches. The major approaches will be examined. It will be explained why in the presence of noise a statistical approach is required. Blind deconvolution will be introduced. The concept of Point Spread Function will be examined in detail. The effect of microscope modality, lens parameters, and specimen parameters on PSF shape will be discussed. Theoretical PSF calculation and PSF measurement using beads will be reviewed with examples. It will be explained why theoretical and measured PSFs can differ from the true system PSF. Blind deconvolution will be further examined as a tool to deal with the uncertainty of the true form of the PSF. Practical guidelines for data acquisition will be reviewed. The relationship between sampling rate and quality of deconvolution results will be explained.

58 • ABRF 2011 — Technologies to Enable Personalized Medicine (R8) Joint Session: Proteomics Research Group (R8b) Metabolomics Research Group 2011 Study (PRG) & Metabolomics Research Group (MRG) W.R. Wikoff1, J.M. Asara3, V.V. Tolstikov 1, P. Aronov2, B. Kesler4, V. Shulaev5, C.W. Turck6 (R8a) PRG-2011: Defining the Interaction between Users and Suppliers of Proteomics Services 1University of California Davis, Davis, CA, United States; 2Stanford University, Stanford, CA, United States; 3Beth D. Hawke1, T.M. Andacht2, M.K. Bunger3, C. Bystrom4, Israel Deaconess Medical Center, Boston, MA, United States; L. Dangott5, H. Molina6, R.L. Moritz7, R.E. Settlage8, 4Thermo Fisher Scientific, Redwood City, CA, United States; C .W. Turck 9 5University of North Texas, Denton, TX, United States; 6Max Planck Institute of Psychiatry, Munich, Germany 1University of Texas MD Anderson Cancer Center, Houston, TX, United States; 2Centers for Disease Control and The ABRF Metabolomics Research Group (MRG) was formed in Prevention, Atlanta, GA, United States; 3RTI International, 2009 and aims to educate research scientists and resource facilities Research Triangle Park, NC, United States; 4Quest in the analytical approaches and management of data resulting from Diagnostics, San Juan Capistrano, CA, United States; comprehensive metabolite studies and to promote the science and 5Texas A&M University, College Station, TX, United States; standardization of metabolomic analyses for a variety of applications. 6Center for Genome Regulation, Barcelona, Spain; 7Institute Last year the MRG conducted a ‘Survey Study’ on the current use of metabolomics technologies and procedures in core facilities. This year for Systems Biology, Seattle, WA, United States; 8Virginia the MRG is organizing a ‘Research Study’ involving a spiked plasma 9 Bioinformatics Institute, Blacksburg, VA, United States; Max sample. The study sample consists of a human biofluid as the matrix, Planck Institute of Psychiatry, Munich, Germany replicating a typical small scale metabolomics pilot experiment that either a core or research laboratory would perform. The sample Over the last ten years the Proteomics Research Group (PRG) has consists of two groups of normal human plasma (NIST plasma ‘Standard undertaken technical studies that have covered a wide range of Reference Material’) with spiked in compounds. There are three issues unique to the rapidly developing field of proteomics and have biological replicates in each group (n = 3 design) with different levels included a range of qualitative and quantitative experiments. The PRG of spiked compounds differentiating the two groups. Participants are studies have resulted in a great deal of attention not only within the asked to determine statistical significance, fold change, and identify ABRF community but also outside as is evident from numerous articles compounds that differ significantly between groups A and B. The design dealing with proteomics methods, procedures and standardization. reflects issues encountered in an actual metabolomics experiment As the field continues to develop, the diversity of instrumentation conducted with human or animal specimens. The study is compatible and laboratory workflows have grown in tandem. Therefore, in the with many methodological approaches in metabolomics, including, PRG2011 study it seemed especially useful to perform a survey to but not limited to LC/MS, GC/MS, NMR, as well as other methods. As help the PRG define future studies based on the current blend of with any metabolomics profiling experiment, the best results would sample types and technologies and obtain a view of emerging trends. be expected using a combination of approaches. The study is the first A survey was created to ascertain three main insights into core facility of its kind in the field of metabolomics and is expected to produce function: 1) How labs interact with their clients, 2) The capacity of labs important information on the strengths and limitations of the various to meet the demands of their clients, and 3) The blend of experimental

platforms and technologies that are commonly used for comprehensive Presentation Abstracts techniques offered to and requested by clients. Survey questions were

metabolite analyses. Research Group designed to obtain information from both users of core facilities and the directors and personnel of core facilities. Questions covered such topics as the type and age of instruments in use, how data is analyzed and presented to client, sources of funding, and emerging proteomics trends. Results are compiled en masse and presented without regard to institution. Early results reveal that about 2/3 of the responders are not ABRF members, and at least one lab still has an operational mass spectrometer that was acquired in 1990!

ABRF 2011 — Technologies to Enable Personalized Medicine • 59 Poster Session Abstracts

102 Designing an Institutional Web-based **ABRF Poster Award Semifinalists Core Facility Management System

A. Hagen1, D. Tabarini2, S. Clisham3, D. John3 101 Center for Genome Research and 1iLab Solutions, LCC, Cambridge, MA, United Biocomputing at Oregon State States; 2Memorial Sloan-Kettering Cancer Center, University New York, NY, United States; 3Institute for Systems 4 C. Rosato, M. Dasenko, A. Girard, M. Peterson, Biology, Seattle, WA, United States; Dana-Farber C. Sullivan, J.C. Carrington Cancer Institute, Boston, MA, United States Center for Genome Research & Biocomputing, The authors and their four institutions collaborated to (i) identify the key Oregon State University, Corvallis, OR, United challenges to core facility management; (ii) identify the requirements States for an effective core facility management system; (iii) design, test and deploy such a system. Through a series of interviews with all participants The Center for Genome Research and Biocomputing (CGRB) Core in the core work flow (customers, core staff, administrators), the team Lab at Oregon State University provides services for fee in genomic identified a number of key challenges, including: (i) difficulty for technologies (DNA sequencing, DNA genotyping (fragment analysis)) researchers in identifying available services; (ii) inconsistent processes and in functional genomic technologies (microarray). We manage a for requesting services; (iii) inadequate controls for approving service Zeiss LSM510Meta confocal microscope as a multi-user instrument; requests; (iv) inefficient processes for tracking and communicating about training is required and assistance is available, both for fee. Sequencing project processes; (v) time-consuming billing practices; (vi) incomplete is provided both for traditional Sanger sequencing on an AB 3730 and revenue capture; (vii) manual reporting processes. The team identified ultra high throughput sequencing on the Illumina Genome Analyzer the following requirements for a system to address these challenges: GAIIX. With the most recent software and reagent upgrades, 2 x (i) ability to support a broad range of core business practices such 150bp runs are now supported with up to 40 million reads per lane as complex quote generation and project management; calendaring/ passing filter. DNA genotyping (fragment analysis) runs on the AB equipment reservation management; sample tracking; complex forms; 3730. Our microarray services include Affymetrix and NimbleGen and import of usage data from hardware; (ii) ability to offer services platforms, and sample labeling, hybridization and scanning are for both internal and external customers, including flexible pricing offered. We purchased ArrayStar microarray analysis software, which and off-site access; (iii) ability to interact with institutional financial is accessible to our researchers through our Biocomputing cluster systems (e.g. SAP, PeopleSoft, Lawson, SunGard Banner) and identify network. The Agilent BioAnalyzer service offers High Sensitivity DNA management systems (e.g. Microsoft Active Directory, LDAP, and other and RNA applications. Our Biocomputing capabilities have expanded. SAML 2.0-compliant services). The team developed and deployed this This past year we reorganized the computing resources into a cloud system across the collaborative partners, as well as other major research architecture. GENOME Cloud is at 507 Processors, 1.5TB Memory institutions. and 300+TB Storage. We process around 20,000 jobs / day on the cloud. We have increased access to our computational resources, 103 RI-INBRE Centralized Research Core decreased analysis turn-around time, and we have added new tools Facility to monitor computational resources. The Biocomputing changes to our High Throughput Sequencing service include removing the IPAR A. Ahmed, N. Nous unit from the network and simplifying data pathways both for data management and researcher analysis capabilities. The CGRB Core Lab University of Rhode Island, Kingston, RI, United also maintains multi-user instruments available to researchers. These States include a Zeiss LSM510Meta confocal microscope, an AB 7500 FAST qPCR instrument includes High Resolution Melting (HRM) capability, a The RI-INBRE Centralized Research Core Facility inaugurated in July

Poster Abstracts Storm 820 Phosphoimager, a Nanodrop, an Invitrogen Qubit, a Genetix 2003, is being supported by the Rhode Island IDeA Network of Q-Pix colony picker, an Axon Genepix 4200A microarray scanner and a Biomedical Research Excellence (RI-INBRE) grant from NCRR /NIH fluorescent plate reader. and by the participating institutions that include: University of Rhode Island, Brown University, Rhode Island College, Providence College, Salve Regina University, and Roger Williams University. This facility is located in the College of Pharmacy at the University of Rhode Island’s Kingston campus. It is equipped with instrumentation in biomedical, pharmaceutical and biotechnological research. The Core Facility is providing access to research instrumentation and training support to RI-INBRE participants as well as other scientists affiliated with academic institutions and the private sector throughout the state of Rhode 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 the equipment. Independent access to the equipment is also available to

60 • ABRF 2011 — Technologies to Enable Personalized Medicine all trained users. In addition, full service access via sample submission shared research resources and services to the university community is provided particularly in ICP-MS, LC/MS/MS and N-terminal protein and to outside investigators. The CLC includes fee-for-service research, sequencing. Technical staff members manage the laboratory and are technology testing and development, and educational components. available to assist in the operation of various instruments and to analyze The Center has seven core facilities, including genomics (DNA samples on a fee-for-service basis. (Supported by NIH-NCRR Grant # sequencing, genotyping, and microarrays), epigenomics, proteomics 1P20RR16457) and mass spectrometry, microscopy and imaging, bioinformatics, bio- IT, and advanced technology assessment. The CLC is part of a New 104 Implementation of Quality York State designated Center for Advanced Technology in Life Science Management in Core Service Enterprise. The mission of the CLC is to promote research in the life Laboratories sciences with advanced technologies in a shared resource environment. Use of the CLC resources and services is steadily increasing due to the B. Hicks, T. Creavalle, J. Dickens, K. Haque, growth in the number and types of cores in the center, to the expansion C. Raley, M.W. Smith of existing services and the implementation of new core technologies, and to the coordinated integration and synergy of services between Genetics and Genomics Group, Advanced the CLC cores. Multidisciplinary support for multi-functional instrument Technology Program, SAIC-Frederick, National platforms is implemented by integrated operations of the CLC core Cancer Institute at Frederick, Frederick, MD, United facilities. Investigators are offered coordinated project consultations States with the directors and staff of all relevant cores during the design, data production and analysis phases of their projects. The CLC is The Genetics and Genomics group of the Advanced Technology involved in establishing and supporting multidisciplinary research Program of SAIC-Frederick exists to bring innovative genomic expertise, projects that involve both intercampus initiatives and multi-institutional tools and analysis to NCI and the scientific community. The Sequencing collaborations. With a concentration of advanced instrumentation and Facility (SF) provides next generation short read (Illumina) sequencing expertise in their applications, the CLC is a key resource for life sciences capacity to investigators using a streamlined production approach. research. The Laboratory of Molecular Technology (LMT) offers a wide range of genomics core services including microarray expression analysis, 106 Searchable Core Facility Database: miRNA analysis, long read (Roche) next generation sequencing, Building Resource Bridges transgenic genotyping, Sanger sequencing, and clinical mutation detection services to investigators from across the NIH. SF and LMT are B. Fleming, T. Hunter working together to bring online the third generation Pacific Bioscience SMRT sequencing platform. As the technology supporting this Vermont Genetics Network, University of Vermont, genomic research becomes more complex, the need for basic quality Burlington, VT, United States processes within all aspects of the core service groups becomes critical. The Quality Management groupworks alongside members of these The VGN Searchable Core Facility Database (http://vgn.uvm.edu/ labs to establish or improve processes supporting operations control corefacilities) is a directory of Core Facilities primarily focused on North (equipment, reagent and materials management), process improvement America but with entries from around the world. It is a tool intended (reengineering/optimization, automation, acceptance criteria for new to foster collaboration and assist cores in growing their user base and technologies and tech transfer), and quality assurance and customer providing networking opportunities. It is populated with Core Facilities support (controlled documentation/SOPs, training, service deficiencies that have voluntarily listed themselves and would like to be contacted and continual improvement efforts). Implementation and expansion of by researchers and other core facilities for potential collaborations. quality programs within unregulated environments demonstrates SAIC- Benefits: Allows researchers to locate resources needed for their studies; Frederick’s dedication to providing the highest quality products and Provides a channel for facilities to collaborate; and Facilitates cores to services to the NIH community. reach financial sustainability. Researchers are able to perform searches online by service offerings, location, association, and key phrases to find a facility that will best meet their needs. Information listed for individual

105 Cornell University Life Sciences Core cores include: short description of core, contact name, email, address, Poster Abstracts Laboratories Center services offered, hyperlink to website, equipment, and date of last , J. VanEe, P. Schweitzer, S. Zhang, revision of information. The data can be exported to an excel readable G. Grills XML file. The database currently lists 292 cores, representing 39 states R. Williams, J. Pillardy, Q. Sun, W. Wang, Y. Li, plus DC, 104 institutions, and 10 associations. D. Betel, T. Stelick, J. Spisak, L. Cote, R. Cameron, H. Wroblewski, B. Hover, L. Zhang, J. Mosher, 107 Dartmouth Genomics Shared Y. Xin, G. Westby, J. Busuttil, S. Monni, Resources R. Sherwood, A.C. Ptak, W. Chen, J. McCardle, C. Bayles, J. Dela Cruz, M. Riccio, R. Bukowski, J. Hamilton, H. Trask, W. Taylor, C. Tomlinson L. Ponnala, C. Myers, H. Singh, M. Howard, J. Flaherty, A. Manocchia, E. Dodge, K. Smith, Dartmouth Medical School, Norris Cotton Cancer C. Aquadro, A. Melnick, T. Brenna, W. Zipfel, Center, Hanover, NH, United States A. Clark, A. Siepel, L. Carr, J.K.C. Rose In order to carry out an accurate diagnosis, prognosis, and/or Cornell University, Ithaca, NY, United States therapeutic assessment for a disease; high-throughput approaches to examine the whole genome and transcriptome are now a necessity for The Cornell University Life Sciences Core Laboratories Center (CLC) modern research. Furthermore, to more fully understand the underlying provides an array of genomics, proteomics, imaging and informatics causes of disease, high-throughput genomics are required to examine

ABRF 2011 — Technologies to Enable Personalized Medicine • 61 global gene expression, regulation, and interactions. To meet these 110 A Web Based Tool for Efficient and the future needs of the research community at Dartmouth, the Management and Use of Research latest technologies in deep sequencing and microarrays are offered. In addition, we offer the following services: Specialized, expensive, high- Core Facilities end instrumentation; Expert staffing; Cost-effective for individual labs; P. Turpen, L. Wilkie, L. Miller Competitive pricing and services with outside sources; On site Norris Cotton Cancer Center facility; Free experimental design consultations; University of Nebraska Medical Center, Omaha, Competitive fee for service charges for all high-throughput approaches; NE, United States and Close proximity to Biostatistics and Bioinformatics shared resources. The need to efficiently and effectively manage research resources 108 A Collaborative Life Cycle Process for is an area of growing concern to both grantor agencies and grantee the Bioinformatics Core Facility institutions. Central administration of core facilities is one way to address some of those concerns, but managing the needs of disparate S. Lin, W.A. Kibbe cores can be a challenge. All research core facilities, irrespective of their emphasis, need to be able to contract for services, bill for those Northwestern University, Evanston, IL, United States services and provide documentation of facility use. While commercial systems are a welcome recent development, most concentrate on The operation of the bioinformatics core facility is constantly challenged sample management data, LIMS, with invoicing and utilization metrics by increasing data volume, emerging technologies, and limited budget. as secondary functions. The shortcoming in these types of offerings is We discuss a Collaborative Life Cycle (CLC) process as a business that they do not readily capture the institution’s or grantor agencies’ management model for this challenging environment. Unlike the needs for use reporting without considerable customization and traditional involvement at the last stage of data analysis, the CLC process corresponding associated costs. We have chosen to create a web based engages the bioinformatics core facility throughout the project with the tool that can facilitate the scheduling/contracting of services, invoicing Pis and the wet lab core facilities. Various tasks of the bioinformatics and fee collection for those services, and address the complex reporting core during the project’s life cycle include: 1) Planning -study design needs of the institution. and statistical power analysis with the PIs 2) Experiment-core wet lab sample tracking and data management 3) Data analysis -data quality control and interpretation in the biological context. Collaborations 111 XSQ: A New Binary Output Format of throughout the project life cycle are critical because the multiple-phase 5500/5500XL Systems process involves numbers of professional disciplines with many skills, , J. Zhang, D. Thomas, P. Suri tools, and procedures. The CLC process will help the bioinformatics C. Yang core facility align the multiple goals, adjust the expectations, mitigate Life Technologies, Carlsbad, CA, United States the potential risks, and improve the end results. The XSQ (eXtensible SeQuence) format was designed to accommodate 109 Promoting Diversity in the Core different run types (standard SOLiD sequencing and exact chemistry Facility call (ECC) runs), simplify indexing samples workflow and support new data types such as ECC in the new 5500 sequencing instrument; these M. Person data cannot be stored in any existing formats (e.g. csfasta & QV.qual) as there are more than one call per position. Other problems with existing The University of Texas at Austin, Austin, TX, formats are the file size, I/O demand, and lack of pairing of reads. The United States XSQ format uses information packing to reduce the file size by 60%, resulting in reduced storage needs and reduced transfer times. It also Diversity is encouraged to counter historical bias against a variety has integrated pairing of reads, so mapping and pairing can happen of groups and has emerged as a positive attribute of the workplace. together much more quickly, rather than having the two reads of a pair However, among pressing economic and scientific outcome pressures, mapped separately and then merged subsequently. The new XSQ file issues not tied to measurable rewards are often overlooked in the core allows users to perform very efficient indexing reassignment and such facility. This poster will present an overview of diversity issues and reassignment only introduces minimal impact to downstream analysis strategies relevant to core facilities. The variety of types of diversity instead of reanalyzing all indexing samples. The hierarchical data format is discussed, including sex, race, ethnicity, nationality, socioeconomic also provides a basic level of partitioning and indexing within the file so background, and sexual orientation. Institutional efforts to encourage that subsets of the data can be retrieved without reading through the Poster Abstracts diversity are described with regards to organizational mandates and entire file. implementation at the level of core facilities. Contrast between written policies and real world practices are noted. A valuable tool available 112 Microsoft Biology Initiative: .NET to the core facility director is self-examination, actively countering barriers that unconscious bias and learned prejudice have created. Bioinformatics Platform and Tools Systematic analysis can identify areas of diversity strength and weakness B. Diaz Acosta in the facility. Strategies for increasing diversity are presented for the hiring process. Practices in the workplace that accommodate diverse Microsoft Biology Initiative: .NET Bioinformatics populations and foster career development are discussed. Finally, Platform and Tools identifying rewards for diversity promotion encourages consistent progress in the face of competing demands. The Microsoft Biology Initiative (MBI) is an effort in Microsoft Research to bring new technology and tools to the area of bioinformatics and biology. This initiative is comprised of two primary components, the Microsoft Biology Foundation (MBF) and the Microsoft Biology Tools (MBT). MBF is a language-neutral bioinformatics toolkit built

62 • ABRF 2011 — Technologies to Enable Personalized Medicine as an extension to the Microsoft .NET Framework—initially aimed at 114 ABRF Affiliates and Chapters the area of Genomics research. Currently, it implements a range of parsers for common bioinformatics file formats; a range of algorithms S.F. Jennings1, G. Grills2, M. Detwiler3 for manipulating DNA, RNA, and protein sequences; and a set of 1 connectors to biological web services such as NCBI BLAST. MBF University of Arkansas at Little Rock, Little Rock, is available under an open source license, and executables, source AR, United States; 2Cornell University, Ithaca, code, demo applications, documentation and training materials are NY, United States; 3Roswell Park Cancer Institute, freely downloadable from http://research.microsoft.com/bio. MBT is a Buffalo, NY, United States collection of tools that enable biology and bioinformatics researchers to be more productive in making scientific discoveries The mission of the ABRF is to advance life sciences core facilities and biotechnology laboratories through research, communication, and 113 NERLSCD: A Model for Regional education. To facilitate this mission, the ABRF has implemented ABRF Networking of Life Sciences Core Affiliates and Chapters and the ABRF Affiliates and Chapters Committee for the following purposes: a) To encourage the establishment, support Directors the operations, and facilitate the coordination of new regional and 1 2 3 special interest groups that have goals related to those of the ABRF in G.S. Grills , M. Detwiler , T. Thannhauser , T. Hunter 4, P.S. Adams5, S.A. Bobin6, support of life sciences shared resources; b) To establish partnerships and collaborate with other existing organizations that have goals related K. Sol-Church7, P. Spatrick8, S. Perkins8 to those of the ABRF in support of life sciences shared resources; c) 1Cornell University, Ithaca, NY, United States; To promote the technologies, research support and administration of 2Roswell Park Cancer Institute, Buffalo, NY, United biomolecular resource facilities; d) To promote the development and States; 3United States Department of Agriculture/ applications of biotechnologies as shared research resources and to Agricultural Research Service, Beltsville, MD, United facilitate the advancement of life sciences research; e) To play a leadership role in networking core laboratories, researchers, and students, States; 4University of Vermont, Burlington, VT, 5 matching those with similar and complementary interests and skills; e) United States; Trudeau Institute, Saranac Lake, To enhance communication on the regional, national and international 6 NY, United States; Dartmouth Medical School, level regarding ABRF activities; to enhance the visibility of the ABRF 7 Hanover, NH, United States; Nemours COBRE in the scientific community; to educate the scientific community about Center for Pediatric Research, Wilmington, DE, the value of the ABRF; and to broaden the number and diversity of United States; 8University of Massachusetts Medical core laboratories and biotechnology laboratories that take advantage School, Worcester, MA, United States of the ABRF research group studies and ABRF membership networking opportunities; and f) To enhance the visibility of the ABRF with funding The Northeast Regional Life Sciences Core Directors (NERLSCD) agencies that support the development, acquisition and application annual meeting is a forum that provides an exceptional opportunity of core facility shared research resources. ABRF Chapters are special for life sciences core facility directors and managers to network with interest groups which may be formed based on common interests colleagues, to learn about biotechnology advances and applications, and/or geographical boundaries and support grassroots networks of and to discuss the challenges and results of regional implementation of individuals who wish to help advance ABRF goals and promote the shared research resources. The NERLSCD meeting was established in mission of shared resource facilities and biomolecular resources. ABRF 2006 as a grass roots effort by core directors. The meeting was held last Affiliates are special interest organizations that are autonomous from year at the University of Massachusetts Medical School in Worcester, the ABRF, have common and complementary interests with the ABRF, MA. The sixth annual NERLSCD meeting will be held at Cornell and have the goal of developing a collaborative relationship with the University in Ithaca, NY, Nov. 9-11, 2011. The goal of the meeting ABRF. Please join us for the ABRF Affiliates and Chapters Open Mic is to facilitate regional networking and sharing of resources and to Session from 6:00 pm to 6:45 pm on Sunday. help reduce regional duplication of costly biotechnology research infrastructure. The meeting provides a structured yet informal setting 115 The Midwest Association of Core for addressing the operational and technical challenges of life sciences Directors Poster Abstracts core laboratories. There are plenary sessions, workshops and discussion forums on financial and management issues facing biotechnology core W. Hendrickson1, P. Hockberger2 laboratories. There are technical workshops on genomics, functional genomics, proteomics, imaging, bioinformatics, bio-IT, and other 1University of Illinois, Chicago, IL, United States; technologies. A core facility poster session offers an opportunity for 2Northwestern University, Evanston, IL, United learning about regional life sciences shared resources and services. States The number and diversity of attendees reflects the broad appeal and usefulness of this meeting for core directors at many institutions in the The Midwest Association of Core Directors (MWACD) was organized in region. The meeting supports a diverse array of core directors and 2010 by a group of scientists at 6 different institutions to foster closer managers who play a crucial role in facilitating advances in knowledge interactions among directors and managers of core facilities throughout and understanding in a wide variety of life sciences research areas. This the Central Plains. The organization shares the same goals as ABRF, regional networking meeting for life sciences core facility directors and it has applied to become a chapter of ABRF. The MWACD differs can serve as an example for the organization, funding, structure, and from ABRF only in that its focus is on regional matters rather than on content of regional meetings for core facility directors in other regions issues of national concern. Towards this goal, the first annual meeting of the United States and in other countries. of the MWACD took place on October 21-23, 2010, at the Crowne Plaza Hotel in Chicago. The goal of the meeting was to provide an opportunity for networking among core directors and managers, to enable interactions with colleagues, sharing of technical advice, and

ABRF 2011 — Technologies to Enable Personalized Medicine • 63 discussions of continuing challenges associated with the operation of 117 Overview of the Agilent Technologies shared research resources and technologies. Keynote presentations SureSelectTM Target Enrichment were delivered by leaders of NIH-NCRR and FASEB, and there were panel discussions on networking, bioinformatics, and information System management systems. There was a poster session, vendor exhibits, and 1 1 1 1 J. Ong , A. Giuffre , S. Joshi , H. Ravi , breakout sessions on 8 different core-related topics. The meeting was C. Pabón-Peña2, B. Novak2, M. Visitacion2, attended by 120 researchers and supported by 17 corporate and not- M. Hamady2, F. Useche2, B. Arezi3, B. Buehler3, for-profit organizations. E. Lin2, S. Hunt2, D. Roberts2, S. Happe1, E. Leproust2 116 Automated Isolation of Genomic DNA from Large Volumes of Whole Blood 1Agilent Technologies, Cedar Creek, TX, United States; 2Agilent Technologies, Santa Clara, CA, 1, S. Krueger1, J. Kennedy2, S. Lee2, E. Vincent United States; 3Agilent Technologies, La Jolla, CA, C. Helt1, A. Bonk2, C. Cowan1 United States 1Promega Corporation, Madison, WI, United Next-generation DNA sequencing has revolutionized the discovery States; 2Hamilton Robotics, Reno, NV, United States of rare polymorphisms, structural variants, and novel transcripts. To A key source for genomic DNA(gDNA) is blood drawn into a standard meet the demand for fast, cost-effective, and accurate genome analysis 10mL Vacutainer® tube. The Promega ReliaPrep™ Large Volume HT methods from small scale studies to large sample cohorts, Agilent gDNA Isolation System integrated on the Hamilton MICROLAB® Technologies has developed the SureSelectTM Target Enrichment STARplus liquid handling workstation provides a unique and System. Available for the Illumina, SOLiD, and 454 NGS sequencing dependable system for isolating genomic DNA from large volumes platforms, SureSelect is a highly robust, customizable, and scalable (3 mL–10 mL) of blood. The novel chemistry and instrumentation system that focuses analyses on specific genomic loci by in-solution resolve many challenges encountered when processing large-volume hybrid capture. In addition, Agilent has introduced SureSelect XT samples in a high-throughput format such as: loss of sample pellets for Illumina and SOLiD, which combines gDNA prep, library prep, during decanting of fluids, transport of full 50 mL tubes to various and SureSelect Target Enrichment reagents in one complete kit. Both locations on a liquid handling robot, and manual re-suspension of SureSelect and SureSelect XT demonstrate high performance, as final DNA pellets. Liquid handler resource constraints were removed measured by capture efficiency, uniformity, reproducibility, and SNP by creationof a new accessory, the ReliaPrep HSM 32 LV instrument, detection. We highlight the utility of the SureSelect system across a which provides heating, shaking and magnetization of samples at wide range of target sizes and genome complexity using pre-designed one deck position. The combination of this device, the MICROLAB catalog libraries targeting cancer gene sets, sequences encoding the STARplus workstation and the ReliaPrep Large Volume HT gDNA kinome, and both human and mouse All Exon content. In addition, Isolation System allows automated recovery of pure gDNA from up to user-defined custom content can be easily developed using the 96 ten milliliter blood samples within 8 hours. We present verification Agilent eArray software with candidate variant coordinates as input. studies demonstrating automated system performance. Comparisons User-defined content can be manufactured on-demand as a custom between the ReliaPrep™ Large Volume HT gDNA Isolation System and SureSelect kit, or combined with pre-defined Agilent catalog content a standard precipitation-based method were made for duplicate blood using the Plus option. We propose a novel approach for variant samples from multiple donors. Yield, purity, and integrity of extracted discovery - using SureSelect catalog designs to uncover candidate gDNAwere assessed using UV absorbance spectroscopyand gel variants, followed by the design of smaller focused custom libraries for electrophoresis. Genomic DNA yields from normal 10 mL whole blood SNP validation and region profiling. By pooling many samples together samples were 200400 µg (depending on white blood cell count) in an per lane or slide, SureSelect multiplexing kits for Illumina and SOLiD eluted volume of 1mL. Recovered DNA exhibited good purity with enable validation across large sample cohorts with substantial cost A260/A280 ratios greater than 1.7 and A260/A230 ratios between 1.8 savings. Accurate post target enrichment pooling is facilitated by the and 2.2. Isolated DNA was suitable for storage and was used in many Agilent Bioanalyzer and QPCR NGS Library Quantification kits which downstream analysis applications. Results of genomic DNA purification ensure equal representation across samples. Further efficiencies are from frozen (hemolysed) blood samples and blood collected using realized using the Bravo Automated Liquid Handling Platform to meet common anticoagulants (EDTA, heparin, citrate)are also comparedto the need for parallel preparation of multiplexed libraries. demonstrate the efficacy of the new system.

Poster Abstracts 118 Gene Synthesis: A Cost-Effective Alternative to Traditional Molecular Cloning

A. Liao, M. Schwartz, H. Lo, J. Zhou, P. Yang GENEWIZ, Inc., South Plainfield, NJ, United States

Gene synthesis is the process of synthesizing a gene in vitro without the need for initial template. Contrary to what many researchers’ beliefs, commercial gene synthesis service is quickly evolving to become a cost- effective alternative to traditional cloning and other molecular biology procedures. The main reasons include: 1) Time savings: Traditional cloning involves a multi-step process that includes cloning strategy design, primer synthesis, PCR, gel extraction, bacteria transformation, and other complex steps. This process requires considerable amount of

64 • ABRF 2011 — Technologies to Enable Personalized Medicine time and human resource that gene synthesis does not. 2) Cost savings: 120 Genome Technology Center at the In most cases, it costs less to order a synthetic gene than it does to order NYU Langone Medical Center: New oligos, cloning kits, and DNA sequencing services. 3) Enhanced DNA performance: Gene synthesis allows for codon optimization which Support for Clinical and Translational has been proven to increase the efficiency of protein expression. 4) Science Convenience: Without the need for a physical template and without , S. Mische design restrictions associated with the traditional cloning process, a J. Zavadil researcher can get a gene of his/her choice by simply supplying the New York University Langone Medical Center, New nucleotide sequence or amino acid sequence. GENEWIZ is a global York, NY, United States CRO that provides a wide range of DNA services, including gene synthesis. GENEWIZ’s gene synthesis service features a 2-3 week To significantly enhance support for clinical and translational research turnaround and expert technical and project management support. within the framework of its CTSI, the NYU Langone Medical Center This informational poster will present case studies of how GENEWIZ’s consolidated the Microarray and DNA Sequencing Cores into a new gene synthesis service benefited researchers who had previously relied Genome Technology Center, a shared resource overseen by the Office on traditional molecular cloning for plasmid construction. for Collaborative Science. The GTC’s team of 4 technical personnel and one faculty level director assists >120 NYULMC laboratories in 119 Nucleotide-Level Variant Analysis of their basic, clinical and translational research. The Sequencing Unit Next-Generation Sequencing Data operates 2 Illumina GAIIs, and a HiSeq sequencer will be added in Q1 Using a Cloud-Based Data Analysis 2011. The GAII capacity is applied to research applications (ChIP-seq, small-RNA-seq and RIP-seq) and to identification of disease-related Pipeline genome-level structure changes and correlates (e.g. RNA-seq of cancer transcriptomes). GTC also has a Roche GS FLX System (454) used for B. Ganter, G. Asimenos, A. Sundquist de novo sequencing of microbial species and for amplicon sequencing DNAnexus, Palo Alto, CA, United States in clinical genetics, patient microbiome diversity, etc. The Microarray Unit operates Affymetrix GeneChip system and high-capacity QPCR To demonstrate the flexibility of a cloud-based solution for analyzing (ABI 7900HT) with automated plate setup and loading for gene and disparate sets of next-generation sequencing data, we looked at carefully microRNA profiling and for SNP genotyping in clinical genetics. The chosen samples across different populations from the 1,000 Genomes GTC cooperates closely with the newly established Center for Health Project (www.1000genomes.org) and conducted an extensive analysis Informatics and Bioinformatics (CHIBI) supported by the NIH/NCRR on two Chinese populations, the “Chinese in Beijing” (CHB) and the CTSA Award. CHIBI provides an HPC facility for sequencing and “Chinese in metropolitan Denver” (CHD), each consisting of 28 exomes. microarray data storage and offers a full range of informatics services. Each dataset was uploaded into the system using raw data files acquired The GTC is committed to regional and nationwide collaborations with from the 1,000 Genomes Project. Using these data and a cloud-based other Cores. GTC participates in the activities of the Genomic Analysis data analysis pipeline, we performed a nucleotide-level variant analysis and Technology Excellence (GATE) Working Group of the Academy for combined with a population allele frequency analysis across all samples Medical Development and Collaboration (AMDeC), particularly in the for the two populations . To identify alleles that are significantly sections of Core Facility Directors, Funding Strategy and Bioinformatics. different across the two populations, a Pearson’s chi-square test was It also contributes to the AMDeC Facilities Instrumentation Resources applied, which resulted in a total of 1.5 Mio SNPs, of which 84 were Services Technologies (FIRST), a real-time online database of nonsynonymous with a p-value of less than 0.01. Interestingly, the genes biomedical research technology and resources available in the New associated with nonsynonymous variants of the Chinese in metropolitan York City area and throughout Northeastern US. Key services of the Denver population were enriched for biological annotations such as GTC are offered to external clients. endocrine system disorder, metabolic disease, cardiac fibrosis, and inflammation (includes ZNF264, RPS6KA2, ROBO2, CRK, MUSK, CBL, 121 Integrated Core Facility Support and CRK, and others). Furthermore, genes usually associated with liver injury were also identified for this population, suggesting the liver is Optimization of Next Generation exposed to toxic agents more so in this population compared to the Sequencing Technologies Poster Abstracts CHB population. The observed genomic differences in these two G. Grills, P. Schweitzer, Q. Sun, J. Pillardy, different Chinese populations living in different parts of the world hint W. Wang, T. Stelick, R. Bukowski, L. Ponnala , towards a potential link between nutrition and different diseases (e.g. J. VanEe heart disease or metabolic diseases). Using this analysis as a case study, we will demonstrate how a scalable computational infrastructure can Cornell University, Ithaca, NY, United States provide researchers and sequencing service providers alike, a cost- effective and secure cloud-based computing platform as a powerful New DNA sequencing technologies present an exceptional opportunity and collaborative technology solution for large scale sequence data for novel and creative applications with the potential for breakthrough analysis and management. discoveries. To support such research efforts, the Cornell University Life Sciences Core Laboratories Center has implemented the Illumina HiSeq 2000 and the Roche 454 GS FLX platforms as academic core facility shared research resources. We have established sample handling methods, LIMS tools and BioHPC informatics analysis pipelines in support of these new technologies. Our genomics core laboratory, in collaboration with our epigenomics core and bioinformatics core, provides sample preparation and data generation services and both project consultation and analysis support for a wide range of

ABRF 2011 — Technologies to Enable Personalized Medicine • 65 possible applications, including de novo or reference based genome critical reagent. The CritRS was put in place in 2004 at Genentech assembly, detection of genetic variation, transcriptome sequencing, to ensure quality, consistency, and traceability of critical reagent small RNA profiling, and genome-wide epigenomic measurements of preparations. Critical reagent analytical characterization is performed methylation and protein-nucleic acid interactions. Implementation of using four platform assays to assess the molecule’s identity and next generation sequencing platforms as shared resources with multi- purity/heterogeneity. Identity is confirmed using Edman N-terminal disciplinary core facility support enables cost effective access and sequencing and MALDI-TOF peptide mass fingerprinting. Purity/ broad based use of these technologies. heterogeneity is assessed by size exclusion chromatography (SEC) and SDS-PAGE. The four platform assays provide sufficient identity 122 Better Outcomes for Our Patients: and purity information for most critical reagents. However, in some Moving Science Forward in the cases additional characterization methods have to be used due to Biomolecular Core Laboratory at the unexpected results, such as observed N-terminal sequence is different from expected sequence, more than one N-terminal sequences are Nemours Center of Pediatric Research present, SDS PAGE shows unexpected band pattern, and SEC can not J. Holbrook, D.L. Stabley, E.C. Hitchens, P. Geller, separate the high and low molecular species of the reagent. In this K. Sol-Church poster we’ll present typical analytical characterization results as well as some unusual findings. Nemours/A.I. duPont Hospital for Children, Wilmington, DE, United States 124 Progress towards Becoming a CLIA- Certified Core Facility Nemours Biomedical Research has a long-standing commitment to scholarly and scientific endeavors directed towards improving the M. McMillan1, M. Gray1, M. Sandoval1, diagnosis and treatment of pediatric medical conditions. Established S. McCann1, S. Gorda2 in 2004 with the support of a COBRE grant from the NIH s National Center for Research Resources, the Nemours Center for Pediatric 1University of Southern California, Norris Cancer Research provides clinical and translational researchers with the tools as Center/Keck School of Medicine, Los Angeles, CA, well as training they need to perform their research. The Biomolecular United States; 2University of Southern California , Core Laboratory (BCL), located at the Alfred I. duPont Hospital for College of Letters, Arts and Sciences, Los Angeles. Children in Wilmington, Delaware, is supported by the Center of CA, United States Pediatric Research. We provide a unique opportunity for Nemours clinicians, research staff, and affiliates at University of Delaware and The USC Norris DNA Core Facility (formerly known as the Microchemical Thomas Jefferson University to develop competitive research projects Core) was created in 1984. It is equipped with an AB3730 DNA in genetics as well as provide essential services in molecular biology sequencer and an AB3900 oligonucleotide synthesizer. It is affiliated and genomics. The mission of the BCL is to facilitate, through state-of- to the Molecular Genomics Core in the Epigenome Center. Our goal is the-arts services and stewardship, discoveries that begin at a molecular to establish the DNA Core Facility as a CLIA-certified laboratory. The level and move rapidly from patient-oriented research to the bedside. rationale is that for Norris Cancer Center clinicians to use our DNA Working in close collaboration with those engaged in research activities, sequencing data in the diagnosis, treatment and prognosis of cancer our goal is to enable fast, effective and productive top quality services patients, the facility must be CLIA (Clinical Laboratory Improvement as well as to match BCL s strengths with our clients needs. This poster Amendments)-certified. We will present our progress towards this goal highlights the services the Biomolecular Core lab offers, including including qualified personnel, identifying genes of medical interest, molecular biology training, a listing of our instrumentation, and core the establishment of appropriate records and the purchase of CLIA- businesses including DNA sequencing, genotyping, gene expression compliant equipment. We plan to file for CAP (College of American and methylation analysis. We will also highlight our newly established Pathologists) accreditation and apply for a Clinical Laboratory License microarray service utilizing the Affymetrix Microarray platform. The (State) as well as submitting a CLIA application (Federal) to the development of this vital core allowed investigators, who previously California Department of Health Services. used extramural services and technologies, to move those projects in house, thus saving both time and money for our clients. 125 Trudeau Institute Molecular Biology Core Facility 123 Critical Reagent Analytical Characterization Program at P. Adams, A.M. Moquin, J.J. Hoffman Poster Abstracts Genentech Trudeau Institute, Saranac Lake, NY, United States , C. Williams, A. Meier, C. Lu, S. Chamberlain Centralization and merging of core facilities is a favorite topic in the P. Motchnik current economy. The Molecular Biology Core Facility (MBCF) at the Genentech, Inc., South San Francisco, CA, United Trudeau Institute has provided a variety of services to the investigators States at the institute while recovering the majority of its expenses since its inception 12 years ago. The MBCF survives by offering an assortment At Genentech, Critical Reagents are internally prepared, recombinantly- of services that are specifically tailored to the Institute mission produced proteins used in GLP/GMP assays in support of regulatory and by personalizing each service to the individual investigator. filings. These proteins are typically extracellular domain constructions, Shared resource equipment and work areas optimize space and growth factors, antibodies and novel hybrid constructions produced instrumentation. Chargeback fees are designed to recover costs in CHO transient or stable cell lines, E. coli or Baculovirus. Genentech’s according to OMB Circular A-21. The MBCF recovers ~ 80% of costs. Critical Reagent System (CritRS) is used to document the process Services provided include DNA sequencing, spectratyping of the T-cell of ordering, manufacturing, characterization, and delivering a repertoire (fragment analysis), production, purification and labeling

66 • ABRF 2011 — Technologies to Enable Personalized Medicine of Major Histocompatibility (MHC) Class I and Class II tetramers for United States Department of Agriculture. The CT unit is comprised Fluorescent Activated Cell Sorter (FACS) analysis, RNA and Protein of four research related components: genetic analysis, proteomics- analysis using “Lab on a Chip” technology, real-time PCR measurement biopolymers mass spectrometry, electron microscopy, and magnetic of gene expression and viral loads, knock-out mouse and Mycoplasma resonance spectroscopy (NMR). In addition, the Research Data Systems, screening, DNA haptenation and recombinant protein expression/ the information pipeline of the CT, provides the means to facilitate data purification for antibody detection, monoclonal and polyclonal distribution to researchers, stakeholders, and the general public. The antibody production and in vivo immunization. A variety of simple availability of integrated resource laboratories assures professional and services such as primer design, primer ordering, stock primers, peptide dependable support to the goals of the ARS community. ordering and Taq production save the investigators time, effort and money. Education and training are provided for all techniques and 128 NYULMC Office of Collaborative for using shared MBCF instrumentation, such as spectrophotometers, Science Cores - Enabling Personalized real-time PCR equipment, tissue prep and image capture/analysis Medicine through Translational equipment. Not only does the MBCF provide the service, the personnel assist in the planning and analysis to maximize proper usage of the Tesearch technique. Services not available in the MBCF are outsourced to other C. Curchoe, T. Winner, J. Salcedo, S. Mische, Core facilities in the area to be sure the quality of service is the highest. D. Levy The MBCF at Trudeau Institute survives through versatility, imagination and customization. New York University, Langone Medical Center, New York, NY, United States 126 Molecular Resource Facility, UMDNJ- New Jersey Medical School The New York University Langone Medical Center (NYULMC) has committed $15 million to ensure that researchers have access to cutting R. Donnelly, S. Kuppasani, K. Dhawan edge enabling technologies. The Office of Collaborative Science enables access to expertise and technology through the centralized University of Medicine & Dentistry of New Jersey, administration of Shared Resource Centers. In partnership with the New Jersey Medical School, Newark, NJ, United NYU Cancer Institute, Center for AIDS Research and Clinical and States Translational Science Institute, we strive to increase collaboration among clinical, translational and basic scientists to support novel science and The Molecular Resource Facility of the New Jersey Medical School was to classify and treat diseases not just by their phenotype, but also by established in April 1995 to enhance the resources available to the their molecular profiles. Here we highlight several Core Resource Labs research community within the medical school. It serves to provide that support translational research at NYULMC. Human biospecimens services to the research community and as a source of information are essential validating mechanistic insights into biological processes on molecular techniques and research strategies involving molecular gleaned from cell lines, animal models of disease, and epidemiological biology. Our facility has developed through the years to be an “all studies. The availability of well-annotated human biospecimens inclusive” nucleic acid facility with limited protein analysis capabilities. is a critical link between basic science and translational research. We provide services in many areas including DNA sequencing, protein The NYULMC Tissue Acquisition and Biorepository Core provides sequencing, peptide synthesis, qPCR and others. In 2009 we began investigators with freshly acquired frozen, normal, and diseased tissue offering High Throughput DNA Sequencing on the SOLiD® instrument. at the time of surgery, as well as pathology-archived, formalin fixed, Our services are available to any laboratory requiring these services. paraffin-embedded (FFPE) specimens, from appropriately consented patients. The Immunohistochemistry and Histopathology Cores 127 United States Department of closely interface with the Tissue Biorepository, providing the means Agriculture/Agricultural Research for clinical and basic research collaborations and the microscopic Service (USDA-ARS) Eastern Regional analysis of clinical research specimens. The Genome Technology Research Center Core Technologies Centers sequencing and microarray units are dedicated to RNA-seq of cancer patient transciptomes, array expression profiling and SNP D. Needleman, A. Nuñez, G. Strahan, D.S. Soroka, genotyping, enabling translation research as well as the preparation of Poster Abstracts W. Damert nucleic acids for molecular profiling, mutational analysis and microRNA screens. The RNAi Core provides an integrated, state-of-the-art, RNA United States Department of Agriculture/ interference (RNAi) based high-throughput screening (HTS) and Agricultural Research Service (USDA-ARS) Eastern offers siRNA/dsRNA screening libraries for the cross-species functional Regional Research Center Core Technologies, characterization of whole genomes in a systematic, comprehensive, and Wyndmoor, PA, United States cost effective manner.

The Core Technologies (CT) unit, located at the Eastern Regional Research Center (ERRC), is a centralized resource of specialized instrumentation and technologies. Its objective is to provide supplementary research data processing, interpretation, analysis and consultation for a broad range of research programs approved by the Agricultural Research Service (ARS), the in-house research arm of the

ABRF 2011 — Technologies to Enable Personalized Medicine • 67 129 Bravo Automated Liquid Handling effective for amplification from damaged RNA template (FFPE and laser Platform for SureSelect Target captured tissue samples) and single-cell input quantities (picograms). The efficacy of Transplex WTA2 amplification for downstream Enrichment applications, primarily qPCR and expression microarray analysis, is 1 2 3 well-documented. It follows that the utilization of next-generation A. Giuffre , M. Visitacion , J. Karbowski , B. Novak2, H. Ravi1, J. Ong1, S. Joshi1, sequencing for gene expression research and diagnostics would be well served by Transplex amplification of RNA isolated from samples of C. Pabón-Peña2, E. LeProust2, D. Roberts2, severely restricted quantity or quality. Strategies for the integration of S. Happe1 Transplex WTA2 with next-generation sequencing are examined, with 1Agilent Technologies, Genomics, Cedar Creek, TX, particular emphasis on elimination of the characteristic fixed primer United States; 2Agilent Technologies, Genomics sequence associated with each amplicon in the amplification library. Division, Santa Clara, CA, United States; 3Agilent Removal of these sites will allow direct entry of the resulting product into the sequencing workflow. Methods under consideration will enable Technologies, Automation Solutions Division, Santa the WTA2 amplicon to feed into the current sample prep protocols for Clara, CA, United States the Illumina GA and GAII, SoLiD 5500/5500xl, and Roche-454 GS FLX/ Junior platforms. Agilent’s SureSelect Target Enrichment products have met the need for a robust and cost-effective approach for systematic resequencing of candidate regions in the human genome and other species. SureSelect 131 Robotic Scripts, Methods, Reagents, is an in-solution method of targeting only the user defined regions and Devices for High Throughput of interest of a genome to identify genetic variants and mutations Automated Production of Next associated with disease.With the advent of SureSelect Target Enrichment Generation Sequencing DNA Multiplexing kits, the ability to achieve targeted enrichment on multiple Fragment Libraries samples in a single sequencing lane is maximized, saving time and money without sacrificing performance. However, the handling of numerous J. Bishop, M. Allen, K. Poulter, W. Zhang, samples can be cumbersome and the possibility for the introduction of M. Landers, D. Mandelman, B. Laubert, A. Harris, sample processing errors greatly increases. Agilent has expanded the R. Bennett SureSelect Target Enrichment System to allow for higher throughput of numerous samples with the Bravo Automated Liquid Handling Platform. Life Technologies, Carlsbad, CA, United States The SureSelect method selects user defined target regions of interest from SureSelect XT prepped genomic DNA libraries by hybridization Recent technological advances have greatly increased both the speed to in-solution biotinylated cRNA probes; these enriched libraries are and throughput of genome and transcriptome sequencing. The pace then compatible for sequencing using the llumina GAII and HiSeq of sequencing has further increased with target-enrichment and platforms. The Agilent automation solution for SureSelect enables library barcoding techniques that allow multiplexing of sequencing processing of up to 96 samples per run in much less time than can runs. However, current manual methods for creating libraries do not be obtained manually. Our SureSelect automation workflow includes scale well, limiting the practical investigation of large numbers of SureSelect XT genomic DNA library construction, hybridization setup, samples. To ease the library-creation bottleneck, we describe here a and automated capture and wash steps. Each of these steps can be set of protocols, robotics scripts, bulk reagents and instrumentation run with minimal user intervention and provide more uniformity than developed to automate the production of up to 96 DNA fragment manual methods. We demonstrate here the throughput potential and sequencing libraries at once. Current protocols for creating sequencing consistency in performance across entire 96-well plates of multiplexed libraries are lengthy, laborious, and not amenable to automation. samples. In summary, SureSelect Automation solution provides an easy We describe here new magnetic bead based methods that produce to use, automated gDNA library preparation and target enrichment libraries with yield, purity, and size-selection comparable or superior system that is a cost-effective approach to analyzing discrete genomic to current column and gel-based protocols. Using these bead-based regions with unprecedented depth, accuracy and throughput. methods we developed a unique combination of optimized adaptor concentrations and clean-up techniques which increase the yield of libraries from small amounts of DNA by several fold. We also describe 130 Modification of the Transplex WTA2 robotic scripts for producing 1-96 libraries simultaneously on two Amplification Product for Next commonly used robotic platforms. These protocols accept 10-8000 ng Generation Sequencing of sheared DNA, calculate and dilute barcode adaptors for each library

Poster Abstracts as necessary, automate all intermediate processing steps, and deliver 1, B. Ward1, D. Fenoglio2 K. Heuermann purified libraries ready for amplification off-instrument. An additional 1Biotech R&D, Sigma Aldrich Corporation, St. Louis, script for post-PCR purification of the libraries is also provided. We MO, United States; 2Biotech Marketing, Sigma- further describe the Library BuilderTM System, comprising a benchtop Aldrich Corporation, St. Louis, MO, United States device and kits of plastic tips, tubes and sealed cartridges prefilled with reagents necessary for producing 1 to 13 DNA fragment libraries Transplex Whole Transcriptome Amplification (WTA2)ä exponentially either with or without size selection. Our analysis of sequencing data amplifies RNA producing a double-stranded cDNA library while shows that libraries produced by all of the above methods are free of precisely maintaining differential levels of individual transcripts in test excess adaptors which interfere with quantitation, are unbiased, of high and reference samples. Though originally designed to amplify nanogram complexity, and free from cross-contamination. The protocols herein quantities of RNA, Transplex WTA2 has been shown to be exceedingly are provided to the community for use or customization.

68 • ABRF 2011 — Technologies to Enable Personalized Medicine 132 Improvements in SOLiDTM Whole 134 GeneMarker® Genotyping Software: Transcriptome Library Preparation Tools to Increase the Statistical Power Workflow to Enable Low Input RNA of DNA Fragment Analysis Amounts C.S. Johathan Liu, D. Hulce, X. Li, T. Snyder-Leiby C. San Jose Hinahon SoftGenetics, LLC, State College, PA, United States Life Technologies, Austin, TX, United States The discriminatory power of post-genotyping analyses, such as The SOLiDTM Total RNA-Seq (STaR-Seq) kit from Life Technologies kinship or clustering analysis, is dependent on the amount of genetic provides a complete workflow for generating directional, random information obtained from the DNA fragment/genotyping analysis. The primed, whole transcriptome libraries from total RNA and fractionated number of microsatellite loci amplified in one multiplex is limited by the RNA. Poly(A) selected RNA is commonly used for expression profiling number of dyes and overlapping loci boundaries; requiring researchers using short read sequencing on the SOLiDTM instrument. The input to amplify replicate samples with 2 or more multiplexes in order requirements in the current STaR-Seq protocol is 100-500ng poly(A) to obtain a genotype for 12-15 loci. AFLP is another method that is selected RNA, input amounts well suited for studies using cell culture limited by the number of dyes, often requiring multiple amplifications or large tissues. However, in circumstances where the available sample of replicate samples to obtain more complete results. Traditionally, is limited, such as in the case of clinically derived material or the need researchers export the genotyping results into a spread sheet, manually to focus the analysis on specific cell populations, there is often too little combine the results for each individual and then import into a third RNA available to perform the cDNA library analysis. Improvements to software package for post-genotyping analysis. GeneMarker is highly the STaR-Seq workflow have been developed and validated pushing accurate, user-friendly genotyping software that allows all of these steps the starting input to 5ng of poly(A) selected RNA, while still maintaining to be done in one software package, avoiding potential errors from high concordance with the current method. Improvements include data transfer to different programs and decreasing the amount of time optimizations in: 1. Reducing RNA fragmentation time to increase RNA needed to process the results. The Merge Project tool automatically fragments within the suitable size range; 2. Altering purification methods combines the results from replicate samples processed with different after RNA fragmentation to minimize sample loss; 3. Adoption of a primer sets. Replicate animal (diploid) DNA samples were amplified bead purification and size selection method to reduce sample loss due with three different multiplexes, each multiplex provided information to gel size selection; 4. Modifying PCR conditions to maximize cDNA on 4 -6 loci. The kinship analysis using the merged results provided library yields without biasing expression profiles. These improvements a 1017 increase in statistical power with a range of 108 when 5 loci enable STaR-Seq to be a viable option for samples with small quantities were used versus 1025 when 15 loci were used to determine potential of total RNA. As the field of next generation sequencing advances, it is relationship levels with identity by descent calculations. These same necessary to meet the need for decreased sample requirements, while sample sets were used in clustering analysis to diagram dendrograms. maintaining strand specificity, and accuracy. The dendrogram based on a single multiplex resulted in three branches at a given Euclidian distance. In comparison, the dendrogram that was 133 RNA-Seq Analysis with NextGENe constructed using the merged results had eight branches at the same Software Euclidian distance.

J. McGuigan, Y. You, C. Liu 135 Hotspot-Mutation Analysis of the SoftGenetics, LLC, State College, PA, United States EGFR/KRAS/BRAF Pathway Using Mutation Surveyor® Software RNA sequencing is a powerful tool for interrogating the entire , D. Hulce, K. LeVan, N. Shouyong transcriptome at once. It allows identification of novel isoforms- J. Liu including gene fusions and alternative splicing- and expression level SoftGenetics, LLC, State College, PA, United States analysis across several orders of magnitude. NextGENe’s new RNA-seq

application can be used to analyze data from any of the three main Hotspot-mutation analysis of the EGFR/KRAS/BRAF pathway (or Poster Abstracts 2nd generation sequencing systems including Roche GS FLXTM and other clinically relevant pathway) can quickly genotype patients as FLX Titanium, Illumina Genome Analyzers, and Applied Biosystems candidates who may respond favorably to specific drug treatments SOLiDTM Systems. Variant detection, expression level analysis, and and therapies or into other groups where treatment options are isoform identification are all performed in one analysis. The tool employs limited and less favorable. Sanger sequencing analysis using Mutation a hybrid approach, combining the use of an exon junction reference Surveyor software provides high-throughput, high-sensitivity variation sequence with the de novo detection of exons. This allows discovery of detection. Increased efficiency can be achieved using flexible and previously un-annotated genes in addition to highly accurate alignment customizable reporting-sequencing results can be organized by to known transcripts. All detected transcripts are reported in addition patient identifiers, variation type (reported or unreported, pathogenic to the information provided by NextGENe’s variant and expression or benign or drug sensitive), by gene/exon/amplicon, or quality reports. Results are shown in the NextGENe Viewer which provides a metrics, and other options. GenBank sequence files from NCBI for high level of visualization for review and editing. EGFR exons 18, 19, 20, and 21; KRAS exons 2 and 3; and BRAF exon 15 were edited to contain reported variations. These reported variations included polymorphisms from dbSNP (downloaded with the 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 data for twelve, simulated (mutations obtained from sequencing reports in the scientific literature), patients were developed and compared

ABRF 2011 — Technologies to Enable Personalized Medicine • 69 to the customized GenBank sequences. Sequencing analysis results **137 Probabilistically Assigning Sites of were grouped by patient-specific identifiers. Any unmatched or low Protein Modification with Scaffold quality data files are identified in the report, indicating which samples require resequencing. Mutations that match reported variations added PTM to the GenBank sequences are highlighted-SNP identifiers or color- M. Turner, N. Vincent-Maloney, B.C. Searle coding of SNP type quickly indicate which variations are pathogenic or drug-sensitive or reported in dbSNP. Unreported variations are not Proteome Software, Inc., Portland, OR, United highlighted and may be benign or variations of unknown significance. States The gene column displays the gene and accession number for that gene used for the analysis. The exon column displays the exon number Accurate interpretation of MS/MS data containing post-translational of the gene, and accession numbers of the mRNA and protein used modifications such as phosphorylation continues to be a difficult problem for the analysis. High-throughput, high-sensitivity variation detection for proteomics researchers. One challenge involves knowing which data coupled with personalized reporting provides robust and economical are reliable for assigning specific amino acid sites of modification and genotyping of patients. separating that from data that are less conclusive. This is particularly difficult when peptide identifications contain multiple possible sites 136 Fully Automated Sample Preparation of modification. Some work towards improving phosphorylation site Methods for High Throughput assignment has been implemented in a software program called Ascore by Beausoleil et al (Nat. Biotechnol. 2006, 24, 1285-1292). Ascore is Transcriptome Analysis by Arrays and more accurate than traditional database search engines at localizing a NGS modification because it only considers the fragment ladder ions that , W. Keilholz, J.D. Heath can differentiate between the two possible sites. Here we present S. Kain Scaffold PTM, a program that extends the Ascore algorithm to consider NuGEN Technologies, Inc., San Carlos, CA, United overlapping data from several peptides simultaneously to improve States confidence in specific site assignments. Scaffold PTM uses the Ascore algorithm to interpret sites of modification on a peptide-by-peptide NuGEN Technologies is a provider of innovative sample preparation basis. Peptide identifications can be read from Scaffold, or any database solutions. As such, we offer a wide palette of systems optimized for search engine that supports the open community standard format, a variety of sample types such as whole blood RNA, small quantity MzIdentML. Once the data has been collated, Scaffold PTM collapses RNA from different sources as well as highly degraded RNA i.e. from the peptide interpretations into protein specific sites of modification, FFPE specimens. The SPIA® (Single Primer Isothermal Amplification) comprised of multiple pieces of overlapping evidence. technology allows rapid linear amplification of input RNA even at picogram level and all of our amplification assays generate microgram 138 Use of Synthetic Transcript Pools to amounts of cDNA, known as SPIA product which is suitable for analysis Evaluate RNA-Seq Performance and i.e. by qPCR. Reagent modules to further prepare the SPIA product Analytical Methods for subsequent analyses on microarrays or massive parallel sequencing complement NuGEN’s current offering. NuGEN targets are compatible P. Whitley, L. Qu, A. Lemire, J. Brockman, with all leading microarray formats and NGS platforms. Automating S. Heater, J. Schageman, J. Gu, K. Lea, C. San Jose, a workflow not only helps maximizing sample throughput but also N. Hernandez, D. Batten, K. Bramlett, D. Ilsley, minimizes variance introduced during the assay. We describe fully C. Mueller, R. Setterquist automated assays on leading liquid handling systems that enable high-throughput target preparation of up to 96 samples using NuGEN Life Technologies, Ambion R&D, Austin, TX, United systems for different sample types. The NuGEN assays are scripted States in a modular form allowing highest flexibility with regard to both, the number of samples processed at a time as well as integration into RNA-Seq and related Massively Parallel Sequencing methods are rapidly the laboratory workflow. Starting from total RNA, the entire process becoming standardized platforms for transcriptome studies . Although through to target preparation for further analysis on microarrays RNA-Seq is now being used routinely, there has not been a thorough or sequencing platforms can be completed within two convenient investigation of fundamental performance metrics using standardized workdays. Basic QC-metrics with these fully automated workflows show reagents and analytics. In order to evaluate the capabilities of RNA- equal or superior performance over manual handling. These methods Seq and associated analysis methods we have used two synthetic

Poster Abstracts are well suited for adoption by core facilities to increase efficiency, RNA spike-in pools that can be added to the RNA sample prior to reduce operator variation, and improve reproducibility and data library preparation and sequencing. The pools consist of 92 synthetic quality. Their compatibility with a range of microarrays and sequencing transcripts, designed by the External RNA Control Consortium (ERCC). platforms further enhance the flexibility for the core labs that can adopt The control pools are designed to enable assessment of both within a single workflow for a range of instruments and applications. and between sample performance. Using sequencing data generated with these controls we have benchmarked several statistical methods designed specifically to assess differential expression of count data. We show that methods which rely on Poisson distributions dramatically underestimate the variance associated with RNA-Seq data. This underestimation causes an inflated False Positive Rate. We also show that various methods for estimating the dispersion parameter in Negative Bionomial tests also vary in their ability to control the Type I error rate. These performance differences are true in the absence of biological variability, suggesting that even technical variability associated with sequencing is not adequately described by a Poisson

70 • ABRF 2011 — Technologies to Enable Personalized Medicine distribution. Additionally, we show that the controls can be used to and FORTRAN development environments without the complexity of evaluate performance of various normalization and scaling methods non-standard dialects or programming models. Thus the performance for RNA-Seq count data. This analysis suggests that traditional scaling of application-specific hardware is achievable with the familiar methods based on the total number of reads result in a significant loss programmability and deployment of a commodity server. In addition to of accuracy and more robust methods are often required. This analysis higher throughput, increased performance can fundamentally improve reveals the importance of using standardized reagents and controls as a research quality by allowing more accurate, previously impractical routine part of the RNA sequencing workflow. approaches. This presentation will discuss the suitability of hybrid- core servers’ advanced architecture and compiler technology for 139 Software Systems for Clinical sequence alignment and assembly applications. For example, the Smith- Research Waterman alignment algorithm is 172 times faster on Convey’s HC-1 than the best software implementation on a commodity server. Such T. Smith1, N.E. Olson1, D. Smith2, C. Mason3 performance speeds research, while reducing energy consumption, floor space, and management effort. Most bioinformatics applications 1 2 Geospiza, Inc. Seattle, WA, United States; Mayo are similarly well suited for this architecture because they have low Clinic, Rochester, MN, United States; 3Weil Cornell data interdependence, which greatly increases performance through Medical College, New York, NY, United States hardware parallelism. Furthermore, small data type operations (four nucleotides can be represented in two bits) make more efficient use of By the end of 2011 we will likely know the DNA sequences for 30,000 logic gates than the data types dictated by conventional programming human genomes. However, to truly understand how the variation models. Bioinformatics applications that have random access patterns between these genomes affect phenotype at a molecular level, future to large memory spaces, such as graph-based algorithms, experience research projects need to analyze these genomes in conjunction with memory performance limitations on cache-based x86 servers. Convey’s data from multiple ultra-high throughput assays obtained from large highly parallel memory subsystem allows application-specific logic to sample populations. In cancer research, for example, studies that simultaneously accesses 8192 individual words in memory, significantly examine 1000s of specific tumors in 1000s of patients are needed increasing effective memory bandwidth over cache-based memory to fully characterize the more than 10,000 types and subtypes of systems. Many algorithms, such as Velvet and other de Bruijn graph cancer and develop diagnostic biomarkers. These studies will use based, short-read, de novo assemblers, greatly benefit from this type high throughput DNA sequencing to characterize tumor genomes and of memory architecture. their transcriptomes. Sequencing results will be validated with non- sequencing technologies and putative biomarkers will be examined in 141 Implementation of GeneSifter large populations using rapid targeted assay approaches. Geospiza is transforming the above scenario from vision into reality in several ways. Improves Workflow Management The Company’s GeneSifter platform utilizes scalable data management within a Core Facility technologies based on open-source HDF5 and BioHDF technologies , J. Boland, J. Bacior, V. Lonsberry to capture, integrate, and mine raw data and analysis results from DNA, A. McCary RNA, and other high-throughput assays. Analysis results are integrated National Cancer Institute, Bethesda, MD, United and linked to multiple repositories of information that include States variation, expression, pathway, and ontology databases to enable discovery process and support verification assays. Using this platform As throughput within sequencing facilities increases, Laboratory and RNA-Sequencing and Genomic DNA sequencing from matched Information Management Systems (LIMS) will become necessary to tumor/normal samples, we were able to characterize differential gene allow laboratory managers and staff to organize workflows, samples expression, differential splicing, allele specific expression, RNA editing, and sample data in a repeatable and reliable manner. At the NCI’s Core somatic mutations and genomic rearrangements as well as validate Genotyping Facility, we have successfully implemented Geospiza’s these observations in a set of patients with oral and other cancers. GeneSifterTM Lab Edition, a commercially available LIMS, for use with our Roche 454 GS FLX sequencer processes. Our current setup consists of 140 Hybrid-Core Computing for High- seven different sample preparation techniques, (six target enrichment Throughput Bioinformatics methods and one whole genome method), which have been divided Poster Abstracts into a series of modular workflows that are arranged in the proper G. Vacek order for each process. Each workflow is then further broken down into specific protocol steps, which are used as guides for the technicians Convey Computer Corporation, Richardson, TX, during protocol performance. Additionally, mastermix preparations, United States reagent lot numbers, file uploads and other pertinent information are input into the system at all relevant protocol steps. Each sample’s data Advanced architectures can deliver dramatically increased throughput is stored on our internal GeneSifterTM server, with the ability to recall for genomics and proteomics applications, reducing time-to- and report it at a later date. Therefore, by developing an extensive completion in some cases from days to minutes. One such architecture, and interconnected setup of workflows in GeneSifterTM, the NCI’s Core hybrid-core computing, marries a traditional x86 environment with Genotyping Facility has successfully increased reliability in sample a reconfigurable coprocessor, based on field programmable gate tracking while decreasing the need for paper- or Excel-base logging array (FPGA) technology. Application-specific instructions executed methods. This project has been funded in whole or part with federal by the coprocessor appear as extensions to the x86 instruction set funds from the NCI, NIH under contract HHSN261200800001E. architecture. This integrated approach provides users familiar C, C++

ABRF 2011 — Technologies to Enable Personalized Medicine • 71 142 Comparison of Data Envelopment the strongest individual correlates of protein concentrations. In a Analysis Models to Identify Potential combined model, characteristics of the coding region and the 3’UTR explained a larger proportion of protein abundance variation than Cancer Biomarker Genes for Colon characteristics of the 5’UTR. Further, we used data from human and six Cancer other organisms (bacteria, yeast, worm, fly, and plant) and established that steady-state abundances of proteins show significantly higher P. Diaz-Candelas, M.E. Sanchez-Pena, C.E. Isaza, M. Cabrera-Rios correlation across these diverse phylogenetic taxa than the abundances of their corresponding mRNAs (p=0.0008, paired Wilcoxon). These Bio IE Lab at University of Puerto Rico, Mayagüez, data suggest strong selective pressure to maintain protein abundances Puerto Rico during evolution, even when mRNA abundances diverge. The absolute protein and mRNA concentration measurements for >1000 human The identification of cancer biomarkers is critical to characterize, genes and for other organisms represent one of the largest datasets detect and understand the illness. In our research group, a novel currently available, and reveal both general trends and specific method based on multiple criteria optimization has been proposed examples of post-transcriptional regulation. to detect potential cancer biomarker genes through the analysis of microarray data. More precisely, the multiple criteria optimization 144 The Application of Targeted problem is approached through a technique called Data Envelopment and Exome Sequencing for the Analysis (DEA). Given a number of genes characterized by at least two Identification of Spontaneous and performance measures, DEA is able to find those genes that are the Induced Mutations in Mice best compromises among these measures. When using microarray data and quantifiers of significant differences of gene expression between D. Hinerfeld, E. Antoniou, S. Daigle, Y. Ding, healthy tissues and cancer tissues, those genes identified through DEA W. Zhang, L. Reinholdt, M. Barter, L. Rowe have been shown to have a high potential to be biomarkers. DEA is known to be efficient due to its structure based on linear optimization. The Jackson Laboratory, Bar Harbor, ME, United Our studies have also shown that DEA can be very effective for the States stated purpose. There are, however, several different DEA formulations that can be used. In this work, a comparison of these formulations is The Jackson Laboratory has established a large collection of presented in terms of number and quality of the selection of genes for spontaneous and N-ethyl-N-nitrosourea (ENU) induced mouse two publicly available colon cancer microarray databases. The results mutants with a wide variety of medically relevant phenotypes. While will provide some light on the different solutions that can be arrived spontaneous mutations can be quite complex, including single to in multiple criteria optimization using DEA, to then establish what is nucleotide polymorphisms (SNPs), transposon insertions, deletions, or most adequate to find cancer biomarkers. inversions, ENU induced mutations are typically SNPs. The traditional method of identifying the causative mutation through genetic mapping **143 Comparing Protein and mRNA and Sanger sequencing of candidate genes has been effective but is time Abundances to Protein Expression consuming, requires large populations of mice and can be expensive. In addition, it is particularly challenging when the mutation is not in Regulation a coding sequence. With a size of over 3 GB, it is still too expensive 1,2 2 2 2 to sequence the genomes of the many mutant strains of interest. The C. Vogel , J.M. Laurent , T. Kwon , S.A. Craig , D.R. Boutz2, H.K. Huse2, K. Nozue3, H. Walia3, combination of array capture for targeted resequencing and/or exome capture followed by high-throughput sequencing on the Illumina GAIIX M. Whiteley2, P.C. Ronald3, S. Abreu Rde4, D. Ko4, has greatly accelerated the pace at which mutations have be identified. S.Y. Le 5, B.A. Shapiro5, S.C. Burns4, D. Sandhu4, In deciding what approach should be employed to identify a specific 4 2 L.O. Penalva , E.M. Marcotte mutation, a number of factors must be considered; 1) Is the mutation 1New York University, New York, NY, United States; spontaneous or ENU induced; 2) Have traditional mapping approaches 2University of Texas at Austin, Austin, TX, United been 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 be States; 3UC Davis, CA, United States; 4University required; This approach has led to the identification of many mutations of Texas at San Antonio, San Antonio, TX, United that result in disease-relevant phenotypes including craniofacial 5 States; NCI-Frederick, Frederick, MD, United States disorders, neurodegeneration, neuromuscular dysfunctions, cholesterol

Poster Abstracts biosysthesis and reproduction. Transcription, mRNA decay, translation and protein degradation are essential processes during eukaryotic gene expression, but their relative global contributions to steady-state protein concentrations in 145 Single-Cell Copy Number Analysis multi-cellular eukaryotes are largely unknown. Using measurements Using the Illumina Genome Analyzer of absolute protein and mRNA abundances in cellular lysate from the , J. Langmore, T. Kurihara, human Daoy medulloblastoma cell line, we quantitatively evaluate the E. Kamberov impact of mRNA concentration and sequence features implicated in J. M’Mwirichia, T. Tesmer, D. Oldfield translation and protein degradation on protein expression. Sequence Rubicon Genomics, Ann Arbor, MI, United States features related to translation and protein degradation have an impact similar to that of mRNA abundance, and their combined contribution CNV, SNP, and mutation analyses of singles cells are important to explains two-thirds of protein abundance variation. mRNA sequence characterize cancer, stem, and embryo cells. We used PicoPlex Whole lengths, amino-acid properties, upstream open reading frames Genome Amplification kits to sequence single human cancer cells, and secondary structures in the 5’ untranslated region (UTR) were single-copy mouse chromosomes, and single bacterial cells using the

72 • ABRF 2011 — Technologies to Enable Personalized Medicine Illumina Genome Analyzer. PicoPlex is a 1-tube, 3-hr, 4-step method 147 Enhanced Multiplexing Capabilities to convert a single cell into cluster-station ready DNA. High quality of Agilent Technologies SureSelectTM sequences with very little background were obtained (0.7% mismatch rates, 90+% mapped reads, <20% ADO, and good paired-end data Target Enrichment System for Next- were typical). Amplification or deletion of regions smaller than 100 Generation Sequencing kb and reproducible SNP and chromosomal breakpoint results were S. Joshi, H. Ravi, A. Giuffre, C. Pabón-Peña, reproducibly determined from single cells sequenced in single lanes. B. Novak, M. Visitacion, M. Hamady, F. Useche, The reproducibility of PicoPlex enables many research and diagnostic J. Ong, S. Hunt, D. Roberts, S. Happe, E. Leproust applications using single cells. Agilent Technologies, Cedar Creek, TX, United 146 Ion Torrent’s Personal Genome States Machine coupled with Fluidigm’s Access Array Provides a Multiplexed Demand has never been greater for revolutionary technologies Sample Approach to Rapid Evaluation that deliver fast, inexpensive and accurate genome information. of Novel Genomic Targets Massively parallel sequencing technologies have enabled scientists to discover rare mutations, structural variants, and novel transcripts at an J. Boland3, J. Myers1, J. Rothberg1, A. May2, unprecedented rate. To meet the demand for fast, inexpensive and D. Roberson3 accurate genome analysis method, Agilent Technologies has developed the SureSelect platform, an in-solutionhybrid selection technology for 1Ion Torrent, South San Francisco, CA, United systematic re-sequencing of user specific genomic regions. With the States; 2Fluidigm, South San Francisco, CA, United implementation of this new technology there is a balancing act of cost, States; 3National Cancer Institute, Bethesda, MD, quality and quantity and it is easier for scientists to sequence entire United States genomes from large sample cohorts. The inexpensive production of large volumes of user specific sequence data is SureSelect’s primary A multiplexed approach to sample processing is essential to maximize advantage over conventional methods. To further reduce costs data return of a single run on a next generation sequencing platform. The and take advantage of the increasing capacity of next-generation ability to process hundreds of samples per day will undoubtedly lead sequencers, such as the HiSeq2000 and the SOLiD4/4hq, we highlight to faster discovery of underlying genetic markers for many of today’s the ability to multiplex DNA samples in a single sequencing lane/slide current diseases. Ion Torrent has invented the first device capable of while maintaining the coverage necessary to confidently make SNP directly translating chemical signals into digital information. The first calls. SureSlelect multiplexing kits have an automation-friendly, easy application of this paradigm-changing technology is sequencing DNA to use protocol where gDNA libraries are uniquely “tagged” and then on Ion’s semiconductor sequencing device. The device leverages combined via mass balance on one flow cell lane/slide. We show high decades of semiconductor technology advances, and in just a few performance across both Illumina and SOLiD multiplexing platforms, years has brought the entire design, fabrication and supply chain as measured by capture efficiency, uniformity and reproducibility. infrastructure of that industry-a trillion dollar investment-to bear on the The multiplexing capabilities SureSelect make it a cost effective way to challenge of sequencing. The result is the first commercial sequencing study human and mouse exome, or any user defined region of interest. technology that does not use light-The Personal Genome Machine, Ion’s When multiplexing HapMap samples, >98% concordance between novel sequencer delivers unprecedented speed, scalability and low SureSelect re-sequencing results and previously determined genotype cost. With the increased speed (2 hour runtime) and accuracy provided is observed. Lastly, we introduce the SureSelect XT kit for preparation by the PGM, we have leveraged our existing multiplexed sample of samples for multiplex sequencing using the Illumina GAII or HiSeq. approach using the Access Array from Fluidigm to amplify 48 samples The SureSelect Multiplexing kit provides the ability to combine across 1 to 48 amplicons, depending on the experiment in question. targeted enrichment with multiplexing, thus maximizing the number of The ability to generate genomic information for 48 samples under samples that can be sequenced at one time, providing optimum time 2 hours using the PGM is critical to quickly confirm whether or not a and cost savings without sacrificing performance. particular novel discovery observed through whole genome or exome Poster Abstracts evaluation is the “hit” you are looking for. For our initial experiments, we 148 Comparison of SYBR Enzymes used amplicons from exons of p53 to demonstrate we could evaluate and Standards in Illumina Library and differentiate known genetic markers between individual samples Quantification within a pool of barcoded samples. Using the Ion Torrent 314 chips, we ran a series of chips that included chips with a single sample up to chips K. Thai, S.S. Levine with 48 samples. MIT BioMicro Center, Massachusetts Institute of Technology, Cambridge, MA, United States

Accurate quantification of libraries generated for Illumina next- generation sequencing is critical to prevent under- and overclustering of the flowcell. Success of qPCR library quantifications depend on the accuracy and reproducibility of standards used in addition to the ability of DNA polymerase to efficiently amplify all adaptor-flanked libraries. We compared the (i) Roche SYBR Green and (ii) KAPA SYBR Fast polymerases and the PhiX standards and KAPA pre-diluted standards to determine the relative importance of these two factors in library quantification by qPCR. We evaluated the success of each method by comparing variation in the number of reads and absolute

ABRF 2011 — Technologies to Enable Personalized Medicine • 73 yield generated from sequencing. Our results indicate that the DNA of transcripts and their expression levels. Current methods for making polymerase from the KAPA SYBR Fast kit is better suited to library sequencer-specific di-tagged DNA fragment libraries for RNA-Seq quantification applications while the Roche SYBR Green kit showed typically comprise preparing rRNA-depleted RNAand either (i) RNA significant variability, possibly related to sample type or insert size. fragmentation, 5’ and 3’ adaptor-ligation, size selection, cDNA synthesis, No differences were observed between the two types of standards and multiple clean-up steps; or (ii) cDNA synthesis followed by cDNA used, making DNA polymerase the determining factor in the successful fragmentation, end-polishing, 5’ and 3’ adaptor-ligation, size selection amplification of each library. and multiple clean-up steps. These methods are generally long and require significant hands-on time. We describe a novel protocol that **149 A New Sequencing Primer and utilizes a unique Terminal-Tagging technology that simplifies the Workflow Increase 5’ Resolution and preparation of directional RNA-Seq libraries from rRNA-depleted or Throughput on HLA Sequencing poly(A)-enriched RNAin about 3 hours, without the need for adaptor ligation, cDNA nebulization or gel purification. The di-tagged cDNA J. Chuu1, S.C. Hung1, S. Berosik1, M. Wenz1, fragments that are generated from this simple, single tube protocol S. Schneider1, P. Ma1 , D. Berchanskiy2, D. Dinauer2 are compatible with different NGS sequencing platforms. Apart from its simplicity, another major strength of this RNA-seq protocol is its 1Life Technologies, Foster City, CA, United States; ability to determine the polarity of the RNA transcripts, which is critical 2Life Technologies, Brown Deer, WI, United States for the annotation of novel genes. Sequencing results obtained from libraries prepared by using RiboZeroTM rRNA-depletion method and High quality and high accuracy are the hallmarks of Sanger re- ScriptSeq™ mRNA-Seq Library Preparation Method show excellent sequencing projects. We have developed a new sequencing primer and directionality with less than 2 % of the sequence reads that map to workflow that improves 5’ sequence resolution, increases throughput, rRNA sequences (28S, 18S, 5.8S and 5S). This reduction in rRNA and reduces hands-on time. The novel sequencing primer chemistry sequence reads improves sequence depth and coverage, and increases produces high quality bases from base 1 on POP-7TM polymer that the percentage of uniquely mapped reads. Further, there is a high previously only could be resolved on the slower POP-6TM polymer. correlation (R2=0.9235) between differentially expressed transcripts The new primer chemistry and workflow also eliminates the need found in the ScriptSeq™ RNA-Seq libraries and the MAQC QPCR panel for a separate PCR clean-up step. These improvements reduce the of genes. entire workflow from PCR to finished sequence data to under 5 hours, compared to 8 hours for the standard workflow. We used our 151 The Agilent Technologies’ enhanced sequencing primer and workflow to investigate feasibility SureSelectTM All Exon Product on Human Leukocyte Antigen (HLA) polymorphisms on twelve DNA samples by using the Invitrogen SeCore® HLA-DRB1 primer set and Portfolio: High Performance Target Group Specific Sequencing Primers. Sequencing reactions generated Enrichment System for Human and with the traditional sequencing primer and with the new sequencing Mouse Exome Sequencing on Illumina primer were electrophoresed on Applied Biosystems 3500xl Genetic and SOLiD Platforms Analyzer using POP-7TM polymer. For each sequencing primer, we compared 5’ resolution and basecalling accuracy and quality. On H. Ravi, A. Giuffre, C. Pabón-Peña, B. Novak, average the traditional primers produced high quality readable S. Joshi, J. Ong , M. Visitacion, M. Hamady, bases by base 25 after the sequencing primer while the new primers F. Useche, J. Eberle, S. Hunt, S. Happe, D. Roberts, produced high quality bases by base 5, and by base 1 in many cases. E. Leproust Because of improved resolution, basecalling accuracy was increased. Agilent Technologies, Stratagene Products Division, This simplified process without a separate PCR clean-up step reduced the overall workflow time by 40%. For HLA genes, obtaining readable Cedar Creek, TX, United States sequence within 5 bases of the primer offers improved polymorphism The dramatic increase in throughput of sequencing data from next- detection and more efficient use of allele specific sequencing primers generation sequencing platforms has enabled scientists to study the for heterozygous ambiguity resolution. In conclusion, the novel primer genome with unprecedented depth and accuracy. Nevertheless, routine chemistry and workflow generates data superior in quality relative to genetic screens in large numbers of individuals continue to remain cost- other currently used solutions and offers significant time savings as well. prohibitive through these approaches. Agilent Technologies’ SureSelect Specific applications of this product are under development and not platform for targeted exome capture, combined with massively parallel intended for clinical use. sequencing, provides a more affordable method to gain novel insights Poster Abstracts into the genetic causes of inherited disorders. In addition, identification 150 ScriptSeq RNA-Seq Library of both common and rare polymorphisms implicated in complex Preparation Method: A Simplified diseases like cancer is greatly facilitated by selectively sequencing the Work-Flow for Directional NGS RNA- protein-coding regions of the genome. In collaboration with the Broad Seq Library Preparation with Whole- and Sanger Institutes, Agilent Technologies has continued to expand Transcript Representation the number of SureSelect target enrichment catalog products in order to enable a more comprehensive view of the protein-coding regions A. Khanna1, R. Sooknanan2, J. Hitchen2, A. Radek2 in humans and model organisms. We discuss the SureSelectHuman All

1 Exon v2 (44Mb) and SureSelectHuman All Exon 50Mb designs. We also Epicentre BIOtechnologies, Madison, WI, United introduce the SureSelectMouse All Exon target enrichment system, 2 States; RiboTherapeutics Inc., Saint Laurent, QC, which improves the ability to study genetic variation between strains in Canada greater detail, and significantly increases the efficiency of screening for causative mutations in N-ethyl-N-nitrosourea (ENU)-mutagenized mice. RNA sequencing is an emerging revolutionary tool for whole- We demonstrate high performance with respect to capture efficiency, transcriptome analysis that provides information about the structure

74 • ABRF 2011 — Technologies to Enable Personalized Medicine uniformity, reproducibility of enrichment, and ability to detect SNPs, in just a few months from ~1 million sensors in the first-generation insertion/deletions, and CNVs across Illumina (Genome Analyzer IIx Ion 314 chips to ~7 million sensors in the second-generation Ion 316 and HiSeq2000) and SOLiD platforms. We highlight the utility of the chips-all while maintaining the same 1- to 2-hour runtime. We will also SureSelect All Exon product portfolio for a wide variety of applications demonstrate that Ion semiconductor sequencing provides exceptional primarily due to the high specificity and excellent cross-platform accuracy, long read length and scalability on a single, affordable bench- sequence coverage. SureSelect All Exon designs also provide a means top sequencing platform. for standardization, consistency of performance, and reliability across multiple laboratories. 154 A Comparison of Post-DNA Sequencing Dye-Terminator Removal 152 PCR-Free Nextera Di-Tagged Protocols DNA Library Preparation for NGS Applications M. Zianni, A. McCoy The Ohio State University, Columbus, OH, United C. Kinross, H. Grunenwald, B. Baas, I. Goryshin, N. Caruccio, M. Maffitt States Epicentre Biotechnologies, Madison, WI, United Capillary electrophoresis, a method for separation of ions based upon States their size to charge ratio, remains in high demand for DNA sequencing. In the process of dideoxynucleotide terminator sequencing, The NexteraTM technology for generating libraries of di-tagged DNA unincorporated nucleotides and other contaminants remaining in the fragments is rapidly becoming the preferred method for massively reaction mixture can cause multiple issues in the electropherograms, parallel DNA sequencing. Despite rapid advances in sequencing such as unincorporated dye peaks, missed base calls, decreased signal instrument throughput, classic library preparation by step-wise ligation strength or a complete lack of data as a result of blocked capillaries. A of adaptors is a time-intensive and throughput-limiting bottleneck. variety of dye-terminator removal protocols exists to clean and purify PCR amplification of libraries prior to cluster generation is also a the sequencing reaction extension products. To determine their quality major concern because of its possibility to reduce library complexity, and reproducibility, six protocols were tested with one large volume particularly in regions of extreme G+C content (high or low), thereby control reaction aliquoted into a 96-well PCR plate. The protocols producing uneven genome coverage and confounding mapping and included ethanol precipitation, gel filtration, and 4 solid phase assembly. In this study, we describe novel modifications of the NexteraTM reversible immobilization procedures with 1 utilizing the surface of a library preparation system to address such library preparation bias plate and the other 3 utilizing magnetic beads. The cleaned and purified by eliminating PCR amplification. Sequencer-ready libraries can be sequencing reactions were processed on the 3730 DNA Analyzer obtained from as little as 200 ng of genomic DNA in 3 hours with (Applied Biosystems), and the contiguous read lengths, QV20+ scores, 90-minutes of hands-on time. Deep sequencing of genomic libraries and signal strengths of the resulting sequences were analyzed with indicates that this system reduces coverage bias and GC bias, as well as Sequence Scanner v1.0 (Applied Biosystems). Based on the results from improves library diversity. two replicate rounds of testing, the gel-filtration protocol provided the longest contiguous read lengths and highest QV20+ scores. 153 Semiconductor Sequencing for Life 155 Simplified Reagents and Workflows J. Myers, J. Rothberg for Robust Sample Preparation of Ion Torrent, South San Francisco, CA, United States DNA, mRNA, and Small RNA

Ion Torrent has invented the first device-a new semiconductor chip- F.J. Stewart, C.L. Hendrickson, L.M. Apone, capable of directly translating chemical signals into digital information. D.B. Munafo, C.R. Meyer The first application of this technology is sequencing DNA. The device New England Biolabs, Inc., Ipswich, MA, United leverages decades of semiconductor technology advances, and in just States Poster Abstracts a few years has brought the entire design, fabrication and supply chain infrastructure of that industry-a trillion dollar investment-to bear on the As yields of data generated by the Illumina, SOLiD, and 454 challenge of sequencing. The result is Ion semiconductor sequencing, sequencing platforms increase, NGS users have transitioned from the first commercial sequencing technology that does not use light, and performing multiple sequencing runs per sample to multiple samples as a result delivers unprecedented speed, scalability and low cost. All per sequencing run. As a result, the bottleneck in sequencing labs has of these benefits are a result of applying a technology that is massively transitioned from data generation to sample preparation, necessitating scalable, as proven by Moore’s Law, to a task that has traditionally used the development of streamlined library construction workflows. We optics-based solutions, which work in a linear fashion: increasing capacity have developed a series of reagents to facilitate the easy preparation requires increasing the number of signals that must be read resulting of numerous samples in parallel, compatible with both manual and in longer run times, higher capital costs and ever more sophisticated automated pipelines. These reagents reduce the amount of labor optics. By contrast, Ion Torrent semiconductor technology can provide required, minimize error in reaction set up, and increase the stability of increases in chip capacity without impacting capital costs or runtime. enzymes used in the construction of libraries for DNA, mRNA and Small Ion Torrent sequencing uses only natural (label-free) reagents and RNA sequencing. As a result, these reagents enable the development takes place in Ion semiconductor microchips that contain sensors which of robust workflows for both individual and high throughput sample have been fabricated as individual electronic detectors, allowing one preparation. sequence read per sensor. We will show how the technology has scaled

ABRF 2011 — Technologies to Enable Personalized Medicine • 75 156 Comparison of Custom Target 157 A Methodology Study for Enrichment Methods: Agilent vs. Metagenomics Using Next Generation Nimblegen Sequencers

K. Bodi1, P.S. Adams2, D. Bintzler3, A. Perera4, D. Grove1, I. Albert1, D. Bintzler2, K. Bodi3, K. Dewar5, D.S. Grove6, J. Kieleczawa7, R.H. Lyons8, M. Bruns1, K. Dewar4, G. Gloor5, T. Johnson6, T. Neubert9, A. C. Noll1, S. Singh10, R. Steen11, J. Kieleczawa7, R.H. Lyons8, T. Neubert9, M. Zianni12 A.G. Perera10, S. Singh6, R. Steen11, M. Zianni12 1Tufts University, Boston, MA, United States; 1Penn State University, University Park, PA, United 2Trudeau Institute, Saranac Lake, NY, United States; 2DNA Analysis, Inc., Cincinnati, OH, United States; 3DNA Analysis, Inc., Cincinnati, OH, United States; 3Tufts University, Boston, MA, United States; 4Stowers Institute, Kansas City, MO, United States, 4McGill University, Montreal, QC, Canada; States; 5McGill University, Montreal, QC, Canada; 5University of Western Ontario, London, ON, 6Pennsylvania State University, University Park, Canada; 6University of Minnesota, Minneapolis, PA, United States; 7Pfizer Research, Cambridge, MN, United States; 7Pfizer Research, Cambridge, MA, United States; 8University of Michigan, Ann MA, United States; 8University of Michigan, Ann Arbor, MI, United States; 9New York University, New Arbor, MI, United States; 9New York University, New York, NY, United States; 10University of Minnesota, York, NY, United States; 10Stowers Institute, Kansas Minneapolis, MN, United States; 11Harvard Medical City, MO, United States; 11Harvard Medical School, School, Cambridge, MA, United States; 12Ohio Cambridge, MA, United States; 12Ohio State State University, Columbus, OH, United States University, Columbus, OH, United States

Over the last four years, we witnessed the tremendous advances in Metagenomics is one of several genomics applications, which has Next Generation Sequencing (NGS) that have dramatically decreased benefited immensely from the high throughput and cost efficacy of the cost of whole genome sequencing. However, the cost of sequencing Next Generation sequencers. And although hundreds of studies on larger genomes is still significant. In addition and depending on the metagenome analysis have been published over the past few years, goal of study, whole genome sequencing creates a large amount of the methodology for conducting them is still very much evolving. additional/auxiliary data that complicates data analysis. There are In this DSRG study we will evaluate the influence of various sample several commercial methods available for isolating subsets of genomes preparation methods, specifically DNA extraction and amplification that greatly enhance the efficiency of NGS by allowing researchers approaches, on data output along with a comparative analysis of Next to focus on their regions of interest. For the 2009-11 DSRG study, Generation sequencing platforms. We will study the effect of these we compared products from two leading companies; Agilent and different experimental and technical strategies on determination of Nimblegen, that offer custom enrichment methods. Both companies sample biodiversity. obtained the same genomic DNA stock and performed DNA capture on the same specified regions. Following capture, the Illumina Genome 158 What Your Blood Has to Say: Analyzer IIx system was used, in two different laboratories, to generate Amplifying Blood RNA for the the sequence data. We present our data comparing in terms of cost, Affymetrix GeneChip® Platform quality, reproducibility and most importantly completeness and depth of coverage. Acknowledgements: We would like to thank Agilent, N. Supunpong Hernandez, T. Barta, C. Willis, Illumina and Nimblegen for all their support in making this study R. Conrad, P. Whitley, K. Bramlett possible. Life Technologies, Austin, TX, United States

Gene Expression measurements from human blood RNA have become an increasingly important research area of focus. A reliable and consistent workflow to obtain RNA measurements from blood would serve to open the clinical arena to gene expression studies as

Poster Abstracts potential diagnostic indicators. The challenge lies in isolating high quality RNA from human whole blood at room temperature without compromising gene expression profiles. To address this challenge, we report a workflow using newly developed MagMaxTM RNA isolation kits for TempusTM stabilized blood and PAXgene® stabilized blood. These two stabilization methods are currently the most widely used methods for blood collection and stabilization in clinics in the United States. High quality RNA generated from these two RNA isolation processes was amplified using the Ambion MessageAmpTM Premier RNA Amplification Kit and hybridized to Affymetrix GeneChip® microarrays. This platform and experimental tools are used, in combination, to demonstrate high quality gene expression profiles from human blood RNA. The reported experimental design includes human whole blood obtained from two donors collected and stabilized in either TempusTM PAXgene® tubes. RNA was isolated using new MagMaxTM RNA isolation

76 • ABRF 2011 — Technologies to Enable Personalized Medicine kits for TempusTM and PAXgene® stabilized blood. Isolated total RNA 160 Electrochemical Simulation was then processed through the GLOBINclearTM-Human kit and then of Covalent DNA Adduct amplified with the MessageAmpTM Premier kit creating a library for hybridization to the Affymetrix Human U133A 2.0 expression arrays. Formation Monitored with Liquid Affymetrix GeneChip® microarray analysis showed parameters within Chromatography/Mass Spectrometry normal limits for expressed genes. Reported resultsof this controlled 2, S. Plattner1, R. Erb1, F. Pitterl1, experiment, support a validated gene expression workflow for blood J. Powers J.P. Chervet2, H. Oberacher1 on Affymetrix expression arrays using a combination of commercial kits for RNA purification from stabilized blood and library amplification 1Institute of Legal Medicine, Innsbruck Medical optimized for hybridization and analysis on expression microarrays. University, Innsbruck, Austria; 2Antec, Palm Bay, FL, United States 159 Performance Comparison of Four Methods Utilized for the Purification DNA adduct is a piece of DNA covalently bond to chemicals. Adducts of Enzymatically-Digested, activate repair processes and, unless repaired prior to replication, may Fluorescently-Labeled PCR Fragments lead to nucleotide substitutions, deletions, and other rearrangements. Generated During T-RFLP Analysis As alterations of the genetic material can cause severe diseases including cancer, inflammation, and neurodegenerative disorders, M. Zianni, J. Panescu, P. Kumar tests on mutagenicity/genotoxicity have become an integral part of risk assessment during the development of any new chemical. A number of in The Ohio State University, Columbus, OH, United vitro tests using different cell lines and in vivo tests mainly using rodents States are available to identify hazardous compounds. The majority of these tests are time-consuming, labor-intensive and hardly automatable. So in The fluorescently-labeled Terminal Restriction Fragment Length search for alternative methods, the usefulness of electrochemistry (EC)- Polymorphism (T-RFLP) assay based on amplified ribosomal DNA liquid chromatography (LC)-mass spectrometry (MS) was evaluated. from bacteria is an inexpensive, widely accessible, effective and well- Generally, EC represents a powerful tool to study in vitro biological established molecular technique for the identification and comparative oxidation processes of a number of different compounds, including quantification of bacterial species in metagenomics. In order to drugs, peptides and proteins. Here, we used EC to initiate adduct facilitate the detection of a large proportion of species in a given formation. The obtained reaction products were separated by LC and sample, it is necessary to maximize the recovery of the fluorescently- detected by MS. Tandem MS experiments were used for structural labeled, restriction enzyme-digested PCR fragments generated confirmation. In a proof of principle study acetaminophen was selected during the process. The post-digestion purification method used to as model compound. Covalent adduct formation was observed for facilitate fragment separation is a critical step in the retention of DNA electrochemical activated mixtures of acetaminophen and guanosine. fragments. Four methods for post-digestion purification were tested Mechanistic studies revealed that adduct formation will only start at in an effort to characterize their integrity, effectiveness, ease of use electrochemical potentials sufficiently high to initiate oxidation of both and potential biases: solid phase reversible immobilization (AMPureTM acetaminophen and guanosine. The stringent necessity of coactivation and CleanSEQTM), contaminant capture (BigDye® XTerminatorTM), sheds a new light on the mechanism of adduct formation, because and dialysis (MilliporeTM Nitrocellulose Membranes). Samples were according to the generally accepted theory activation of the adduct- collected from four different oral sites in each of 16 patients. Each forming agent should be sufficient. Chromatographic separation sample was divided equally and the DNA was isolated with two distinct enabled the differentiation of four isomeric forms. Their connection to methods. PCR reactions were carried out with two paired universal acetaminophen was proven in dose-response experiments. EC/LC/MS primers for the 16S gene that were labeled with VIC and FAM, purified represents a fast, simple, convenient and functional tool to study DNA with AMPureTM and digested separately with HhaI and MspI restriction adduct formation which has great potential to complement the existing enzymes. From each of the digestions, identical aliquots were purified battery of mutagenicity/genotoxicity tests. with the four different methods. The DNA fragments were analyzed on the Applied BioSystemsTM 3730 DNA Analyzer using identical

**161 Quantitative miRNA Expression Poster Abstracts conditions, followed by data analysis with GeneMapper® v4.0. Based on preliminary data analysis, CleanSEQTM is the superior purification Analysis Using Fluidigm Microfluidics method because it resulted in the most numerous peaks recovered per Dynamic Arrays sample with a wide distribution of sizes from approximately 50 to 1200 J. Jen, J. Sung Jang, V.A. Simon, R.M. Feddersen, bp. F. Rakhshan, D.A. Schultz, M.A. Zschunke, W.L. Lingle, C.P. Kolbert. Mayo Clinic Micorarray Shared Resource and Biospecimens Accessioning Processing Shared Resource, Rochester, MN, United States

MicroRNA (miRNA) is a small non-coding RNA that can regulate gene expression in both plants and animals. Studies showed that miRNAs play a critical role in human cancer by targeting messenger RNAs that are positive 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 throughput qPCR-based microfluidic dynamic array technology (Fluidigm). We compared the results to hybridization-based microarray platforms

ABRF 2011 — Technologies to Enable Personalized Medicine • 77 using the same samples. We obtained a highly correlated Ct values rapidly increasing in the field of RNA research. Our aim has been to between multiplex and single-plex RT reactions using standard qPCR develop library preparation methods and tools that aid in the reliable assays for miRNA expression. For the same samples, the microfluidic generation of libraries for next generation sequencing from total RNA. technology (Fluidigm 48.48 dynamic array systems) resulted in a left- Reported here are results from the development of the Ambion® shift towards lower Ct values compared to those observed by standard RNA-Seq Library Construction kit optimized for sequencing on the TaqMan (ABI 7900HT, mean difference, 3.79). In addition, as little as Illumina® next generation sequencing instruments. We show results 10ng total RNA was sufficient to reproducibly detect up to 96 miRNAs from two protocols utilizing the same reagents that allow generation at a wide range of expression values using a single 96-multiplexing of RNA-Seq libraries targeting either the small RNA fraction of total RT reaction in either FFPE or FF samples. Comparison of miRNAs RNA, or the whole transcriptome which includes transcripts larger than expression values measured by microfluidic technology with those 100 base pairs. Results are reported from Illumina® Genome Analyzer obtained by other array and Next Generation sequencing platforms II sequencing of both small RNA and transcriptome libraries with a showed positive concordance using the same samples but revealed focus on mapping to the miRBase and RefSeq references respectively. significant differences for a large fraction of miRNA targets. The qPCR- We also demonstrate the use of External RNA Control Consortium array based microfluidic technology can be used in conjunction with (ERCC) transcripts as spike-in controls for transcriptome libraries multiplexed RT reactions for miRNA gene expression profiling. This that aid in quality control of the library generation procedure and approach is highly reproducible and the results correlate closely with aid in downstream data analysis. The library construction technology the existing singleplex qPCR platform while achievingmuch higher embedded in the Ambion® RNA-Seq Library Construction kit enables throughput atlower sample input and reagent usage. It is a rapid, cost researchers to analyze the transcriptome of their research samples in effective, customizable array platform for miRNA expression profiling a precise, sensitive and robust manner while maintaining information and validation. However, comparison of miRNA expression using regarding the genomic DNA strand to which the RNA transcript maps different platforms requires caution and the use of multiple platforms. utilizing the Illumina® Genome Analyzer II sequencing platform. The workflow and results reported here demonstrate new commercially 162 Apollo 200 Fully Integrated DNA available options for library construction enabling small RNA and HID System: Multi Channel Results in transcriptome profiling and novel discovery using next-generation Under 2 Hours sequencing technology.

S. Jovanuvich, O. El-Sissi, H. Franklin, B. Nielsen, 164 A Case Study: Molecular Profiling of S. Pagano, R. Belcinski, G. Bogdan Breast Cancer from Formalin-Fixed, IntegneX, Inc, Pleasanton, CA, United States Archival Material — Gene Expression Profiles from FFPE Samples with The ideal solution to meet the requirements of modern human Improved RNA Decrosslinking identification is an automated, DNA-based hu man identification Technology system that processes samples rapidly and at low cost. IntegenX™ Inc. 1 2 3 will describe the Apollo 200™ DNA HID System. The Apollo200 is R. Jaggi , S. Quabius , G. Krupp the first fully automated sample-to-answer system for STR based HID. 1University Bern, Bern, Switzerland; 2UKSH The system is based on integration of the company’s proprietary and University Klinik, Kiel, Germany; 3AmpTec GmbH, patented technologies as well as its rapid in-house microfluidic chip Hamburg, Germany prototyping. Reagents in disposable cartridges are loaded on the system with up to four buccal swab samples, the sample processing is We have developed a novel demodifaction/decrosslinking protocol initiat ed and a CODIS compatible profile is ready in less than two hours for RNA recovery from archival (FFPE) material. The resulting FFPE with no further user interaction. The Apollo 200 integrates all of the RNA quality is superior to RNA obtained with other commercial sample handling steps starting from buccal swab(s) or blood, through FFPE RNA isolation kits: larger RNAs can be recovered, and RT- cell lysis, DNA extraction, amplification, separation, and detection. qPCR data demonstrate less variability and lower Cq values. This Further, the system uses rapid PCR chemistry, on-board capillary FFPE RNA is suitable for differential gene expression measurement electrophoresis, and integrated la ser induced fluorescence detection. by qPCR, high concordance with parallel RNA samples from fresh- Initial data from testing conducted by IntegenX and customers will be frozen tissues was observed. Prognosis of breast cancer is determined shown. **Apollo 200™ is a trademark of IntegenX, Inc. by clinicopathological and molecular factors. We developed and validated molecular scores reflecting the hormone status (ER, PGR,

Poster Abstracts 163 Transcriptome Analysis Using Next- HER2 scores) and the proliferation status (PRO score) of breast cancer Generation Sequencing Technology cells. The scores can be combined to an overall RISK score. Molecular scores are independent prognostic parameters, they were validated in K. Bramlett, K. Lea, L. Qu, P. Whitley, postmenopausal patients with estrogen receptor positive breast cancer. J. Schageman, J. Gu Multivariate analysis revealed that PRO and RISK scores outperform conventional parameters (histological grading and Ki-67 labeling Life Technologies, Carlsbad, CA, United States index). Molecular scores are based on routine pathological material, High throughput RNA sequencing (RNA-Seq) is becoming increasingly testing can be implemented easily into routine diagnosis. utilized as the technology of choice to detect and quantify known and novel transcripts. Multiple next-generation sequencing (NGS) platforms are available that enable transcriptome profiling through RNA-Seq workflows. Demonstrations of the power of RNA-Seq to profile the well annotated transcriptome and also identify novel transcribed regions, gene fusions, and even identify novel classes of RNA are

78 • ABRF 2011 — Technologies to Enable Personalized Medicine 165 Benchmarking miRNA Expression tumorigenesis. However, currently there is no good method to Levels in Degraded RNA Samples systematically study miRNA expression in FFPE samples on next generation sequencing platforms. We have designed and developed Using Real-Time RT-qPCR and a ligation-based miRNA detection method to capture small RNA Microarray Technologies sequences in FFPE samples and convert them into templates suitable TM 5, S.V. Chittur1, S. Tighe2, V. Nadell3, for sequencing on the SOLiD System. Total RNA was isolated from J. Holbrook matched lung adenocarcinoma FFPE and snap frozen tissues using an R. Carmica4, K. Sol-Church5, A.T. Yeung6 Ambion RecoverAllTM kit. A PureLinkTM miRNA Isolation kit was used 1State University of New York at Albany, Albany, to enrich the small RNA fraction in these total RNA samples. Library NY, United States; 2University of Vermont, preparation using a SOLiDTM Total RNA-Seq kit with modified protocol Burlington, VT, United States; 3Ohio University, was performed on the enriched RNA followed by sequencing on TM Athens, OH, United States; 4The University of SOLiD system. Our results show that small RNA extracted from FFPE samples was successfully converted to small RNA libraries. Very similar Texas Medical Branch, Galveston, TX, United 5 mapping statistics were obtained from matched FFPE and fresh-frozen States; Nemours/A.I. duPont Hospital for Children, samples after SOLiDTM sequencing. A good correlation of miRNA 6 Wilmington, DE, United States; Fox Chase Cancer expression pattern was also observed. This suggests that miRNA Center, Philadelphia, PA, United States molecules are less affected by sample degradation and RNA-protein crosslink. This study provides a foundation for miRNA expression The Nucleic Acid Research Group (NARG) has conducted experiments analysis on SOLiDTM system using FFPE samples in cancer and other to determine the impact of RNA integrity and priming strategies on diseases. cDNA synthesis and Real-Time RT-qPCR. As a continuation of the RNA integrity theme, this year’s study was expanded to evaluate the impact of RNA integrity on priming strategies for the analysis of nine miRNA **169 Structural Insights into the targets using Real-Time RT-qPCR. The nine targets were selected Mechanism of microRNA Modulated based on data obtained by the Microarray Research Group (MARG) Viral Translation and represent groups of miRNAs that are expressed at low, medium, or 2, E.A. Pham1, P. Pang1, M.A. Winters1, high levels in the First Choice human brain reference RNA sample. The S. Patel 2 1,3 two RT-qPCR priming strategies tested in this study include the miRNA M. Eckart , J.S. Glenn TaqMan assay (Megaplex) of ABI and the RT2 miRNA qPCR assay of 1Department of Medicine, Stanford University Qiagen/SA Biosciences. The basis for the ABI assay design is a target- School of Medicine, Stanford, CA, United States; specific stem-loop structure and reverse-transcription primer, while 2Protein and Nucleic Acid Core Facility, Stanford the Qiagen design combines poly (A) tailing and a universal reverse transcription in one cDNA synthesis reaction. For this study to assess University School of Medicine, Stanford, CA, United 3 both RT methods, samples that were used as templates were human States; Palo Alto Veterans Administration Medical brain reference RNA that has been subjected to controlled degradation Center, Palo Alto, CA, United States using RNase A to RIN (RNA Integrity Number) values of 7 (good), 4 (moderately degraded), and 2 (severely degraded). In addition to MicroRNAs (miRNAs) are small non-coding regulatory RNAs that control this Real-Time RT-qPCR data, the same RNA templates were further a vast array of cellular processes by repressing mRNA translation. Liver- analyzed using universal poly (A) tailing followed by hybridization expressed miR-122 is a miRNA that has been co-opted by hepatitis C to Affymetrix miRNA GeneChips. We present some insights into RT virus (HCV) to enhance viral translation. Recently, miR-122 antagomir priming strategies for miRNA and contrasts qPCR results obtained using therapy in non-human primates has been shown to suppress HCV different technologies. viremia; this proof-of-concept study demonstrates the considerable potential of this novel antiviral strategy. The mechanism by which miR- 122 modulates HCV translation, however, is unclear. To examine the 168 MicroRNA Analysis Using RNA structural changes that miR-122 exerts on the HCV internal ribosomal Extracted from Matched Formalin- entry site (IRES), we developed an advanced Selective 2’-Hydroxyl Poster Abstracts Fixed Paraffin-Embedded (FFPE) and Acylation analyzed by Primer Extension (SHAPE) method of analyzing Fresh Frozen Samples on SOLiDTM RNA architecture. SHAPE determines RNA secondary structure at System single-nucleotide resolution, with an accuracy far superior to other mapping methods. Using the above strategy, we show that binding of K. Lea, J. Gu, E. Zeringer, S. Heater, J. Schageman, miR-122 to one of its target sites within the 5’ UTR of HCV induces a C. Mueller, K. Bramlett conformational shift in the HCV IRES at the distant AUG translation start site. Surprisingly, binding of miR-122 to its second target site in HCV Life Technologies, Carlsbad, CA, United States is mediated by a number of non-canonical base-pairings. Mutation of Archived formalin-fixed paraffin-embedded (FFPE) specimens the 3’ half of miR-122 (tail) disrupted these non-canonical interactions represent excellent resources for biomarker discovery, but it has been and its ability to induce a conformational shift at the AUG start site. a major challenge to study gene expression in these samples due to We also observed that, in vitro, the first miR-122 target site in HCV mRNA degradation and modification during fixation and processing. is part of a putative triple-strand RNA motif. These results provide MicroRNAs (miRNAs) regulate gene expression at post-transcriptional the first demonstration that the tail of this liver-encoded miRNA can level and are considered as important regulators of cancer progression. directly alter the RNA conformation of the HCV IRES, and thereby Next generation sequencing technologies such as SOLiDTM provide provide new insights into the mechanism by which miR-122 influences an ideal method for measuring the abundance of miRNA molecules viral translation. We also show that the 5’ UTR of HCV contains a triplex in different cancer stages and provide insightful information on structure important for viral translation.

ABRF 2011 — Technologies to Enable Personalized Medicine • 79 170 Development of ERCC RNA Spike-In possible separation. For tryptic digests a 200 Å stationary phase is Control Mixes superior to a 300 Å due to its two-fold increase in surface area while 1000 Å are required for the separation of proteins. L. Qu, A. Lemire, K. Lea, D. Batten, S. Jian Gu, P. Whitley, K. Bramlett 172 Biological Application of Constrained Peptides Life technology, Carlsbad, CA, United States E. Murage, M. Castro, H. Fazeli The National Institute of Standards and Technology (NIST) hosted External RNA Control Consortium (ERCC) has been working since Biosynthesis, Inc., Lewisville, TX, United States 2003 to generate a common set of RNA controls that can be used in gene expression measurements. These controls have been designed Peptides are attractive therapeutic agents for many diseases. However, to mimic natural eukaryotic mRNA sequences with the ability to be poor cell permeability, short half life in vivo due to rapid enzyme used across multiple measurement platforms. These controls open-up degradation has long been a major drawback in their clinical application. the possibilities for technical evaluation of multiple gene expression In order to overcome this, modification of peptides backbone has systems including real-time quantitative PCR, one-color and two-color been of most interest. In particular, synthetic cyclic peptides are being microarray systems, next generation sequencing platforms, as well as investigated as potential drug candidates due to their improved cell serve as an in-process quality control check for library amplification permeability, enzyme stability, high receptor affinity and selectivity. For processes. The long standing history of Ambion® as the RNA instance, hydrocarbon stapled peptides has been shown to bind and Company creates a great opportunity to formulate the ERCCs controls inhibit the NOTCH1 transcription factor. NOTCH proteins have been into useful mixes that can be spiked into RNA after isolation. Each mix shown to play a pivotal role in cellular differentiation, proliferation contains 92 of the ERCC controls in a mixture that spans over 6 logs of and apoptosis. Mutations in NOTCH1 have been linked with diseases dynamic range. The 92 transcripts in each mix are further divided into like T-cell acute lymphoblastic leukemia.1 Thus constrained peptide 4 sub-pools that can be utilized to evaluate fold-change measurements assemblies would provide a good opportunity to explore the protein- of gene expression between the two mixes. TaqMan® Gene Expression protein interactions due to the increased binding affinity compared assays targeting the 92 transcripts are utilized to monitor progress to the linear counterparts. As a result of the attractive biological through the transcriptome library preparation steps required to properties offered by these modified peptides there is a growing create a next generation sequencing (NGS) library from RNA. We demand for constrained peptides in the current drug discovery demonstrated the use of TaqMan® assays to inform a researcher research. To support the unmet need for custom made constrained of library quality prior to continuing with an expensive and time- peptides, Biosynthesis Inc. is now offering stapled peptides and lactam consuming next generation sequencing experiment. These controls bridge constrained peptides. 1. Raymond E. M., Melanie C., Tina N. D., are platform agnostic and provide informative data for multiple library Cristina Del Bianco, Jon C. A., Stephen C. B., Andrew L. K., D. Gary G., generation methods targeting various gene expression measurement Gregory L. V., James E. B., Nature 2009, 462, 182-188. platforms from RNA. ERCC RNA spike-in control mixes from the NIST traceable ERCC plasmids has great potential in the hands of our 173 The University of Texas at Austin customers to open-up new capabilities in understanding variability — Protein and Metabolite Analysis in RNA preparation, RNA library preparation, determining detection Facility limits of measurement systems, and informing downstream analysis. As such, we report here the first in a potential line of RNA control products M. Gadush, H. Lo, F. Geigerman, M. Mercado, leveraging the certified plasmid sequences from NIST. M. Person

171 Effects of Particle Porosity on the University of Texas at Austin, Austin, TX, United Separation of Larger Molecules States , D. DiFeo, H. Jurgen Wirth, A. Gooley The Protein and Metabolite Analysis Facility at the University of Texas R. Freeman at Austin is a joint effort of the College of Pharmacy, Center for Research SGE Analytical Science, Austin, TX, United States on Environmental Disease (CRED), and the Institute for Cellular and Molecular Biology (ICMB). Services and collaborative research are The pore structure of a chromatographic stationary phase accounts offered for the detection, characterization and quantification of for the vast majority of the surface area responsible for the separation. biomolecules. The Facility’s goals are to provide sensitive protein Poster Abstracts The pore diameter influences the overall surface area and with it the identification and modification analyses, to provide custom peptide capacity of the column but also limits the size of the analyte the column syntheses, to offer services for the identification and quantification of can or should be used for. In adsorption chromatography the limiting metabolites, nutrients and xenobiotics, to implement novel analytical effect of the pore diameter is further enhanced by adsorbed analyte methods, to improve the sensitivity of existing analyses, to provide molecules partially blocking the pore structure. In the analysis of large consultation on the selection and implementation of analytical methods, molecules pore diffusion becomes a crucial parameter in the efficiency to offer training in the usage and applications of the instrumentation, of the column. A number of models deal with hindered mass transfer in and to provide technical expertise in support of individual research porous systems. Effects of particle porosity on the separation of larger goals. The ICMB portion of the Core contains an ABI Procise 492 cLC molecules are discussed and examples for the separation of small, protein sequencer, a Protein Technologies Inc. Symphony peptide medium and large analytes on various pore size stationary phases are synthesizer, two Bio-rad Duoflows and a GE Heathcare AKTA protein given. Pore size is shown to be an important parameter when analyzing purification systems, two Beckman System Gold HPLC systems, a larger molecules. By selecting the right pore size for a task the capacity Berthold Technologies Mithras luminescence and fluorescence detector, and the mass transfer trade-off can be optimized to achieve the best an Invitrogen gel electrophoresis set-up, an Art Robbins Instruments

80 • ABRF 2011 — Technologies to Enable Personalized Medicine Phoenix crystallography robot and a LC-MALDI-TOF/TOF (an ABI 4700 of six scientific platforms: Cell Imaging (confocal and fluorescence with a LC Packings Ultimate Nano-LC system with a Probot spotting microscopy; walk-up), Proteomics (2-D, DiGE and fluorescent protein robot). In the College of Pharmacy, the Core has an Applied Biosystems analysis; walk-up), Automation and High throughput screening 4000 Q-trap LC MS/MS system with ESI, APCI and nanospray sources (Pinning robot and liquid handler; full service), Protein expression and coupled with a Shimadzu LC-20AD HPLC system, ThermoFinnigan LCQ antibody production (in collaboration with local animal facilities; full ion trap mass spectrometer with ESI, APCI and microspray interfaces service), Genomics (real-time PCR; walk-up), and Data storage/analysis combined with a Michrom Magic 2002 HPLC system, a ThermoFinnigan (cluster, server and workstations). Users get in-depth consultation Trace MS GC-quadropole with EI positive, negative CI and selected ion for proposed projects, and can obtain training in any of the walk-up monitoring (SIM), an ABI Voyager-DE Pro MALDI-TOF and a Bio-rad aspects of the facility, or take advantage of the full-service platforms. Bioplex 200 fluorescent microbead array system. CIAN is designed to facilitate training, enhance interactions, as well as share and maintain resources and expertise. 174 Microscopy and Imaging Facility, Cornell University 177 Danforth Center: Proteomics & Mass Spectrometry Core Facility R. Williams, C. Bayles, J. Dela Cruz, M. Riccio, R. Doran, W. Zipfel L. Hicks, S. Alvarez, B. Zhang, Z. Liu, H. Wang, J. Fazlic Cornell University, Ithaca, NY, United States Danforth Center, St. Louis, MO, United States The Microscopy and Imaging Facility of the Cornell University Life Sciences Core Laboratories Center (CLC) provides an array of shared The Proteomics & Mass Spectrometry Facility at the Donald Danforth research resources and services relating to optical microscopy, Plant Science Center (http://www.danforthcenter.org/pmsf/) is fluorescence, and whole animal imaging. The mission of the facility is equipped with state-of-the-art technologies for the detailed study of to provide cutting edge technologies and high quality services that will a wide range of biomolecules. The facility provides both full- and self- significantly contribute to life sciences research, training and education service capabilities to both internal and external clients at competitive programs. rates. The facility offers fast, high quality specialized analytical services including: protein extractions, liquid chromatographic separations; high 175 Utilization of Flow Cytometry in resolution 1D/2D gel electrophoresis; gel image analysis and protein Personalized Medicine expression analysis; high-throughput protein spot excision; in-solution and in-gel protein digestion; high-throughput protein identification; P. Lopez accurate protein molecular weight analysis; protein covalent/non- covalent complex analysis; biomolecule interactions (surface plasmon New York University, New York, NY, United States resonance); small molecule separation/structure determination; and protein post-translational modification analysis. Major instrumentation Flow cytometry is a multiparametric data-gathering tool with an includes: LTQ Orbitrap Velos (Thermo Scientific), QSTAR XL Q-TOF, enormous, well-established array of applications for intact cells, two 4000QTRAPs, two 6520 Q-TOFs (Agilent), 5975C GC-MS organelles, functionalized beads, lysed cell contents or supernatants. (Agilent), TriVersa NanoMate (Advion), Chip Cube (Agilent), two 1200 Multiparametric analysis using state-of-the-art cytometers has the HPLCs (Agilent), 3 nanoflow HPLCs (LC Packings/Eksigent), System potential to yield a distribution of cellular phenotypes with high Gold HPLC (Beckman Coulter), two Shimadzu HPLCs (Shimadzu), dimensionality (up to 20 parameters per cell simultaneously) specific UPLC (Waters), Biacore2000,3100 OFFGEL fractionator (Agilent), 1D to an individual disease state or system-wide perturbation. Purified and high resolution2D gel electrophoresis systems (BioRad/Amersham populations of atypical and/or cells representative of the disease state Biosciences), Typhoon 9410 (Amersham Biosciences), GelPix (Genetix), can be collected as the first step towards biomolecular characterization. and MultiProbe II (Perkin-Elmer). Protein intact mass, identification and Flow cytometers may be outfitted with multiwell-plate accessories to characterization are a few of the many applications that the facility provide single-cell capture of specified cells with correlation to each performs. For proteomics applications,two Q-TOFs andthe LTQ cell’s individual phenotype (indexed sorting), or rapid sampling from Orbitrap Velos instruments are well-suited for analyzing both small Poster Abstracts microtiter plates (plug-flow). An exciting new cytometric innovation peptides and large proteins. The LTQ Orbitrap Velos can be set up may allow determining the full signature of biomarkers from individual with the TriVersa to automate direct infusion of samples to perform cells . The massively multiparametric mass cytometer analyzer makes exact mass measurements for molecular formula determination or use of the high sensitivity and resolution of mass spectroscopy to facilitate targeted analysis of modifications. The LTQ Orbitrap Velos record the elemental composition as well as metal-tagged biomarker and the 6520 Q-TOF are also used for online LC-based quantitative distribution of individual cells at a rate of up to 1000 cells per second. proteomics (iTRAQ and label-free). The 4000 QTRAP systems serve as powerful instruments for targetedmetabolite analyses (e.g. plant 176 Cell Imaging and Analysis Network hormones, pABA, etc.), and the GCMS and one LC-QTOF are used for (CIAN) - Multi-Platform Resources metabolomics profiling initiatives. and Services

E. Küster-Schöck, J. Lacoste, G. Lesage, S. Bunnell, H. Han McGill University, Montreal, QC, Canada

The Cell Imaging and Analysis Network (CIAN) provides services and tools to researchers in the field of cell biology from within or outside Montreal’s McGill University community. CIAN is composed

ABRF 2011 — Technologies to Enable Personalized Medicine • 81 178 Proteomics and Mass Spectrometry Biomolecular interaction analysis (Surface Plasmon Resonance – Biacore). Applications in Biomedical Research Each of our services is staffed and supported by highly experienced and dedicated scientists. Beyond merely making available facilities and C. Diaz, M. Chow, R. Zheng, C. Silva-Sanchez, services, the PAN facility also enables methods development, and new J. Koh, S. Chen applications development, designed to meet the needs of the research community requiring the services. We will present research examples University of Florida ICBR Proteomics Facility, where the PAN facility played a significant role in the application of Gainesville, FL, United States these technologies to basic science projects.

Proteomics and mass spectrometry have provided unprecedented 180 University of Nebraska Medical tools for fast, accurate, high throughput biomolecular separation and Center Mass Spectrometry and characterization, which are indispensable towards understanding the biological and medical systems. Studying at the protein level Proteomics Core Facility allows researchers to investigate how proteins, their dynamics and M. Wojtkiewicz, P. Ciborowski modifications affect cellular processes and how cellular processes and the environment affect proteins. The mission of our facility is to provide University of Nebraska Medical Center, Omaha, excellent service and training in proteomics and mass spectrometry NE, United States to UF scientists and students. Here we present our capabilities in proteomics and other analytical services. The tools include a gel-based The UNMC Mass Spectrometry and Proteomics Core Facility offers a 2D-DIGE (Two Dimentional Difference Gel Electrophoresis) and gel- broad range of services, such as ESI and MALDI protein identification free iTRAQ (Isobaric Tags for Relative and Absolute Quantitation). using Mascot and Sequest Algorithms, iTRAQ-based quantitative Along with our capacity of separating thousands of proteins and proteomics, MRM protein quantitation, phosphoproteomics profiling characterizing differential protein expression, we have a suite of state- and molecular weight determination for proteins, peptides and small of-the-art mass spectrometers available for biomedical sciences and molecules. The facility is equipped with LTQ Orbitrap ETD, LTQ Velos, advanced technology research, including a tandem time-of-flight (4700 4800 MALDI TOF-TOF, 4000 Q TRAP, all with supporting nano-LC Proteomics Analyzer, AB), quadrupole/time-of-flight (QSTAR XL, AB), systems. Although the majority of users are investigators from UNMC, and hybrid quadrupole-linear ion-trap (4000 QTRAP, AB). These we also provide services for other outside academics and corporations. instruments are mainly used for protein identification, posttranslational For further information, visit our website: www.unmc.edu/mspcf. modification characterization and protein expression analysis (e.g., Mass Western). Our facility is also set up to provide Edman de novo N-terminal 181 UCLA Molecular Instrumentation protein sequence analysis and Biacore biomolecule interaction analysis. Center- Proteomics and Mass We are fully set up to synthesize and purify peptides and have a good Spectrometry Facilities track record with this service as well. Proteomics and mass spectrometry are useful in large-scale suvey of proteome for hypothesis generation as M. Sondej, W. Yan, R.J. Alvarado, G.A. Khitrov, well as in detailed analysis of target proteins for hypothesis testing. Our G. Czerwieniec, J. Strouse services also include accurate molecular weight analysis, MRM-based protein screening and targeted metabolite profiling. To ensure success University of California at Los Angeles, Los Angeles, and maximize productivity, the facility offers education, consultation, CA, United States data processing and reporting, and support of grant application. The UCLA Molecular Instrumentation Center (MIC) is a state-of-the- art campus-wide facility dedicated to enabling the use of modern 179 The Protein and Nucleic Acid (PAN) instrumentation in molecular characterizations (www.mic.ucla.edu). The Facility at Stanford University UCLA Molecular Instrumentation Center is housed within and managed through the Department of Chemistry and Biochemistry and is composed S. Patel, M. Eckart, N. Kosovilka, A. Sanchez, Y. Tran, P. Walker, R. Winant, E. Zuo of five divisions: J.D. McCullough Laboratory of X-ray Crystallography, Magnetic Resonance Facility, Materials Characterization lab, W. M. Beckman Center, Stanford University, CA, United Keck Proteomics Center and Mass Spectrometry (MS) Laboratory. States The MIC operates as an open access center where qualified users are encouraged to perform their own sample analysis under the training and guidance of the MIC personnel and is available to researchers at

Poster Abstracts The Protein and Nucleic Acid (PAN) Facility (http://pan.stanford.edu) at Stanford University’s Beckman Center is a multifaceted biotechnology UCLA, other academic institutions and commercial enterprises. The fee-for-service laboratory providing services to the Stanford scientific UCLA Proteomics Center and Mass Spectrometry Laboratories have community, other non-profit and biopharmaceutical organizations. The five Ph.D. level staff memberswho are experts in sample preparation, Facility’s mission is to be adaptable and responsive to the changing 2-D gel and other electrophoresis techniques, bioinformatics, and needs of biomedical research by providing basic science investigators mass spectrometry. The equipment for the UCLA Proteomics Center continued access to key tools and applications in an efficient and cost includes Bio-Rad electrophoresis cells for running 1- and 2-D gels; Bio- effective manner. The Facility offers a diverse array of instrumentation Rad Fx Fluorescence Imager and GS-800 Densitometer for imaging; and technical capabilities in Molecular Genetics and Protein Analytics. DIGILAB Genomic Solutions ProPicII spotcutter; Thermo LTQ FT MS In Molecular Genetics the services include Gene Expression and with Eksigent NanoLC-2D HPLC; Thermo LTQ Orbitrap XL MS with Genotypring (Affymetrix microarray), quantitative methylation and Eksigent NanoLC-2D HPLC and a Bruker SolariX-hybrid Qq-FTMS mutation analysis (PyroMark), qPCR, DNA sequencing and fragment equipped with a 15 Tesla Magnet System. Our Bioinformatics resource analysis, Nucleic Acid QC (Agilent Bioanalyzer) and Oligonucleotide center hosts a number of qualitative and quantitative software for 2-D synthesis. For protein characterization the laboratory offers Protein gel and mass spectrometry data analysis and hardware such as a linux Sequencing, Peptide Synthesis, Protein Identification (MALDI) and cluster and servers for MS database searching and data storage.The MS

82 • ABRF 2011 — Technologies to Enable Personalized Medicine Laboratory is located next door and works closely with the Proteomics 184 New MAbPac Phases for Monoclonal Center. Their mass spectrometers includes Applied Biosystems-MDS Antibody (MAb) Variant Analysis Sciex 4000 Q Trap with Autosampler; Applied Biosystems Q-Star Elite Quad-TOF Hybrid LC/MS/MS system; Applied Biosystems Voyager- G. Gendeh, S. Rao, Y.X. Hou, X. Liu, Y. Agroskin, DE STR MALDI-TOF; Thermo Finnigan LCQ Deca Ion Trap MS with C. Pohl Autosampler and PDA; Agilent 6890-5975 GC-MS with Autosampler and Waters LCT Premier with ACQUITY UPLC and Autosampler. Dionex Corporation, Sunnyvale, CA, United States

182 Microscale Thermophoresis: MAbs generally exhibit complex heterogeneity including glycosylation, oxidation, phosphorylation, amino-terminal modifications, incomplete Interactions of Proteins, Small processing of the C-terminus, and asparagine deamidation. These Molecules, Nucleic Acids, and Vesicles variations in composition could impact their efficacy, stability, and safety. Monitoring and reporting such variations in therapeutic proteins is S. Duhr, P. Baaske required by the FDA and other regulatory agencies. Two new MAbPacTM NanoTemper Technologies GmbH, Munich, phases were developed to meet these needs. The MAbPac SCX-10 is a Germany newly designed strong cation-exchange column for the characterization of heterogeneity of MAbs. This is a complementary addition to the This work gives an overview on a new Technology for the measurement existing ProPac® WCX-10 column that provides high resolution and of biomolecule interaction that is termed Microscale Thermophoresis orthogonal selectivity for MAb charge variant analysis. The MAbPac (MST). The term Microscale Thermophoresis refers to the directed SCX stationary phase is based on nonporous, highly cross-linked styrenic movement of molecules in optically generated microscopic temperature type polymeric media with a proprietary hydrophilic coating. Sulfonic gradients. This thermophoretic movement is determined by the entropy acid functionality is added through controlled radical polymerization of the hydration shell around the molecules. Almost all interactions grafting. These particles exhibit a wide range of pH stability with high between molecules and virtually any biochemical process related to selectivity and minimal band spreading. The MAbPac SEC-1 is a new a change in size, stability and conformation of molecules alters this size-exclusion chromatography (SEC) column specifically developed for hydration shell and can be quantified. Such changes allow quantification characterization of monoclonal antibody (MAb) aggregates, enzyme of binding affinities of proteins, nucleic acids and small molecules as digested fragments, and other size-based separation applications. The well as measurement of enzymatic activities with MST. In addition MAbPac SEC column is based on high-purity, spherical, porous (300 also functional studies of small molecule inhibitors are possible. The Å), 5 μm silica covalently modified with a proprietary diol hydrophilic microscopic temperature gradient is generated by an IR-Laser, which layer. This stationary phase can handle both high- and low- salt eluents is strongly absorbed by water. The readout method of the interaction as well as mass spectrometry compatible eluents. The MAbPac column analysis is based on fluorescence: intrinsic fluorescence of proteins can is packed into a nonmetallic, biocompatible PEEKTM column housing be used as well as proteins expressed with GFP/YFP/RFP and also dye to eliminate metal contamination from the column hardware that can labeled biomolecules. In this presentation we will describe the technical compromise MAb separations. The stationary phase is designed to details and the benefits of the Microscale Thermophoresis technology minimize undesired nonspecific interactions between proteins and the platform. We will show examples for interaction measurements ranging stationary phase. Various applications with relevant comparisons along from protein -ribosome, protein -protein, small molecule -receptor with a demonstration of the ruggedness of these new phases are shown binding to studies where the interactions between receptor containing in this poster. vesicles and proteins are analyzed. 185 The VGN Proteomics Module: A 183 Reduction of Sample Carryover in Transferable Laboratory Module for Proteomics LC-MS Experiments Undergraduates

G. Gendeh, M. Karsten, E.J. Sneekes, R. Swart J. Murray

Dionex Corporation, Sunnyvale, CA, United States Vermont Genetics Network, University of Vermont, Poster Abstracts Burlington VT, United States Relevant biomarkers are often present at concentrations near or below the detection limit of current analytical methods. Despite this challenge, The Vermont Genetics Network (VGN) Outreach Core’s mission is several biomarker candidates have been identified and moved into the to bring cutting-edge technology and knowledge to undergraduates validation phase. The increase in sensitivity of analytical methods and at colleges throughout the state of Vermont. The VGN Proteomics mass spectrometry, in particular, over the past years is the reason for Outreach project initiated in the fall of 2009 exposes undergraduates this accomplishment. However, a sensitivity increase alone is insufficient in the state of Vermont to proteomics technology using hands- to accurately identify potential biomarkers; carryover reduction is also on laboratory experiences. We provide all teaching materials, important to ensure a marker is actually present in the sample being laboratory materials and if necessary equipment for colleges within analyzed. Therefore, reduction of carryover has received increased the state to run the module. All materials become the property of the attention from the proteomics community. recipient institution upon completion of the laboratory module. The undergraduate students learn about this cutting edge technology and gain new skills that we believe will help them with their future scientific careers. In this transferable module, students learn how protein expression in yeast is changed after exposure to oxidative stress or an environmental toxin. Total protein is then harvested and prepared for 2D gel analysis. Proteins with differential expression are isolated from the 2D gel and prepared for Mass Spectrometry at the UVM

ABRF 2011 — Technologies to Enable Personalized Medicine • 83 Proteomics Core Facility. The data is processed and students examine 3-hr, 4-step process. Although the read coverage is poor in a single lane, their results and use bioinformatics tools to further understand the the reproducibility of the reads allows single cells to be compared for biological implications of the results. The students than present their SNP and CNV genotype, mutations. The same amplified samples can findings, describe specific proteins that showed differential expression. be used for PCR and microarray analysis, including genome-wide SNP A hypothesis is presented explaining the biological relevance of the genotyping, mutation, and copy number analysis. PRC amplification or protein expression change along with a plan for testing this hypothesis. target enrichment can be used for high accuracy and coverage single- This work was sponsored by Grant Number P20 RR16462, from the cell genomic analysis. IDeA Networks of Biomedical Research Excellence (INBRE) Program of the National Center for Research Resources (NCRR), a component of 189 PRG-2011: Defining the Interaction the National Institutes of Health (NIH). Between Users and Suppliers of Proteomics Services/Facilities 186 Quantitative Western Blotting with Amersham ECL Prime D.H. Hawke1, T.M. Andacht2, M.K. Bunger3, C.E. Bystrom4, L.J. Dangott5, H. Molina6, M. Winkvist, S. Grimsby, K. Söderquist, R.L. Moritz7, R.E. Settlage8, C.W. Turck9 A. Marcusson 1University of Texas MD Anderson Cancer Center, GE Healthcare Bio-Sciences AB, Uppsala, Sweden Houston, TX, United States; 2Centers for Disease Control and Prevention, Atlanta, GA, United Western blotting is a well established technique used to study proteins 3 from a wide variety of sources. The technique is used throughout the States; RTI International, Research Triangle 4 life sciences from basic research to medical diagnostic applications. Park, NC, United States; Quest Diagnostics, San Western Blot is at best considered as semi-quantitative and hence Juan Capistrano, CA, United States; 5Texas A&M limited to studies involving large protein differences.Here we University, College Station, TX, United States; demonstrate the use of a new ECLTM reagent, AmershamTM ECL Prime 6Center for Genome Regulation, Barcelona, in a number of typical Western Blotting applications. The results Spain; 7Institute for Systems Biology, Seattle, WA, demonstrate that, Amersham ECL Prime can be used for detection of United States; 8Virginia Bioinformatics Institute, low abundant proteins, that signals are very stable over time and cover Blacksburg, VA, United States; 9Max Planck Institute a broad dynamic range. These features make Amersham ECL Prime of Psychiatry, Munich, Germany highly suitable for accurate quantitative analysis. Over the last ten years the Proteomics Research Group (PRG) has 187 New Approaches to Quantitative undertaken technical studies that have covered a wide range of issues Western Blotting unique to the rapidly developing field of proteomics. These studieshave included a range of qualitative and quantitative experiments. The PRG M. Winkvist, Å. Hagner-McWhirter, studies have resulted in a great deal of attention not only within the K. Söderquist, S. Grimsby ABRF community but also outside as is evident from numerous articles dealing with proteomics methods, procedures and standardization. GE Healthcare Bio-Sciences AB, Uppsala, Sweden As the field continues to develop, the diversity of instrumentation and laboratory workflows have grown in tandem. Therefore, for the Fluorescent detection in Western blotting offers high sensitivity, broad PRG2011 study it seemed especially useful to perform a survey to dynamic range and stability of signals. This makes it highly suitable for help define future studies based on the current blend of sample types quantitative Western blotting. Here we show how fluorescent Western and technologies and obtain a view of emerging trends. A survey was blotting can be used for simultaneously detection of up to three created to ascertain three main insights into core facility function: 1) different proteins on the same blot at the same time and for detection How labs interact with their clients, 2) The capacity of labs to meet the of proteins of the same molecular weight without stripping and re- demands of their clients, and 3) The blend of experimental techniques probing. We also demonstrate how fluorescent Western blotting with offered to and requested by clients. Survey questions were designed to 3 layer probing can be used to increase sensitivity and thereby enables obtain information from both users of core facilities and the directors detection of very low abundant proteins. Finally we demonstrate that and personnel of core facilities. Questions covered such topics as the it is possible to compare the total protein amount to the target protein type and age of instruments in use, how data is analyzed and presented by using Deep PurpleTM protein staining prior to ECLTM PlexTM Western

Poster Abstracts to client, sources of funding, and emerging proteomics trends. Results blotting. are compiled en masse and presented without regard to institution.

188 Rubicon PicoPlex-NGS Kits Available 190 GlycoMaster — Software for for Sequencing Single Cells Using the Glycopeptide Identification with Illumina Genome Analyzer Combined ETD and CID/HCD Spectra , T. Kurihara, E. Kamberov, J. Langmore , L. Xin J. M’Mwirichia, T. Tesmer, D. Oldfield P. Shan Bioinformatics Solutions Inc., Waterloo, ON, Rubicon Genomics, Ann Arbor, MI, United States Canada Rubicon Genomics has released its PicoPlex-NGS kits to prepare single Objective: To automate the data analysis for the identification of cells for NGS analysis on the Illumina Genome Analyzer. These kits glycopeptides with combined ETD and CID/HCD fragmentation. The enable single eukaryotic or prokaryotic cells to be lysed, DNA extracted inputs for GlycoMaster software include the raw mass spectrometry and amplified, and adapted for paired-end sequencing in a 1-tube,

84 • ABRF 2011 — Technologies to Enable Personalized Medicine data of a Thermo Orbitrap instrument and the list of proteins in reduction and alkylation or separation of the different chains. We used the sample. The list of proteins can be identified from the same 1,5-diaminonaphtalene (DAN) as matrix because of its reductive and mass spectrometry data by using a database search method. The ISD enhancing properties. The samples were mixed with matrix solution software uses the signature ions of simple sugars in the HCD spectra and dried at ambient air on the MALDI sample plate. In a second to identify the HCD-ETD spectrum pairs of glycopeptides. Then experiment, the same samples were prepared using sDHB (super DHB), the ETD spectrum in each pair is used to identify the glycopeptide which is known to be a good ISD matrix but without reduction capacity. sequence, the glycosylation site and glycan mass. The output of The samples in sDHB didn’t generate any ISD fragmentation due to the the software is an excel file that contains information about the intact disulfide crosslinks in IgG. In DAN, the same samples generated identified glycopeptides, including glycan composition, mass error, rich ISD spectra containing fragments from both the heavy and the light glycan components, glycosylation site and glycopeptide sequence. chain of the antibodies. The ISD spectra permitted reading through A score is associated with each identification result that can be used the cysteine residues in the sequences implicating the disulfide bridges to sort the identifications according to confidence. GlycoMaster was were reduced by DAN. Up to 60 residues of the different chains of tested with an ETD-CID dataset containing 3561 MS and 2738 MS/ various mAbs were matched and validated, respectively. A dedicated MS spectra from tryptic digest of reduced and alkylated 12 standard software tool allowed a very fast interpretation of the spectra. Overall protein mixture containing 3 glycoproteins ( human serotransferrin, the method takes only a few minutes from sample preparation, chicken ovalabumin and ovomucoid) from Sigma. Glycopeptides were spectrum acquisition and processing to the result in form of sequence enriched on cellulose column or by using ZIC-HILIC spin columns from annotations in the ISD spectra. EMD. Purified glycopeptides were analyzed by LTQ Orbitrap Velos ETD. PEAKSTM Studio Suite was used for spectral preprocessing and **193 Developing a Redox-Sensitive Red protein identification. GlycoMaster reported 161 identified HCD-ETD Fluorescent Protein Biosensor spectrum pairs of glycopeptides. 95 of 161 were manually validated. 68 glycopeptides were reported by GlycoMaster, and 40 glycopeptides I. Magpiong1, N. Koon2, S.M. Yei2, A.J. Risenmay2, were validated. K. Kallio2, S.J. Remington2 1Mount Holyoke College, South Hadley, MA, 191 Development of an Automated 2 Method for Antibody Purification United States; University of Oregon, Portland, OR, and Analysis United States Redox environments are of particular interest, especially in the G. Gendeh, W. Decrop, R. Swart mitochondria with its highly reducing environment and its role as the Dionex Corporation, Sunnyvale, CA, United States central processing unit of apoptosis. Monitoring of mitochondrial redox environments is crucial to the study of apoptotic disorders. Reporting The screening and analysis of monoclonal antibodies can be an of the thiol/disulfide status in live cells was made possible with the extremely time consuming task, especially as the many of the workflows development of redox-sensitive green fluorescent protein (roGFP). We used today require many manual steps. This situation is fully remedied aim to develop a red version redox-sensitive fluorescent protein (roRFP). by the technology offered by the MAb Analysis platform from Dionex Expanding the array of redox-sensitive proteins with a red version will - a single platform can screen a large number of MAbs and, in a second enable simultaneous visualization of multiple reducing intracellular step, provide detailed analytical information, including charge variant compartments. mKeima is a monomeric red fluorescent protein that analysis and aggregate assessment. This unique technology allows absorbs light maximally at 440nm and emits red light at 620nm. This drug discovery laboratories to develop MAb therapeutics much more large Stokes shift is dramatically decreased in acidic environments. By quickly than before, and bring these important new therapeutics to following protocol similar to that used in the development of roGFP, market in a shorter time frame. surface residues at key positions were changed to cysteines and random mutagenesis was performed on varying excitation species of 192 Fast QC of Intact Monoclonal mKeima. Mutants were screened and a ratiometric variant of mKeima Antibodies Using MALDI-Top-Down was identified (roRFP2) which exhibits changes in its spectral properties Sequencing as a result of changes in the thiol/disulfide equilibrium. Preliminary Poster Abstracts fluorescence spectroscopy measurements of roRFP2 indicate a highly A. Resemann, R. Paape, L. Vorwerg, D. Suckau reducing redox potential of -330mV indicating it may be a useful probe in reducing subcellular compartments such as mitochondria or Bruker Daltonics, Bremen, Germany in the cytoplasm. By employing vector recombination of shuttle vector PYX142, we successfully targeted roRFP2 in vivo to the mitochondria Antibodies are playing an important role in drug development in the and cytoplasm of Saccharomyces cerevisiae. Expression of roRFP2 was last years. The function of therapeutic monoclonal antibodies (mAbs) is visualized using fluorescence microscopy. Thus, through mutagenesis directly depending on their structure including terminal modifications and residue substitution we successfully created a red version redox- like C-terminal lysine excision and N-terminal pyroglutamylation of sensitive biosensor that tested effectively as a ratiometric indicator the heavy chain. Here, we describe a method for very fast and simple and expressed in the mitochondria and cytoplasm of S. cerevisiae. validation of the terminal sequences of heavy and light chain of intact Moreover, the redox potential of roRFP2 is significantly more negative monoclonal antibodies using MALDI-Top-Down-Sequencing (MALDI- than the widely used roGFPs. TDS) using in-source decay (ISD). Intact mAb (IgG) in a concentration of 1 mg to 5 mg/ml was directly mixed with matrix solution without prior

ABRF 2011 — Technologies to Enable Personalized Medicine • 85 194 PeaksDB: New Software for combination with pI filtering to allow greater confidence for peptide Substantially Improved Peptide identifications. IDSieve considers the SEQUEST parameters Xcorr and ΔCn to assign statistical confidence (false discovery rates) to Identification from Orbitrap ETD the peptide matches. The distribution of predicted pI values for Mass Spectrometry peptide spectrum matches (PSMs) is considered separately for each 1, J. Zhang2, L. Xin2, B. Shan2, W. Chen2 immobilized pH gradient isoelectric focusing fraction, and matches B. Ma with pI values within 1.5 times inter-quartile range (within pI range) 1University of Waterloo, Waterloo, ON, Canada; are analyzed independently of matches outside the pI ranges. We 2Bioinformatics Solutions, Inc., Waterloo, ON, tested the performance of IDSieve and Peptide/Protein Prophet on the Canada SEQUEST outputs from 60 immobilized pH gradient isoelectric focusing fractions derived from mouse intestinal epithelial cell protein extracts. Objective: To substantially improve the peptide identification sensitivity Our results demonstrated that IDSieve produced 1355 more peptide and accuracy from the Orbitrap ETD data with computational methods. spectrum matches (or 330 more peptides) than Peptide Prophet using Method: The algorithm takes full advantage of the characteristics comparable false positive rate cutoffs. Therefore, combining pI filtering of the Orbitrap ETD data, including: (1) high mass resolution of the with the appropriate statistical significance measurements allows for a precursor ions, and (2) the distributions of different fragment ion higher number of protein identifications without adversely affecting types in the MS/MS scans. For the first characteristic, a pre-search step the false positive rate. We further tested the performance of pI filtering is conducted to determine the precursor mass error distribution. This using ID Sieve when samples were prefractionated using either pH does not only make the precursor mass more accurate by a software range 3.5-4.5 or 3-10, and either 24cm or 7cm IPG strips. recalibration, but also allows the use of the mass error as an important feature in the peptide-spectrum matching score function. For the 196 Proteomics-Based Molecular Model second characteristic, the frequencies of different fragment ion types for Prediction of Heterogenic at different precursor charge states are statistically learned, and used Treatment Response and Discovery in the score calculation. Moreover, the precursor-related ions in the of New Cell Survival Mechanisms of MS/MS spectra are removed. Additionally, the score function makes use of the similarity between a database peptide and the de novo Basal-Like Breast Cancer sequencing result. Result: PeaksDB was compared against three other T. Hemström, Y. Lyutvinskiy, R. Zubarev search engines: MSGF-DB, Mascot, and ZCore. The same shuffled decoy database was appended to the target database and searched together Karolinska Institutet, Stockholm, Sweden to estimate the false discovery rate (FDR) of each individual engine. The same search parameters were used for all engines except that MSGF- We aim to build a proteomics-based model for prediction of drug DB does not support variable PTMs. If no variable PTM is allowed, the response and discovery of new signaling-related mechanisms numbers of identified peptides of different engines at 1% FDR are: explaining the heterogeneity in treatment response observed in the PeaksDB (2356) > MSGF-DB (2147) > Mascot (1459) > ZCore (1030). group of basal-like breast cancer (BLBC), which is a group of breast If a few common PTMs are allowed, the numbers change to PeaksDB cancer associated with a relatively short survival of patients. A panel (3501) > Mascot (2677) > MSGF-DB (2147) > ZCore(1125). Conclusion: of cell lines of BLBC origin will be subjected to conventional as well as PeaksDB substantially improved the sensitivity and accuracy of peptide molecular pathway specific anti-cancer drugs followed by MTT-assay identifications on Orbitrap ETD data. At 1% false discovery rate, viability assessment. The range of drug concentrations will cover the PeaksDB identified 1.3 to 1.6 times as many peptides as Mascot 2.3. maximal plasma drug concentration of patients. Focus will be on cell lines exhibiting a high or low sensitivity to treatment. Drug responses of 195 IDSieve: Protein Identification such cell lines will be further examined in terms of cell cycle distribution and cell death levels and based on these assessments relevant drug Using Peptide pI Filtering of MS/MS concentrations and exposure times will be chosen. Analysis of protein Data for Improved Confidence in expression in cells exposed to relevant treatment conditions will be Identifications performed by LC-MS/MS. By way of key node analysis of protein expression data cellular signaling involved in sensitivity or resistance to 1, K.D. West1, X. Zhang1, J.L. Bundy2, N.R. Garge drugs will be hypothesized and validated by means of anti body-based J.L. Stephenson, Jr.2, B.J. Cargile3, M.K. Bunger1 techniques, experimental modulation of signaling and analysis of cell 1RTI International, Research Triangle Park, NC, cycle and cell death. Thereby we will add two more dimensions, which Poster Abstracts United States; 2Centers for Disease Control and are the drug response-related changes in activity of signaling pathways Prevention, Atlanta, GA, United States; 3Sapient and functional morphological changes, such as apoptotic cell death, to gene expression-based models of treatment response. Drug response Proteomics, Morrisville, NC, United States data in terms of viability, protein expression, and cellular signaling will The main challenge of tandem mass spectrometry based proteomic be integrated in a computerized model. By focusing on a well defined analysis is to correctly match the tandem mass spectra produced to the subgroup of breast cancer exhibiting clinical heterogeneity in anti- correct peptides. However, the large number of protein sequences in cancer drug response we have a reasonable good chance to develop a database increases the chances of a false positive identification for a fine-tuned model for prediction of treatment response and identify any given peptide match. Here we present an automated algorithm currently unknown drug resistance mechanisms, which may involve new called IDSieve that utilizes target-decoy database search strategy in possible predictive markers and drug targets.

86 • ABRF 2011 — Technologies to Enable Personalized Medicine **197 Multi-Functional Superparamagnetic lysate easily generates 500,000 peptides. The separation of these Iron Oxide Particles as Cancer highly complex peptide samples is one of the major challenges in analytical chemistry. The main strategy to improve the efficiency of Therapeutic Agents packed columns is either to increase column length or to decrease 1 1 1 the size of the stationary phase particles. However, to operate these A. Narayanan , P.M. Gannett , J.A. Barr , R.L. Carroll2, S.L. Yedlapalli2 columns effectively, the LC conditions need to be adjusted accordingly. Naturally, the on-line coupling to MS systems has to be taken into 1Robert C. Byrd Health Sciences Center, account in the optimization process. Here, the authors report on the Department of Basic Pharmaceutical Sciences, performance of nano LC columns operating at ultrahigh pressure. West Virginia University, Morgantown, WV, The effects of column parameters (particle size and column length) United States; 2C. Eugene Bennett, Department of and LC conditions (gradient time, flow rate, column temperature) Chemistry, West Virginia University, Morgantown, were investigated with reversed-phase (RP) gradient nano LC. High- resolution LC-MS separations of complex proteomic peptide samples WV, United States are demonstrated by combining long columns with 2 μm particles Superparamagnetic iron-oxide nanoparticles (SPIONs) are magnetic and long gradients. The effects of LC parameters on performance and nanoparticles may be useful for early detection and treatment of the influence on peptide identification are discussed. cancers. SPIONs provide therapeutic drug-loading capabilities and targeting specificity through the use of antibodies or receptor specific **199 Comprehensive Biomarker Discovery tags. These versatile particles are prime candidates for improving Platform Reveals Qualified MRM current means to the treatment of cancer and potentially other Assay for Prediction of INF/Ribavirin diseases. This multi-disciplinary project includes various milestones Treatment Response in Hepatitis C and multiple aims: i) identify and optimize SPION design parameters Patients that optimize aqueous stability and maximize amphiphilic character, ii) evaluate SPION drug storage and release characteristics, and J.W. Thompson1, L.G. Dubois1, K. Patel2, iii) demonstrate binding and entry into targeted cells. The specific J.E. Lucas1, J. McCarthy1, J.G. McHutchison2, aim of this project is the coupling of cell-specific targeting agents to M.A. Moseley1 SPIONs. An antisense oligonucleotide against Survivin mRNA was synthesized by solid-phase DNA synthesis with an amino-terminated 1Duke Institute for Genome Sciences and Policy, linker on the 3’ end (5’CCCAGCCTTCCAGCTCCTTG-(CH2)6-3’NH2). Durham, NC, United States; 2Duke Clinical Research SPIONs were prepared and then coated with a copolymer containing Institute, Durham, NC, United States surface carboxylic acid groups (-COOH). The carboxylic acid groups were activated by treatment with N-((3-dimethylamino)-propyl)-N- The current standard of care for chronic Heptatitis C (CHC) results in ethyl carbodiimide and coupled to oligonucleotides via the –NH2 sustained viral response in only half of patients and also has significant terminus to the –COOH groups resulting in the formation of an amide side effects. Improved pre-treatment clinical predictors of response linkage between the –COOH groups of the SPION and the –NH2 of are needed to individualize treatment and select individuals most the antisense oligonucleotide. Circular dichroism (CD) studies were appropriate for new therapy. We have identified 10 peptides to 6 performed to quantify/optimize coupling and to demonstrate antisense proteins which can differentiate non-responders from responders with Survivin duplex formation was not inhibited by the presence of the high accuracy based on pre-treatment samples. Serum samples were SPION. CD results were correlated with agarose gel electrophoresis randomized, immunodepleted (MARS 14), and digested with trypsin data and demonstrated oligonucleotides coupling to the SPION and prior to LC-MSE or LC-MRM analysis. LC-MSE analyses were performed that the SPION did not significantly alter duplex formation. Future on a Waters nanoAcquity LC and QToF Premier, while MRM analyses studies will target cellular absorption and antisense binding to Survivin were performed on nanoAcquity and Xevo TQ. Rosetta Elucidator® was mRNA using confocal microscopy (Supported, in part, from NIH used to quantify all LC-MSE data. PLGS v2.4 was used to make peptide GM081348 grant and the WVU Research Corporation). identifications. MRM method generation and sample quantitation was performed with Skyline v0.6. The response signature was initially Poster Abstracts 198 Ultrahigh-Performance Nano LC-MS/ identified from the analysis of 96 samples (Duke Hepatology Clinical MS Analysis of Complex Proteomic Research database) by LC-MSE on a QToF mass spectrometer, using sparse latent factor regression analysis. This yielded a group of almost Samples 400 candidate peptides which were part of 3 metaprotein predictors; these peptides were then curated based on set selection criteria to G. Gendeh, E.J. Sneekes, B. de Haan, R. Swart give 86 target peptides for MRM analysis. MRM analysis of the original Dionex Corporation, Sunnyvale, CA, United States 96 samples yielded a final group of 10 peptides which maintained Bonferroni-corrected statistical significance for predicting treatment Determination of the proteome and identification of biomarkers are response. The assay has been qualified by a blinded analysis of an all- required to monitor dynamic changes in living organisms and predict comers clinical trial sample set (PEDS-C, NIH, n=51) using the MRM the onset of an illness. One popular method to tackle contemporary method, which yielded an AUROC of 0.91 with a sensitivity of 0.828 proteomic samples is called shotgun proteomics, in which proteins are and specificity of 0.786. The analysis of an additional CHC clinical trial digested, the resulting peptides are separated by high-performance cohort (Chariot, n = 243) is ongoing. This presentation will utilize CHC liquid chromatography (HPLC), and identification is performed with treatment response as a case study on the critical analytical, statistical, tandem mass spectrometry. Digestion of proteins typically leads and clinical cohort requirements to successfully perform biomarker to a very large number of peptides. For example, digestion of a cell discovery and verification.

ABRF 2011 — Technologies to Enable Personalized Medicine • 87 200 Discovery of Yersinia pestis and based on the thermophoresis of molecules, which provides information Yersiniophage Peptide Targets for about molecule size, charge and hydration shell. We validated the method using immunologically relevant systems including human Use in Multiple Reaction Monitoring interferon gamma and the interaction of calmodulin with calcium. The Methods affinity of the small-molecule inhibitor quercetin to its kinase PKA was 1, S. Bearden2 determined in buffer and human serum, revealing a 400-fold reduced L. Luna affinity in serum. This information about the influence of the biological 1Division of Vector-Borne Infectious Diseases, matrix may allow to make more reliable conclusions on protein Centers for Disease Control and Prevention, functionality, will facilitate more efficient drug development, and may Fort Collins, CO, United States; 2Toxicology and allow for sensitive diagnostics in complex biological samples. Environmental Research and Consulting, The Dow Chemical Company, Midland, MI, United States 202 2-D DIGE Analysis of Multicellular Tumor Spheroids in Evaluation of Aims: We present the discovery of functional yersiniophage and Breast Cancer Treatment Yersinia pestis (Y. pestis) peptide targets for monitoring bacteriophage- based amplification processes and species-specific identification of V. Ruddat1, M. Winkvist2, S. Grimsby2, Yersinia pestis, the etiological agent of plague. Methods: For protein A. Monazzam3, K. Nyamekye3, confirmation and peptide target discovery, ultra performance liquid Å. Hagner-McWhirter2 chromatography and hybrid tandem mass spectrometry was utilized 1 2 to identify proteolytic cleaved peptides generated from digestion of GE Healthcare, Piscatway, NJ, United States; GE whole phage and bacterial lysates. Peptide discovery was accomplished Healthcare Bio-Sciences AB, Uppsala, Sweden; with a UPLC NanoAcquity interfaced into a Q-TOF Premier (Waters 3Uppsala Applied Science Lab, GE Healthcare, Corporation) for full scan and MS/MS experiments. Targeted Uppsala, Sweden development utilized the MRM initiated detection and sequencing (MIDAS) workflow design software (Applied Biosystems) to create in Many cancers can be diagnosed using positron emission tomography silico MRM transitions which optimized collision energies for trypsin (PET) and PET can also be used to monitor how effective various cleaved peptides from the proteins of interest. The analytical column treatments are in individual patients. Tumor spheroids are cancer cells utilized was an UPLC bridged ethyl hybrid (BEH) 1.0 x 50 mm reverse grown on agar coated dishes forming a 3D structure. They are widely phase C18 (1.7-µm particle size) interfaced into a 4000 Q TRAP for used in preclinical cancer research, where the multicellular tumor MRM triggered MSMS. Results: We screened yersiniophage phiA1122, spheroid model is considered biologically and physiologically similar R, V, and Y proteins for conserved peptide targets for utilization in to in vivo grown tumors. In this study we have used Two-dimensional routine mass spectrometry monitoring of the phage amplification Difference Gel Electrophoresis (2-D DIGE) analysis to gain more insight process. We also identified murine toxin and F1 antigen peptide targets in differences in protein expression as a result of drug treatment that are unique to Yersinia pestis, which upon lysis, can be utilized for of multicellular tumor spheroids. 2-D DIGE can be used to identify confirmatory identification of Y. pestis. Conclusions: Major and minor possible new biomarkers for development of new PET tracers and drug capsid protein peptides will serve to enhance quantifiable metric targets. Combining 2-D DIGE and PET results can be used for improving measurements of the amplification process for rapid yersiniophage diagnosis and treatment. diagnostic capabilities and concomitant determination of the presence of viable Y. pestis bacilli. Moreover, the targeted murine toxin peptide 203 N-Glycosylation of Antibodies directly confirmed the presence of Y. pestis at 26°C and can be applied Characterized by Mass Spectrometry: as an additional tool for zoonosis surveillance capabilities in a clinical An Integrated Software Approach laboratory setting. Significance and Impact of Study: Applications utilizing these peptide targets identified in this work may be beneficial U. Schweiger-Hufnagel, A. Asperger, in emergency response situations requiring pre A. Resemann, D. Suckau

**201 Protein-Binding Assays in Bruker Daltonics, Bremen, Germany Biological Liquids Using Microscale Antibodies represent one of the most important classes of Thermophoresis glycoproteins playing a central role in the immune response of living

Poster Abstracts organisms. Furthermore, there is a growing interest in recombinant 1, P. Baaske1, C. Wienken2, S. Duhr antibodies as potential biotherapeutic agents. The analysis of the 2 2 M.J. Willemsen , D. Braun N-glycosylation pattern present on antibodies is challenging due to its 1NanoTemper Technologies GmbH, and Center heterogeneous structure. The glycan profile is highly dependent on for Nanoscience, University Munich, Munich, the process by which a recombinant glycoprotein is generated, such as host organism and growth conditions. Changes to the glycosylation Germany; 2University Munich, Physics Department, pattern can significantly alter biological function. To characterize the Munich, Germany N-glycosylation pattern of a recombinant antibody, a bottom-up Protein interactions inside the human body are expected to differ from approach was pursued. Tryptic digests of antibody samples were the situation in vitro. This is crucial when investigating protein functions separated by nano-LC and analyzed by MALDI mass spectrometry. An or developing new drugs. In this study, we present a sample-efficient, integrated software approach allowed a detailed characterization of free-solution method, termed Microscale Thermophoresis (MST), that the glycosylation pattern and visualization of the relevant mass spectra. is capable of analysing interactions of proteins or small molecules in LC-MALDI-TOF/TOF analysis of the digested antibody provided, biological liquids such as blood serum or cell lysate. The technique is in addition to the nearly complete sequence coverage of the non- glycosylated peptides, a detail-rich picture of the highly complex

88 • ABRF 2011 — Technologies to Enable Personalized Medicine pattern of N-linked glycans in form of the respective N-glycopeptides. lane size standard. Methods included importing raw data files to the Targeted analysis of potential glycopeptides significantly increased the software and physically identifying reference peaks in the samples information content. Integrated glycoprotein analysis software tools known to have the same size. The program uses this information to (ProteinScape 2.2) allowed identification of glycan modifications and calibrate from one capillary to another. Characteristics of the aligned interactive result validation. In this process software facilitated the data (such as relative size, peak height, peak area, peak ratios) were initial characterization of the antibody as well as the subsequent quality exported in an excel sheet. Ninety six raw data files from 4 dye capillary control tasks. electrophoresis were analyzed. Peak height, height ratio, area, area ratio, and relative sizes were determined for all samples. These values 204 Magnetic ZIC-HILIC Beads can be used to determine characteristics such as number and relative Enrichment for Neutral and Acidic size of degradation products or other macromolecules, such as DNA Glycopeptides binding carbohydrates commonly functioning in gene regulation.

A. Resemann1, J. Wohlgemuth2, S. Andrecht2, 206 Analysis of Complex Oligosaccharides A. Schneider1, U. Schweiger-Hufnagel1, D. Suckau1 from Glycopeptides and 1Bruker Daltonics, Bremen, Germany; 2Merck KGaA, Glycoproteins Using MSn Spectra and Darmastadt, Germany Oligosaccharides Spectral Library

Glycosylation is the most abundant protein posttranslational modification F. Xiang and is involved in many relevant biological processes and crucial to the Shimadzu Biotech, Pleasanton, CA, United States understanding of many diseases. In depth analysis of glycosylation sites is difficult, however, as glycopeptides exhibit a significant micro Glycosylation is a common post-translational modification to cell heterogeneity at glycosylation sites. In addition, ion suppression surface and extra cellular matrix proteins as well as to lipids. Unlike effects require selective methods for glycopeptide enrichment. Mass proteins and nucleic acids that are linear polymers of amino acids spectrometric analysis of glycopeptides is challenging because both and nucleotides respectively, with linkages at only one position, the peptide as well as the glycan moiety have to be elucidated for a full carbohydrates can adopt complex branched structures with individual structural understanding. We used Fetuin, Asialo-Fetuin and Alpha-1- monomeric units linked at one of several sites. A detailed analysis Acidglycoprotein to equally representing sialylated and non-sialylated of complex carbohydrate structures has been explored with mass glycosylic structures. In addition, monoclonal antibodies were analyzed spectrometric techniques, and still presents challenge for the analyst. as a dedicated example for pharmaceutical QC. Proteins were digested This presentation will describe a systematic approach of carbohydrates with trypsin and glycopeptides were enriched using a dedicated ZIC- and glycoconjugates analysis with MSn techniques. Oligosaccharides, HILIC glycocapture beads in combination with an optimized buffer cleaved from glycoproteins (by hydrazinolysis or enzymatically), were system (EMD Chemicals Inc.). The glycopeptides were analyzed using characterized using a hybrid MALDI Ion Trap / TOF mass spectrometer. ESI ion trap MS for glycoprofiling and MALDI-TOF/TOF-MS for in High mannose, biantennary and triantennary oligosaccharides were depth characterization of the glycopeptides. For database searches, analyzed using MS, NS2, MS3 and MS4 modes. Intact oligosaccharides an integrated software approach was used: protein searches of the were analyzed in MS mode using a cooling gas to prevent fragmentation. glycopeptide MS/MS spectra were performed for obtaining the amino Individual precursor ions were isolated in the trap, subjected to acid sequence of the glycopeptide, and searches in glycan databases fragmentation with Argon, to provide MS2 data. Product ions were based on the same glycopeptide MS/MS spectra were carried out selected for further fragmentation, which was achieved by increasing to complete the characterization of N-linked glycopeptides. In the the energy for collisionally induced dissociation. In MS mode, the singly current study, two important features of glycoprotein analysis are charged sodium adduct form of these molecules was detected, which shown: (1) The employed integrated software approach allowed the is typical of the analysis on conventional MALDI mass spectrometers. glycan identification in a similar way as peptide identification. The In MS2 mode, high mannose oligosaccharides readily lost the core important step of interactive result validation was facilitated by a suite N-acetylglucosamine residues, whilst MS3 and MS4 modes were used of dedicated data and result viewers. (2) Compared to MS analysis of to sequentially fragment the product ion corresponding to the residual native glycoprotein digests, the enriched samples allowed to detect branched mannose oligomer. In MS2 mode analysis of biantennary Poster Abstracts more glycopeptides and permitted the acquisition of higher quality and triantennary structures also fragmented losing disaccharide units, MS/MS spectra. For MALDI-TOF/TOF-MS analysis, linear positive ion such as the galactose - N - acetylglucosamine units that define each mode detection of precursor ions proved to be highly suitable for the antennary branch, or core fucosylated - N - acetylglucosamine units. analysis of even multi-sialylated glycopeptides. MS3 of selected MS2 products ions could be used to differentiate fragments generated from either the reducing or non-reducing ends. 205 Fragment Analysis of Carbohydrates Cross-ring cleavages were also observed during fragmentation in MS2, Following Capillary Electrophoresis and the relevant product ions could be used to differentiate branched structures by further fragmentation in MS3 mode. Many tandem mass T. Snyder-Leiby, D. Hulce, F. Li, X. Li, C.S. Liu spectrometric experiments have been revealing that oligosaccharides might have characteristic signal intensity profiles, depending on the SoftGenetics, LLC, State College, PA, United States glycosidic linkage and branching structures. In addition to the MSn capability of the platform, there is a library of observational mass spectra Depending on the macromolecule size and configuration, migration acquired from structurally defined oligosaccharides. The presentation rates through capillary electrophoresis vary greatly. Internal size will show the enhancement of carbohydrate identification by utilizing standards for capillary electrophoresis of the same macromolecule may comparison procedure of the signal intensity profiles of MSn spectra not be readily available. The Macromolecule Tool in GeneMarker® between the analyte and structurally defined oligosaccharides in the aids with analysis of macromolecule fragments without an internal library.

ABRF 2011 — Technologies to Enable Personalized Medicine • 89 207 A Unique Workflow for Glycoprotein 209 Optimization of Data-Dependent Characterization from Sample Parameters for LC-MS/MS Protein Preparation to MS/MS Spectral Identification Interpretation D. Johnson, R. Orlando 1, R. Lee2 J. Albanese Complex Carbohydrate Research Center, University 1AB SCIEX, Foster City, CA, United States; 2ProZyme, of Georgia, Athens, GA, United States Inc., Hayward, CA, United States A typical bottom-up protein identification workflow involves Protein glycosylation is a complex dynamic post-translational proteolytic digestion followed by identification of the resulting peptides modification, which is used by an organism to regulate a number of by LC-MS/MS using data-dependent acquisition (DDA). Recent important functions. Variable composition, linkage, branching and developments in chromatography, such as uHPLC and superficially anomericity of the constituent monosaccharides in combination with porous Fused-core particles, offer significantly improved peptide the general heterogeneity due to the indirect, non-template control resolutions. The narrow peak widths, often only several seconds, can of their biosynthesis are the basis of the structural complexity of permit a 15 minute LC run to have a similar peak capacity as a 60 glycoprotein glycans. This poster presents a complete workflow for minute run using a traditional HPLC approach. In theory these larger glycoprotein characterization comprising sample preparation for peak capacities should provide higher protein coverage and/or more glycan release, glycan separation using graphitized carbon separation protein identifications when incorporated into a proteomic workflow. coupled to MALDI spotting, automated MS and MS/MS analysis However, we initially observed a decrease in protein coverage when MALDI-TOF analysis and spectral interpretation with SimGlycan® implementing one of these faster chromatographic approaches, and Software. The workflow has been first optimized using commercially the more we optimized the LC separation the worse or MS results. available immunoglobulin G from human serum and a glycan library Careful data inspection revealed that the MS/MS spectra were of low of defined bisecting and none-bisecting glycan structures. The IgG quality because the automated MS/MS events were occurring on the sample contains both different protein sequence isoforms (IgG1-3) and tail of the chromatographic peaks. In other words, our new separation different glycan isoforms. The results demonstrated that, the RGSPS kit strategy was “too fast” for our DDA settings. These observations led us with InstantABTM labeling provides a fast and robust glycan release to develop a general strategy to optimize DDA settings. Method: Data and labeling method including sample clean up in less than 2 hours was acquired using an Agilent 1100 Capillary LC system, using Halo prior to mass spectrometric analysis. The combination of the LC MALDI Peptide ES-C18 columns online with ESI ion trap MS detection on a Workflow with the nanoflow hypercarb column enables better glycan Thermo-Fisher LTQ MS with a Michrom captive spray interface. Data separation and therefore more specificity for isomeric glycans structures was searched with Mascot, and ProteoIQ (Nusep, Athens GA) was used characterization. High energy CID MS/MS spectral comparison of the for data comparisons. Results/Conclusions: We have demonstrated that released and/or labeled glycans with the control samples provides a with DDA optimization these higher resolution separations provided greater number of MS/MS fragments than electrospray CID MS/MS by this new LC strategy do indeed lead to superior results in the analysis spectra or MALDI post source decay (PSD) spectra for more detailed of individual proteins, simple protein mixtures, and complex proteomic information. Next, this robust workflow will be tested and expanded samples. For instance, we are able to decrease or LC-MS/MS analysis on therapeutically interesting antibodies. Studying the heterogeneity (run to run) time from 100 minutes to 20 minutes without the loss of of glycosylation patterns obtained from cell culture influences can help protein coverage or protein identification. understand efficacy, binding affinity, specificity and pharmacokinetic properties. 210 Increasing Protein Identification Using Coupled Chip Based NanoLC 208 Analysis of Intact Proteins in Columns Biotechnology N. Hebert1, J.B. Young1, R. van Soest1, R. Freeman, D. DiFeo, H.J. Wirth, A. Gooley D.W. Neyer1, C.L. Hunter2 SGE Analytical Science, Austin, TX, United States 1Eksigent Technologies, Dublin, CA, United States; 2AB SCIEX, Foster City, CA, United States The demand for separation techniques for intact proteins is increasing with the introduction of a new generation of high resolution mass Poster Abstracts Nanoflow liquid chromatography coupled with nano-electrospray spectrometers which are able to measure the mass of small to medium (nanoLC-MS) is the method of choice for sensitive peptide and protein size proteins very accurately. Liquid chromatography is a valuable tool analysis for proteomics research. We recently reported on a new for separating these proteins prior to the MS analysis. Intact protein microfluidic platform (cHiPLC-nanoflex) for nanoLC-MS applications. chromatography is most commonly used in a top-down approach in In addition to delivering easy-to-use, dead-volume-free connections to proteomics or to determine expression levels during recombinant microfluidic devices, the platform’s cHiPLC columns provide excellent protein synthesis. The size of the protein molecule results in very column-to-column separation reproducibility. We have reported low diffusion coefficients and therefore slow mass transfer in and out previously(1) that while increasing only gradient time or column length of the pore system. A sufficiently large pore diameter is required to can improve peak capacity, using a longer column in combination minimize the effects of restricted pore diffusion. We show examples of with increasing gradient time is the most effective way of obtaining the separation of intact proteins on a column packed with 3µm C8 silica higher peak capacity. In this presentation we report on how the dead- with 1000Å pore size. The molecular weight of the protein examples volume free connections of the cHiPLC Nanoflex platform allow for reach from ribosomal proteins (<40kDa) over monoclonal antibodies the coupling of two 15 cm long chip columns for improved resolution. (~150kDa) to intact membrane proteins derived from mouse liver. The proposed set-up makes it easy to switch between single and dual column mode depending on application requirements. An additional

90 • ABRF 2011 — Technologies to Enable Personalized Medicine advantage of using two separate 15 cm columns rather than one 30 loading flow rate is decoupled from the gradient flow rate. Here we cm column is that sample matrix components that are not retained on investigate the role sample trap column injection has on analyte the first column (e.g. salts) can be directed to waste, instead of entering retention and overall chromatographic performance. Using a direct flow the nanospray source/mass spectrometer. We have investigated the nano-LC pump with the ability to deliver flow rates ranging from 50 nl/ effect of increased resolution through doubling column length on the min to 20 ul/min coupled to an autosampler and commercially available number of peptides/proteins identified in a digested cytosolic fraction peptide standards and protein digests, we evaluated the relationship of a human cell lysate using various gradient lengths. An ABSCIEX between flow rate, analyte concentration and analyte composition to TripleTOF 5600™ MS was used with an Information Dependent determine the effect on chromatographic performance. Acquisition method consisting of a TOF MS survey scan at >30 000 resolution, followed by 20 MS/MS in a second at >15 000 resolution. 214 Spatial Proteomics: A New LC- All data was processed using ProteinPilot™ Software 4.0 with integrated MS/MS Tissue Imaging Workflow false discovery rate (FDR) analysis. 1 Remco van Soest*, David W. Providing Protein Identities and Their Neyer; Jia Eng Siow, and Phil Paul, Eksigent Technologies, Dublin, CA; poster ASMS 2008: “Improving resolution in nanoLC separations for Distribution in Tissue proteomics using ultra high pressures”. C. Lübbert, M. Schürenberg, M.Becker, R. Paape, D. Suckau 211 Using 200 µm ID cHiPLC Columns for Increased Sample Throughput in Bruker Daltonics, Bremen, Germany Peptide Quantitation MALDI-Imaging of proteins in tissue sections has established itself as a powerful new approach to biomarker discovery and histopathological R. van Soest1, N. Hebert1, D.W. Neyer1, J.B. Young2 research in recent years. However, the lack of direct identification strategies continues to be an obstacle preventing its broader use in 1Eksigent Technologies, Dublin, CA, USA; 2AB Proteomics studies. Initial studies that utilized in situ digestion followed SCIEX, Concord, ON, Canada by MALDI-MS/MS analysis typically provided 5-50 peptide IDs of only 1-5 high abundant proteins. Here we introduce a novel proteomics Nanoflow liquid chromatography coupled with nano-electrospray technology that combines the spatial information with the routine (nanoLC-MS) is the method of choice for sensitive peptide and protein identification of proteins from tissue sections. Highly resolved protein analysis. We recently reported on a new microfluidic platform (cHiPLC- digests are generated by applying trypsin onto two subsequent tissue nanoflex) for nanoLC-MS applications. In addition to delivering sections by supersonic nebulization. One of the sections is then analyzed easy-to-use, dead-volume-free connections to microfluidic devices, by MALDI imaging mass spectrometry at up to 50 µm spatial resolution the platform’s cHiPLC columns provide extreme column-to-column yielding a list of 200-2000 peptide signals per image. Peptides are separation reproducibility. The column ID of choice for best sensitivity extracted from the other sections entire surface and submitted to is 75 µm, using a flow rate of 200 nl/min. While providing excellent routine LC-MS/MS analysis, in our case using MALDI-TOF/TOF. The sensitivity, the low flow rate reduces sample throughput because identified peptide list is then filtered by the peaklist from the image. of gradient delay in the nanoLC system itself, the autosampler valve All matching peaks in the image can then be assigned to a protein and and sample loop and the connecting tubing between injection valve, the co-localization of 2 or more tryptic peptides confirm their protein column and nanospray source. One way to address this is to use a larger association. We analyzed rat brain and testis/epididymis using the new ID column at proportionally higher flow rate. While this will reduce Spatial Proteomics approach typically yielding peptide distributions at sensitivity when the injection volume is kept equal, delay times can be the 50-100 µm level. In brain, more than 100 peptides were identified greatly reduced. In this presentation we will show data demonstrating and more than 20 proteins localized without the need for MS/MS the throughput increase when using 200 µm vs. 75 µm ID cHiPLC analysis directly from the tissue. The intensity, co-localization of 2 or columns while reporting as well on the sensitivity trade-off, and the more peptides and the degeneracy of molecular weight of peptide- effect thereof on peptide quantitation. to-protein mapping were used as primary validation tools beyond significant mascot scores from peptide identification. As an extension 212 Flow Rate, Concentration and of the established top-down imaging strategy, this bottom-up Spatial Poster Abstracts Saturation: Investigating the Proteomics approach may facilitate the identification and simultaneous Fundamentals of Sample-Trapped localization of a much greater number proteins than it was previously Column Injection possible.

C. Marshall-Waggett, H. Svobodova, A. Berg, 215 Increasing the Sample Loading G. Valaskovic Capacity for Peptide Analysis by LC- MS/MS Using 150 µm ID Packed Tip New Objective, Inc., Woburn, MA, United States Columns Sample trapped column injection is an injection strategy commonly , H. Svobodova, C. Marshall-Waggett, employed in nanobore LC/MS based analysis of complex peptide A. Berg G. Valaskovic mixtures. The approach of sample trapped column injection provides several inherent advantages. By effectively desalting and concentrating New Objective, Inc., Woburn, MA, United States samples on-line, sample traps improve analytical column longevity and throughput. Additionally, the ability to load samples onto a trap A predominant workflow for qualitative proteomics has been “GeLC- column at a much higher flow rate than is feasible for a typical 75um MS,” a combination of 1- (or 2-D) gel electrophoresis with reverse-phase ID nanobore analytical column is a particularly attractive feature of this nanoflow liquid chromatography mass spectrometry (nLC-MS/MS). The approach. Duty cycle can be significantly improved when the sample limited protein quantity isolated from a single gel band coupled with

ABRF 2011 — Technologies to Enable Personalized Medicine • 91 column loading capacity maximums necessitate the use of 75 µm ID 217 Detection of p53 Phosphorylation packed columns for optimal sensitivity. However, limitations on sample and Oligomerization Using Proximity injection volume, gradient and flow characteristics, and excessive delay volume hinder throughput. Novel methods for fractionating complex Ligation Assay biological samples with higher loading capacities and more efficient S.M. Chen, K. Huwiler, R. Bruinsma, B. Marks, recovery, such as novel solution phase tube-gel fractionation and B. Schweitzer, T. Settineri, M. Shannon, D. Ruff others, demand a column format which maximizes on the extended dynamic range of these emerging techniques. Packed tip columns with Applied Biosystems, part of Life Technologies, a larger ID (150 µm to 200 µm) facilitate higher sample loading capacity Foster City, CA, United States and enable higher flow rates for improved cycle time while maintaining the optimal sensitivity realized in the nanobore packed tip columm The tumor suppressor protein p53 is one of the most studied format. Using peptide standards, single protein digests and whole yeast architects of transcription control in cells. Recent reports detail digests improvements in cycle time and sample loading capacity using crucial tertiary structural requirements for p53 influence in 150 µm ID packed tip columns are demonstrated. transcription regulation complexes. These findings indicate the tetrameric assembly of p53 protein is essential for stabilization 216 Comparing Global and Targeted Lipid and localization of the protein. This event is critical for DNA binding and Fatty Acid Shotgun Profiling of and transcriptional activation. Post-translational modifications (PTM) Brain Tissue Extracts by NanoESI- have been linked to activation of p53 function in response to genotoxic stress. In particular, phosphorylation of the p53 Ser-15 is a critical event Infusion in the genotoxic pathway cascade. We investigate the interconnection 1 2 2 2 between this PTM and p53 higher order structure formation. We J. Albanese , B. Simons , G. Impey , E. Duchoslav , K. Koisten3, K. Ekroos3 employ a novel antibody binding assay scheme to elucidate details of the multimeric status of p53 in both recombinant p53 protein 1AB SCIEX, Foster City, CA, United States; 2AB experiments and in cells undergoing genotoxic responses. This SCIEX, Concord, ON, Canada; 3Zora Biosciences, approach utilizes the Proximity Ligation Assay (PLA) to simultaneously Espoo, Finland query the role of Ser-15 phosphorylation and the multimeric state of p53. By use of a phospho-Ser-15 specific monoclonal antibody PLA The growth in lipidomics research is uncovering a need for complete probe, we quantify proximity of two individual phospho-Ser-15 motifs and comprehensive workflows for identifying and quantifying lipid in experimental samples. Our results demonstrate that phospho-Ser15 species from biological extracts. In choosing analytical methods promotes multimerization of p53 protein both in vitro and in cells for lipidomics, different yet complementary mass spectrometry responding to genotoxic stress. Further studies using PLA to explore the approaches can provide a more complete and comprehensive data p53 protein-protein interactome should reveal a more complete view set leading to a detailed characterization of lipid molecular species of the downstream interplay facilitated by the tetrameric conformation. from complex extracts. A preliminary strategy carried out as global “shotgun” tandem mass spectrometry by direct infusion electrospray 218 Investigating the Robustness of a New ionization or LC-MS/MS analysis, uses information dependant MS/MS Microfluidic Device scanning in both polarities for unbiased lipid profiling. The second approach involves multiple lipid-class-specific precursor ion and C. Hughes, J. Langridge, T. McKenna neutral loss scanning whose resulting spectra can be used directly to identify and characterize lipids and fatty acids in tissue-derived lipid Waters Corporation MS Technologies Centre, extracts. The multiple precursor ion scanning (MPIS) methods have Manchester, United Kingdom been published and recently reviewed by Ekroos et al, describing the advantages of targeted MPIS techniques for generating comprehensive Successful assembly of the components used in nanoscale LC requires lipid arrays from small sample volumes. Fully characterizing these lipid some level of expertise in order produce good chromatography components by high quality MS/MS for fatty acid chain length and with minimal band broadening.Microfluidic devices, where double bond positioning is a critical step for understanding their chromatographic separation is performed on a chip mounted close to biological implications in cell signaling and lipid-initiated disease a MS inlet, enable significant reduction in dead volumes and enable progression. Taking advantage of the speed, selectivity, and sensitivity optimal chromatography to be achieved by the novice user. Here, we of hybrid triple quadrupole technology, whole lipid extracts from demonstrate the robustness and reproducibility of a Waters Nanotile

Poster Abstracts rat brain tissue can be analyzed by direct nanoESI infusion for in coupled to a new Time of Flight MS. We made 35 injections of an E. depth glycerophospholipid profiling - achieving both qualitative and Coli tryptic digest, containing 50fmol spikes of four proteins, onto a quantitative data (with the use of synthetic lipid internal standards) in nanoAcquity UPLC system coupled to a Trizaic source.The Trizaic very fast analysis times. Lipid species identification and quantitation consists of a ceramic tile containing a trapping column, analytical is carried out using LipidViewTM Software enabling post acquisition column and emitter. Trapping flow rate was set to 8uL/min for 1.8mins processing of precursor ion, neutral loss, MRM, and MS/MS data via lipid and the analytical reversed phase separation was performed at a flow database searching and accurate peak integration. We present robust rate of 450nL/min, changing the acetonitrile composition from 3 to targeted and global workflows for the identification and quantitation of 40% over 90minutes, with 30minute re-equilibration. Emitter eluent glycerphospholipids in total lipid extracts from rat brain tissue. was analysed using a ToF MS operating in MSe mode. In this mode, the MS performed alternate low and elevated energy scans, the low energy providing precursor ion information and the elevated energy providing fragment ion information. Data was processed using ProteinLynx Global Server where the ion information was collated using retention time information in order to associate precursors and fragments. Data was then searched using a non-redundant database and the internal spike

92 • ABRF 2011 — Technologies to Enable Personalized Medicine allowed absolute quantitation. Comparing chromatograms from various 220 Identification of Various Pink-Red stages of the 70-hour experiment shows excellent reproducibility. The Pigments Formed by Reacting Various average number of E. Coli proteins identified was 389±17, in which 325 replicated in 20 out of the 35 injections. Moreover, the absolute Amino Acids with Onion (allium cepa quantitation measurements using the ADH spike for identified proteins L.) Thiosulfinates Using HPLC with with >3 peptides is 285±7ng and the quantitation measurement for one Diode Array Detector and Tandem of the other protein spikes, Enolase, shows a CV of 7%. Mass Spectrometry 1, E. Lee2, K. Yoo2, D. Russell1, B. Patil2 219 Effective Semi-Automated Extraction Y. Rezenom of Intact Mitochondria from Solid 1Laboratory for Biological Mass Spectrometry, Tissues Using Gentle Mechanical Department of Chemistry, Texas A&M University, Homogenization and Pressure Cycling College Station, TX, United States; 2Vegetable Technology & Fruit Improvement Center, Department of Horticultural Science, Texas A&M University, 1 1 2 V. S. Gross , G. Carlson , E. Freeman , College Station, TX, United States A.R. Ivanov2, A. Lazarev1

1 During the processing of onion, pink-red colored pigments are often Pressure BioSciences, Inc., South Easton, MA, formed. The process is believed to be a multistep process including 2 United States; HSPH Proteomics Resource, Harvard enzymatic and non enzymatic reactions. In order to investigate this School of Public Health, Boston, MA, United States process, we developed a reaction system, where pink-red pigments (‘pinking’) can be formed by reacting amino acids with onion Impaired mitochondrial function has been linked to many diseases, thiosulfinate formed by reacting an isolated garlic alliinase and (+)-S-1- such as stroke, heart disease, cancer, Type II diabetes and Parkinson’s propenyl-L-cysteine sulfoxide (1-PeCSO) in the natural onion juice. The disease. Mitochondria-enriched preparations are needed for unknown pink-red pigments formed during this process were separated proteomic and metabolomic studies that may provide crucial insights and detected using a high-performance liquid chromatography (HPLC) into tissue-specific mitochondrial function and dysfunction, and and a diode array detector (DAD) at 515 nm. Fractions collected from answer fundamental questions of cell energetic and oxidative stress. this separation were further analyzed using liquid chromatography Mitochondria extractions from whole tissue samples are typically (LC) and tandem mass spectrometry. Similar head group structure, two performed using Potter-Elvehjem homogenizers or similar labor- 3,4-dimethyl pyrrolyl rings that were cross linked by allyl group, was intensive manual disruption methods that require extensive operator determined for all conjugate-pigments formed from different amino experience, and often result in damage to fragile organelles and high acids based on the accurate and tandem mass spectrometry. However, sample-to-sample variability. Here we describe a semi-automated the tail group attached to the N-terminal of pyrrole ring differed for method that uses a novel gentle mechanical homogenizer (The PCT each pink-red pigment depending on the amino acid used. In addition, Shredder) and hydrostatic pressure to release intact mitochondria in most cases more than one pink compound were identified for the from fresh rat tissues with minimal hands-on time. Pressure Cycling same amino acid used. We presumed that the complexity of the pink- Technology (PCT)-based tissue homogenization is conducted under red pigments was due to the involvement of 21 natural amino acids and controlled thermodynamic conditions (time, temperature and pressure) other derivatives of the products. Finally, we suggest that the pinking leading to more reproducible results. The quality of mitochondria process in crushed onion is very similar to the greening process in preparations was characterized by electron microscopy, 2D PAGE and crushed garlic, emphasizing that thiosulfinates from flavor precursors respiration assays. Our data demonstrate that mitochondria extracted and free amino acids are absolutely necessary during the discoloration. by the PCT sample preparation system (PCT-SPS) are intact, functional, and exhibit a protein pattern comparable to control samples isolated using a conventional Potter-Elvehjem homogenizer. The resulting 221 Improvements in Data-Dependent mitochondria-enriched samples were also subjected to trypsin Acquisition for Enhanced Protein digestion followed by nanoLC-MS/MS analysis on an LTQ-Orbitrap. Identification Proteomic and pathway profiles of mitochondria samples prepared Poster Abstracts , N. Kitagawa, W. Tang, J. Roark, using the novel extraction technique were compared to those extracted C. Miller using a conventional manual method to demonstrate the purity of J. Satulovsky, P. Perkins mitochondrial preparations extracted using the novel PCT-SPS method. Agilent Technologies, Palo Alto, CA, United States

Comprehensive proteome analysis can be very challenging due to complexity and range of protein concentrations. Techniques such as 2D LC, pI-based fractionation and gel electrophoresis are typically employed to increase separation efficiency as a strategy for obtaining more peptide MS/MS spectra and thus increasing the number of proteins identified. In data-dependent mode, efficient and comprehensive protein identification depends on several conditions during the acquisition, among them: the number of precursors examined per unit time, selection of the most promising precursors for fragmentation, and application of the appropriate fragmentation conditions to yield the highest quality product ion spectrum during acquisition. This work describes changes to the precursor selection and fragmentation steps to select the precursors most likely to produce good quality peptide MS/MS spectra, and to increase the quality of those spectra.

ABRF 2011 — Technologies to Enable Personalized Medicine • 93 222 UHR-Q-TOF Analysis Can Address information. This data enables a sum formula generation for known Common Challenges in Targeted and and unknown target compounds.Additionally, optionally acquired MS/ MS data can extend the capabilities for structural elucidation. Mass Untargeted Metabolomics accuracy, resolution and isotopic fidelity are independent of the TOF- 1 1 2 2 MS acquisition rate. Therefore, these instruments can be coupled to gas A. Barsch , G. Zurek , D. Krug , R. Müller chromatography, which typically delivers narrow peak width requiring 1Bruker Daltonics, Bremen, Germany; 2Universität fast MS scan speeds. Corynebactrium glutamicum, a gram positive, non- des Saarlandes, Saarbrücken, Germany toxic bacterium, is used in the industrial production of amino acids like lysine and glutamate. C. glutamicum can be grown on different carbon Here, we present an ESI-UHR-Q-TOF based analysis of myxobacterial sources. Glucose is metabolised via glycolysis and the tricarboxylic acid secondary metabolites, which permits to solve several challenges (TCA) cycle, whereas propionate is catabolised through the methylcitric frequently encountered in metabolite profiling studies. Myxobacteria acid pathway. The prpD2 gene encodes a 2-methylcitrate dehydratase are promising producers of natural products exhibiting potent which is involved in the degradation of propionate. Metabolic profiling biological activities, and several myxobacterial metabolites are currently of Corynebacterium glutamicum delta-prpD2 extracts of cells grown under investigation as potential leads for novel drugs. However, the on glucose or glucose and propionate analyzed by GC-APCI-TOF-MS myxobacteria are also a striking example for the divergence between revealed several compounds elevated in cells grown on propionate. the genetic capacity for the production of secondary metabolites Identification of 2-methylcitric acid and alanine using accurate mass and the number of compounds that could be characterised to date. and isotopic pattern information in MS and MS/MS spectra provided a Wildtype and mutant strains were analyzed concerning the production proof of concept for the identification of target compounds using high patterns of known metabolites and with regard to the discovery resolution MS technology. of new metabolites. Sample throughput: Since mass accuracy and resolution of TOF instruments are independent of the acquisition rate, 224 Biomarker Discovery Using New they are perfectly suited for a coupling to UHPLC separations. These Metabolomics Software for hyphenations enable a reduction of analysis time in combination with a high chromatographic resolution and therefore permit an increased Automated Processing of High sample throughput. The UHR-TOF analysis revealed that an acquisition Resolution LC-MS Data rate of up to 20Hz did not compromise the achieved mass accuracy or 1, S. Hnatyshyn2; M. Reily2; P. Shipkova2; resolution. Targeted and untargeted metabolite profiling: Acquisition of D. Peake T. McClure1; M. Sanders1 full scan accurate mass spectra enable the targeted screening for known compounds e.g. from the class of DKxanthenes based on very selective 1Thermo Fisher Scientific, San Jose, CA, United high resolution EIC (hrEIC) traces with small mass windows of 1.0 - 0.5 States; 2Bristol Myers Squibb, Princeton, NJ, United mDa. A comparison of several datasets following a “comprehensive States feature extraction” combined with a statistical analysis permits an untargeted discovery of novel biomarkers using the same data files Robust biomarkers of target engagement and efficacy are required in as for the targeted analysis. Identification: Even a mass accuracy of 0.1 different stages of drug discovery. Liquid chromatography coupled ppm is not sufficient for an unambiguous formula identification for m/z to high resolution mass spectrometry provides sensitivity, accuracy values above 500. A combination of accurate mass data and isotopic and wide dynamic range required for identification of endogenous pattern information in MS and MS/MS spectra can extend this m/z range metabolites in biological matrices. LCMS is widely-used tool for for reliable formula suggestions. Examples for novel metabolites from biomarker identification and validation. Typical high resolution LCMS Myxobacteria will be shown. profiles from biological samples may contain greater than a million mass spectral peaks corresponding to several thousand endogenous 223 Metabolic Profiling of a metabolites. Reduction of the total number of peaks, component Corynebacterium Glutamicum Delta- identification and statistical comparison across sample groups remains prpD2 by GC-APCI High Resolution to be a difficult and time consuming challenge. Blood samples fromfour Q-TOF Analysis groups of rats (male vs. female, fully satiated and food deprived) were analyzed using high resolution accurate mass (HRAM) LCMS. A. Barsch1, G. Zurek1, M. Persike2, J. Plassmeier2, All samples were separated using a 15 minute reversed-phase C18 K. Niehaus2 LC gradient and analyzed in both positive and negative ion modes. Data was acquired using 15K resolution and 5ppm mass measurement 1 Poster Abstracts Bruker Daltonik GmbH, Bremen, Germany; accuracy. The entire data set was analyzed using software developed 2Centrum für Biotechnologie, Universität Bielefeld, in collaboration between Bristol Meyers Squibb and Thermo Fisher Germany Scientific to determine the metabolic effects of food deprivation on rats. Metabolomic LC-MS data files are extraordinarily complex Metabolomics studies based on Gas chromatography -Mass and appropriate reduction of the number of spectral peaks via spectrometry (GC-MS) are well established and typically employ identification of related peaks and background removal is essential. A electron impact (EI) ionisation. Target compounds of interest can single component such as hippuric acid generates more than 20 related be identified by comparison to commercial or public databases. peaks including isotopic clusters, adducts and dimers. Plasma and Unfortunately, many possible biomarkers detected in metabolic urine may contain 500-1500 unique quantifiable metabolites. Noise profiling experiments cannot be identified due to the lack of reference filtering approaches including blank subtraction were used to reduce spectra for a majority of biologically relevant compounds. Therefore, the number of irrelevant peaks. By grouping related signals such as many possible biomarkers remain “unknowns” up till now. Hyphenating isotopic peaks and alkali adducts, data processing was greatly simplified gas chromatography with high resolution TOF-MS technology with soft by reducing the total number of components by 10-fold. The software atmospheric pressure ionisation (APCI) can preserve the molecular processes 48 samples inunder 60minutes. Principle Component ion information and delivers accurate mass and isotopic pattern Analysis showed substantial differences in endogenous metabolites

94 • ABRF 2011 — Technologies to Enable Personalized Medicine levels between the animal groups. Annotation of components was still widely used substrates for the study of protease activity. The accomplished via searching the ChemSpider database. Tentative preparation of peptide-pNA however, presents several technical assignments made using accurate mass need further verification by challenges. Firstly, the amino group of pNA has a low nucleophilic comparison with the retention time of authentic standards. property due to the electron-withdrawing effect of the nitro group. Secondly, the poor solubility of a p-nitroanilide intermediate and lastly, 225 On-Line Electrochemistry/MS - A coupling in solution phase by DCC, azide or active ester, commonly Powerful Tool for Rapid Prediction of used techniques are not effective. Here we report the development Phase I and II Drug Metabolism of two novel supports for facile solid phase peptide syntheses, namely, Wang-resin and Rink Amide-resin conjugated with a pNA J. Powers1, J. Purkerson1, A. Kraj2, M. Eysberg2, analog, 5-amino-2-nitrobenzoic acid (Anb5,2). Based on a paper by J.P. Chervet2 Hojo, et al. in which they described the introduction of Anb5,2 to a p-methylbenzhydrylamine (MB) resin; we successfully coupled Anb5,2 1Antec, Palm Bay, FL, United States; 2Antec, to either Wang or Rink Amide resin using the TBTU method in the Zoeterwoude, The Netherlands presence of p-dimethylaminopyridine (DMAP). Anb5,2-Wang or Rink Rmide resin is then coupled to a Fluorenylmethyloxycarbonyl (Fmoc) The use of Electrochemsiry is a complementary approach to traditional containing amino acid. Peptide synthesis can subsequently proceed methods such as in vivo (human, rodent) or in vitro (liver microsomes) using Fmoc synthesis strategy. The use of this pNA analog containing metabolism studies, and delivers the oxidative metabolic fingerprint of resins circumvents the tehcnical difficulties stated above. These resins a (drug) molecule in a very short time. The acquired mass spectra are also greatly facilitates the synthesis of peptide-pNA-like chromogenic presented in simple 2 dimensional or more illustrative 3 dimensional substrates for protease research. plots, so-called MS voltammograms. A MS voltammogram visualizes the ion abundance versus m/z as a function of applied potential to the **227 Validation of an Ion Mobility electrochemical cell. With a MS voltammogram the optimal potential Hydrogen/Deuterium Exchange Mass can be determined for electrochemical generation of the desired metabolite for further research, e.g., a phase II metabolism study (i.e. Spectrometry System adduct formation). It is a quick method to identify reactive pathways K. Fadgen2, T. Wales1, M. Stapels2, M. Eggertson1, of the compound of interest. Additionally, electrochemistry allows to J. Engen2 trace the reactive metabolite conjugates with targets (e.g., proteins, glutathione) without matrix interactions in contrary to traditional 1Northeastern University, Boston, MA, United methods. A dedicated software program has been developed to States; 2Waters Corporation, Milford, MA, United automate and simplify the MS voltammogram acquisition. The program States controls the syringe pump, the potentiostat and triggers the acquisition of mass spectra at the designated cell potentials. The total acquisition Hydrogen/deuterium exchange mass spectrometry (HXMS) has proven time needed for recording of a full MS voltammogram can be as short to be a useful analytical method for the study of protein dynamics and as 5 minutes. Amodiaquine an anti malaria agent was chosen as one of changes to protein conformation. Rapid chromatographic separations the model drugs to investigate oxidative metabolism using the on-line at 0°C must be utilized to preserve the deuterium label during LC/ EC/MS system with automated MS voltammogram acquisition The easy MS analysis. Unfortunately, fast, low temperature LC separations are and fast Electrochemical conversion of Amodiaquine into its 4 major not very efficient and can cause spectral overlap for large proteins. phase I metabolites will be presented. In a second step Glutathione The addition of a gas phase, ion mobility separation (IMS) into the (GSH) is added to the electrochemically generated metabolites to form HXMS workflow inserts an orthogonal separation that occurs on the the appropriate GSH-metabolite adducts, mimicking phase II reactions. millisecond timescale after the LC step without causing any detrimental All known adducts were successfully formed and identified with MS. impact on analysis of deuterium levels. An improved MS system has Additionally, MS voltammograms of other drugs and xenobiotics (e.g., recently been developed that is capable of higher resolution gas-phase acetaminophen, amiodarone, irinotecan) are presented. The data mobility separations. The improved MS platform was combined with demonstrate that hyphenation of electrochemistry with electrospray a fully automated ultra performance liquid chromatography (UPLC) mass spectrometry provides a versatile and user-friendly platform system developed for HXMS. To evaluate the effect of the new MS Poster Abstracts for rapid and cost efficient screening of target compounds (drugs, platform on deuterium recovery, HXMS experiments were performed xenobiotics, etc.) in phase I and phase II metabolomics studies. using glycogen phosphorylase B as a model protein. Replicate studies of deuterium labeling reactions ranging from 10 seconds to 100 226 Facile Solid-Phase Synthesis of minutes were evaluated. No significant deviation in deuterium levels Peptide-p-Nitroanilide (pNA) Analog was observed when using either mobility (HDMSE) or non-mobility Containing Conjugates Using a Novel (MSE) separations and the standard deviation of deuterium uptake Wang or Rink Amide Resin for replicate analyses, for HDMSE and MSE separations were in good agreement with one another. Using the newly developed protocol, an X. Wang1, C. Po2, J. He2, A. Hong2 HXMS study of a monoclonal antibody was performed to compare deuterium recovery using a 6 or 12 minute chromatographic separation. 1AnaSpec, Fremont, CA, United States; 2Eurogentec Even though chromatographic resolution was reduced with the faster Group, Fremont, CA, United States gradient, HDMSE allowed several overlapping peptides to easily be interrogated. In addition, the shorter analysis time improved deuterium Proteases play a key role in literally all biological processes, and are of recovery for the sample. The application of this additional level of great interest, especially to the pharmaceutical industry. Colorimetric separation will be essential in future studies of very large proteins in based Peptide-p-Nitroanilide conjugates (peptide-pNAs), with which chromatographic efficiency is expected to be suboptimal. absorbance at approximately 408 nm, have historically been and are

ABRF 2011 — Technologies to Enable Personalized Medicine • 95 228 Differential Expression of Proteins in focusing patterns between the two conditions is clearly visualized with Lung Cancer Using Difference in Gel the Bioanalyzer protein assay. The combination of OFFGEL and lab-on- chip protein analysis allows separation and identification of isoforms Electrophoresis (DIGE) based on pI and molecular weight. Protein isoforms differing only by 1 2 2 a few kDa in apparent molecular weight were successfully separated F. Masri , P.Beckett , K.S. Aulak and enriched by OFFGEL electrophoresis for further downstream 1University of Kalamoon, Faculty of Pharmacy, analysis by LC/MS-MS. The combination of OFFGEL, Bioanalyzer, Syria; 2Amersham Biosciences, Cleveland Clinic and mass spectrometry is thus an efficient combination for detailed Foundation, Cleveland, OH, United States characterization of recombinant proteins such as mAb’s. Background: Lung cancer remains the leading cause of cancer-related 230 Top-Down Mass Spectrometric mortality worldwide. Early detection of lung cancer is problematic due Sequence Analysis of 116 kDa E. coli to the lack of a marker with high diagnosis sensitivity and specificity. ß-galactosidase The purpose of this study was to develop techniques to identify the differential expression protein profiles between tumor and tumor- A. Resemann, R. Paape, L. Vorwerg, D. Suckau free of lung cancer tissues. Methods: 2-dimensional differential in- gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ Bruker Daltonik GmbH, Bremen, Germany ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to analyze four samples of lung cancer tissue (3 replicates each). Most Top-Down mass spectrometric studies performed so far focused Results: From optimized 2DE image, A total of 2561 spots were detected on proteins in the 20-50 kDa range, even monoclonal antibodies are and 427 spots of these were differentially expressed (p<0.01). 40 spots comprised of protein chains smaller than 50 kDa. However, the interest were subjected to mass spectrometry including overexpressed proteins in large protein top-down analysis increases as many therapeutic and underexpressed proteins. Some proteins were the products of protein drugs have larger molecular weights. In this work we evaluated oncogenes and others were involved in the regulation of cell cycle the capability of top-down mass spectrometry to provide N- and and signal transduction such as Annexin III, Selenium binding protein, C-terminal sequences from large proteins such as antibodies and the glyceraldehydes-3-phosphate dehydrogenase, cathepsin D and 116 kDa single chain protein ß-glycosidase from E. coli (1023 amino catalase. Conclusion: These data are valuable for mass identification acid residues; ß-Gal). Intact protein samples at concentrations of 1 of differentially expressed proteins involved in lung carcinogenesis, mg to 5 mg/ml were directly mixed with matrix solution without prior establishing human lung cancer proteome database and screening reduction and alkylation or separation of the different chains: for intact molecular marker to further study human lung cancer. Using the DIGE mass detection with sDHB or sinapinic acid, for ISD with sDHB or DAN. approach, we were able to find many proteins that were expressed The samples were mixed with matrix solution and dried at ambient air differently due to the disease state (tumor and tumor-free). on the MALDI sample plate. In a second experiment, the protein was analyzed after top-down protein-LC purification to simultaneously analyze by-products of the commercially available protein. A Dionex 229 Separation and Detection of PepSwift monolithic capillary column was used for LC-fractionation. Protein Charge Isoforms with a The eluate was deposited onto the MALDI sample plate and DAN Combination of OFFGEL- and Lab- matrix or sDHB was added for Top-Down measurements via in-source on-Chip Electrophoresis and Mass decay (ISD) and intact molecular weight determinations, respectively. Spectrometry The ISD spectrum of ß-Gal provided a straight sequence readout of 60 N-terminal and 50 C-terminal residues plus it permitted the assignment 2 1 2 2 R. Salowsky , S. Babu CV , C. Wenz , A. Ruefer of the N-term Met residue being properly removed. The LC-MALDI 1Agilent Technologies, Bangalore, India; 2Agilent analysis provided a number of side products that are currently Technologies, Waldbronn, Germany investigated.The ISD spectrum of ß-Gal was analyzed using a new QC software tool against a set of defined protein variants, which permitted Separation and analysis of charge heterogeneity in recombinant even the automatic assignment of the correct protein N- and C-terminal protein or monoclonal antibody (mAb) production is a prime quality structures. This work highlights the capability of MALDI-ISD based control step in the biopharmaceutical industry.This step is often carried analysis even for large proteins on a routine basis. out by a combination of separation techniques followed by mass spectrometric detection. Two dimensional gel electrophoresis (2D-GE) **231 The Poor-Man’s Fractionation of

Poster Abstracts is unrivalled in terms of resolution but is a tedious procedure. Here Cellular and Organellar Proteomes we present a combination of two easy methods that separate proteins at the Stages of Lysis and Proteolytic in analogy to 2D-GE according to pI and molecular weight (kDa) with Digestion, Eliminating the Use of high reproducibility for the analysis of mAb’s and recombinant proteins Analytical Fractionation Techniques followed by mass spectrometry (MS) analysis. For the 1st dimension, OFFGEL electrophoresis was used. This method takes advantage of the E. Freeman, A.R. Ivanov impressive resolving power of immobilized pH gradient (IPG) gels but in contrast to conventional IEF delivers samples in liquid-phase. Fractions Harvard School of Public Health, Cambridge, MA, with charged isoforms in solution can directly be analyzed by MS. For United States the 2nd dimension, a highly sensitive microfluidic on-chip protein sizing method was employed. This method allows protein separation from 5 to Current methods and technologies to analyze complex proteomes 250 kDa and offers a sensitivity equivalent or better than silver staining rely on the use of costly instrumentation and separation tools on top and a linear dynamic range across four orders of magnitude. The charge of labor-intensive methods in order to more thoroughly characterize heterogeneity in a mAb sample was evaluated under native conditions the representative proteins. Common methods to characterize and with addition of a mild detergent (Tween-20). The difference in proteomic specimens include geLC-MS, 2D-gel electrophoresis,

96 • ABRF 2011 — Technologies to Enable Personalized Medicine MuDPIT, and off-gel electrophoresis (OGE) ahead of LC-MS/MS. response proteins obtained from the mouth swab, we suspected We have evaluated an alternative method that does not require that the pulmonary infection was caused by Mycobacterium. PCR additional sample fractionation on additional equipment, meanwhile assay followed by direct sequencing of the PCR products confirmed minimizing alterations introduced by analytical techniques. For both the presence of Mycobacterium sp. in the mouth swab. Until now, whole human cells and isolated mitochondria, the proposed method immunoassays have been the only way to detect an active immune involved exposure of the pelleted cells or organelles to sequence- response and infer infection in historical samples, but these were grade trypsin in order to obtain peptides and proteins cleaved from plagued by low specificity and sensitivity. However, we demonstrate the cell and organelle surfaces. The “stripped” cells and mitochondria here the feasibility of incorporating global protein profiling in the were also lysed and their contents digested. Additionally, total lysates diagnosis of infection from archeological samples. Protein signatures of cells and mitochondria not exposed to trypsin ahead of lysis were obtained from these samples could be extremely useful in determining generated and tryptically digested. The obtained proteomic profiling the status of infection while genomic-based assays can be used to results were compared to those acquired using commonly employed detect the identity of the pathogen. fractionation techniques (OGE and SDS-PAGE). Resulting digests from all protocols were analyzed using LC-MS/MS, and all were analyzed 233 A New Technology for Highly for the proportion of unique and differentially enriched proteins Efficient and Reproducible Albumin achieved with each. Our results showed that the inexpensive and Depletion from Whole Serum simplistic fractionation method allowed for similar or superior depth of proteomic profiling as well as a substantial increase in the number C. Bolcato, H. Anderson, M. Maust, G. Boyce, of unique identifications of membrane-bound and extracellular matrix M.J. Powell related proteins. Additionally, the method provides information about spatial localization and differential abundance of proteome subsets Protea Biosciences, Morgantown, WV, United States in the cell or organelle architectural compartments. These results suggest that a very straightforward approach for the preparation of One of the major problems faced in the analysis of the human serum cellular and organellar specimens prior to proteomic characterization proteome is the broad dynamic range of its protein constituents. can be reproducibly and reliably utilized by biological and medical High abundance proteins, such as human serum albumin (HSA) and laboratories that have no previous analytical biochemistry expertise. gamma-immunoglobulin (IgG), which together comprise 75% of total serum protein content, inhibit the analysis of lower abundant proteins 232 Detecting Immune System Response of interest. Affinity-based depletion strategies are often employed to selectively remove these high abundance proteins in order to enrich Proteins in a 500 Year-old Inca the lower abundant proteins and facilitate their analysis. Traditionally, Mummy antibody-based schemes are used to produce affinity-binding ligands 6, A. Corthals1, L. Davalos2, for HSA and IgG. Variability in the production of these antibodies leads A. Koller to a wide variety of specificities and selectivities. Thus, the efficiencies D.W. Martin3,4, R. Rieger3, E.I. Chen3,5 for antibody-based depletion strategies for the selective removal 1City University of New York, John Jay College of of high abundance proteins in human serum can vary broadly, from Criminal Justice, Department of Sciences, New York, 70 to 95%, and can dramatically affect the reproducibility of human NY, United States; 2Department of Ecology and serum proteome studies. Here, a new approach to affinity-based serum Evolution and Consortium for Inter-Disciplinary albumin removal is presented. Instead of utilizing a traditional antibody- based depletion approach, the ProteaPrep albumin depletion capture Environmental Research, State University of New ligand is a recombinant bacterial protein that is highly purified for York Stony Brook, Stony Brook, NY, United States; robust, efficient removal of albumin from human serum samples. The 3 Stony Brook Proteomics Center, State University recombinant ligand technology produces binding constants with of New York Stony Brook, Stony Brook, NY, United human serum albumin (Kd ~ 1 × 10-11M) that are significantly States; 4Department of Medicine, Division of stronger than antibody-based methods, which have binding constants Hematology, State University of New York Stony that range from Kd’s of 1 × 10-6 to 1 ×10-8 M. The results

Brook, Stony Brook, NY, United States; 5State show that the functionalized beads bind and remove albumin from Poster Abstracts University of New York Stony Brook, Department of serum in a highly efficient and rapid manner. Depletion efficiencies are Pharmacological Sciences, Stony Brook, NY, United >99% for the selective removal of HSA from serum samples in less than States; 6State University of New York Stony Brook, 20 minutes. The recombinant bacterial protein ligands are shown to be Stony Brook, NY, United States effective for the depletion of albumin from the sera of other species, including mouse, rat, pig, horse, cow, and monkey. Disease detection in ancient human samples currently relies on genomic-based assays, which are error prone due to contamination and 234 Qualitative and Quantitative cannot distinguish between active and latent pathogenic infection. On Characterization of Proteomes Using the other hand, protein-based assays such as global protein profiling Ion Mobility Mass Spectrometry with offer complementary alternatives for the pathological diagnosis of Data Independent Acquisition archeological specimen. The discovery of three Inca mummies in 1998, perfectly preserved in the permafrost of the high Andes, allowed us to M. Stapels, K. Fadgen, S. Geromanos, J. Langridge analyze mummy samples by protein-based and genomic-based assay. A buccal swab from one of the 500 year old mummy was analyzed by Waters Corporation, Milford, MA, United States shotgun proteomics to detect the protein profile. Among the identified A central goal in a proteomics study is to fully characterize a sample both proteins, we found a signature of proteins indicating an immune qualitatively and quantitatively. A data-independent analysis yields response to a bacterial infection at the time of the mummy’s death. reproducible fragmentation and peak area information for peptides in Based on the external visible symptoms and the gamut of immune

ABRF 2011 — Technologies to Enable Personalized Medicine • 97 a complex mixture (1). The addition of ion mobility into this analysis TDS data was performed using Bruker’s BioTools software. By means of inserts an orthogonal, gas-phase separation, occurring in the millisecond LC-MALDI-TDS, one of the by-products, detected at 16.6kDa, turned timescale, which is poised nicely between chromatographic and TOF out to represent a truncated version of the expected TAU protein, mass spectrometry timescales. Peptides and their corresponding containing the sequence range [1-161]. C-terminal ISD fragments fragment ions are aligned by chromatographic retention time in data- yielded unambiguous evidence for the correct assignment of the independent analyses. When ion mobility is used, peptides and their truncation site. Another unexpected by-product, being approx. 130Da fragment ions also share the same mobility drift time which dramatically lower in mass than the expected protein product, was lacking the improves specificity. In this study, eukaryotic and prokaryotic samples N-terminal methionin residue. This was concluded from the assignment were analyzed by one-dimensional (1D) and 2D chromatography using of the respective N-terminal fragments in the ISD spectrum. As shown high-low pH RP-RP separations, both with and without ion mobility above, LC-MALDI-TDS turned out to be very efficient with regard to separation. The number of proteins identified in a sample doubled by analysis time and lab work required, especially when comparing the using a five fraction 2D separation compared to 1D chromatography. method to alternative approaches like Edman sequencing or geLC Utilizing the mobility separation yielded an additional increase in workflows, i.e. SDS-PAGE with subsequent LC-MS/MS analysis of the the number of proteins and peptides by at least 20%, without any digested proteins. additional instrument time. The amount of information obtained from a sample depended on the amount loaded as well as the time dedicated 236 Novel Proteomic Analysis of to analysis of the sample. For one prokaryotic sample, 552 proteins Esophageal Inflammation were reproducibly identified with 1D chromatography without ion mobility from 0.75 ug of material. Loading 5 ug and performing 5 K. Jonscher1, A. Kendrick1, S. Fillon2, fractions, along with ion mobility, yielded 1260 reproducible protein Z. Robinson2, J. Masterson2, S.J. Ackerman3, identifications. Stoichiometry between proteins was determined by G.T. Furuta2 comparing the average intensity of the top three peptides to every protein to that of an internal standard (2). Agreement was found 1Department of Anesthesiology, University of between the experimental stoichiometric ratios and those found in Colorado Denver, Aurora, CO, United States; literature for many protein groups. 1. Geromanos, S.J., et. al. Proteomics. 2Department of Pediatrics, University of Colorado 2009 (6):1683-95. 2. Silva, J.C., et. al. Mol Cell Proteomics. 2006 (1):144- Denver, United States; 3Digestive Health Institute, 56. University of Illinois at Chicago, Chicago, IL, United States 235 Characterization of Unexpected By-Products in a Recombinant TAU Introduction: Esophageal inflammation associated with eosinophilic Protein by Capillary LC Coupled to esophagitis (EoE) and gastroesophageal reflux disease (GERD) require MALDI-Top-Down-Sequencing (LC- invasive endoscopy and biopsy for diagnosis and testing efficacy of MALDI-TDS) treatments. In the 1970’s, the Enterotest, a weighted gelatin capsule filled with and attached to a 90 cm long nylon string, was introduced as a A. Asperger2, B. Kovacech1, A. Kovac1, D. Suckau2 minimally invasive method to identify intestinal infections. Patients tape the string end extruding from the capsule to their cheek and swallow the 1Institute of Neuroimmunology of Slovak Academy capsule; the string subsequently deploys into the small intestine and the of Sciences, Bratislava, Slovakia; 2Bruker Daltonics, capsule released. After incubation for 15 min to overnight the string is Bremen, Germany pulled back out of the mouth. Here we present pilot data demonstrating that secretions adhering to the string may be recovered and analyzed Compared to Edman sequencing, MALDI-TOFMS based top-down using shotgun proteomics. Methods: Strings from the distal esophagus sequencing of proteins (MALDI-TDS) is much faster, cheaper, can of patients with GERD and normal controls (n=2/group) were cut into 2 deliver significantly longer sequence readout from both, protein N- cm sections and proteins recovered by incubating strings in lysis buffer. and C-terminus, and has no limitations in case of terminally modified Tandem mass spectrometry analysis using a high-capacity quadrupole proteins. These features make MALDI-TDS an extremely appealing ion trap was performed on trypsin-digested samples and data were method for the QC analysis of recombinant protein products, for searched with X!Tandem through the LABKEY interface. Spectral instance biopharmaceuticals. Direct MALDI-TDS analysis requires counting provided relative quantitation of proteins between samples. purified, homogeneous protein samples. However, coupling MALDI- Results: After removal of isoforms, 34 proteins common to all string TDS to upfront LC separation allows to analyze mixtures of proteins. samples (2 each from 2 patients/subjects) were identified. Annexin A1, Poster Abstracts Since recombinant protein products often contain unexpected keratins 16, 4 and 13, and MUC5B were most highly upregulated in heterogeneities, LC-MALDI-TDS represents a promising method esophageal secretions from GERD subjects as compared with normal to characterize such undesired by-products. We describe here the controls. In addition, functional clustering revealed that anti-apoptotic characterization by LC-MALDI-TDS of unexpected by-products proteins and those involved in extracellular signaling were present in occurring in a recombinant TAU protein. Initial MALDI-TOF analysis only GERD and not normal controls. Conclusion: The Esophageal String of the protein sample revealed at least two minor components being Test can be used to sample esophageal secretions in health and disease. different from the expected protein mass. To separate these sample Host response proteins are present in the esophageal lumen during constituents, capillary LC was used. The LC fractions were deposited inflammation. This work was supported by R21-AI079925, Thrasher on a MALDI target pre-spotted with sDHB matrix. Subsequently, Foundation, CURED Foundation, Pappas Foundation and the Colorado MALDI-ISD spectra of the LC separated compounds were acquired Clinical Translational Scientific Institute (CCTSI) NIH/NCRR Grant using a Bruker Ultraflex III MALDI-TOF/TOF. Analysis of the MALDI- Number 5UL1RR026314-02.

98 • ABRF 2011 — Technologies to Enable Personalized Medicine 237 Multi-Site Assessment of ProteoRed 238 In Depth Characterization of Protein Plasma Reference Sample for Changes Induced by Glucose Benchmarking LC-MS Platform Limitation Using Complementary Performance Ionization Techniques

A. Campos1, K. Asanov1, E. Oliveira1, N. Colome2, B. Müller1, W. Jabs2, M. Behrens2, D. Suckau2, S. Martínez-Bartolomé3, J-P Albar3, F. Canals2, C. Baessmann2, K. Niehaus3, T. Noll1 ProteoRed ISCIII Consortium4 1Institute of Cell Culture Technology, Faculty 1Proteomics Platform, Barcelona Science Park, of Technology, Bielefeld University, Bielefeld, Barcelona, Spain; 2Proteomics Laboratory, Germany; 2Bruker Daltonik GmbH, Bremen, Medical Oncology Research Program, Vall d Germany; 3Proteome and Metabolome Research, Hebron Institute of Oncology VHIO Vall d Hebron Faculty of Biology, Bielefeld University, Bielefeld, University Hospital, Barcelona, Spain; 3Centro Germany Nacional de Biotecnologia-CSI, Madrid, Spain; 4ProteoRed ISCIII Consortium, Madrid, Spain Comprehensive quantification of specific changes in biological systems in response to a certain treatment or perturbation is one of the most One of the missions of the Spanish Proteomics Network (ProteoRed important but also among the most challenging tasks in proteomics. ISCIII) is to assist its proteomics core facilities in evaluating their Different kinds of separation techniques, mass spectrometer types, capabilities to perform qualitative and quantitative proteomics identification strategies, and quantification strategies can be applied analysis. In 2010, the ProteoRed’s Sample Collection and Handling for this purpose. Here, we investigate the benefits of combining the Group designed a moderately complex plasma standard reference results obtained by two main ionization techniques used in proteomics, sample primarily to be used for routine quality assurance monitoring ESI and MALDI. Results are derived with the aid of a bioinformatics of laboratory instrumentation, as well as inter-laboratory performance platform, which integrates different proteomics techniques. Human assessment, and development and validation of novel technologies. cell cultures obtained from the periventricular region of the brain The ProteoRed Plasma Reference (PPR) sample is a subset of highly were grown under normal and under glucose limitation conditions. abundant well-characterized human plasma proteins with a number To investigate the changes induced by the glucose limitation, stable of isoforms, in addition to 4 spiked-in proteins, altogether distributed isotope labeling with amino acids in cell culture (SILAC) was used. over 5 orders of magnitude in concentration. The PPR sample was After labeling, samples were mixed and fractionated. 40µg of recently stress tested in the latest ProteoRed Multicenter Experiment the cytosolic protein fraction was separated by 1D-SDS PAGE, which (PME6) that counted with the participation of 17 proteomics facilities was cut into 10 bands. Each band was digested and then analyzed with using a wide range of LC-MS platforms. Although each laboratory nanoscale liquid chromatography (nLC) coupled to an ESI-ion trap was allowed to use its own favorite methodology, we requested the and a MALDI-TOF/TOF, respectively. The use of ESI-MS in addition sample be analyzed in a single LC-MS run in experimental triplicate to MALDI-MS resulted in a 46% increase in protein identification (3 different digestions).Evaluation of the results submitted by the and a 124% increase in peptide identification. Combining data of study participants revealed moderate discrepancies at the peptide both ionization techniques, 664 proteins consisting of 4658 peptides identification level, and poor overlap at the protein identification were identified. Peptides identified with ESI-MS are significantly more level. In an attempt to identify the source of such irreproducibility, hydrophobic than peptides identified with MALDI-MS. An analysis raw data of 8 laboratories (24 LC-MS runs) were reanalyzed centrally of the amino acid composition of the identified peptides revealed a using a standardized data analysis pipeline, which included protein significant bias of ESI-MS for aliphatic and hydroxyl-containing amino inference using ProteinProphet software. We found that the majority acids while MALDI-MS favors peptides with aromatic, basic and amide of protein identification discrepancies across submitted reports of group-containing amino acids. The results show, that the use of nLC- these 8 laboratories were due to inconsistencies on how data analysts ESI-MS in addition to nLC-MALDI-MS greatly improves the number and computational tools group and/or infer proteins. Immunoglobulin of identified and quantified proteins and peptides. The experiment

variable chain identifications were particularly conflicting throughout revealed little changes in the cytosolic proteome of cells growing under Poster Abstracts identification lists, even in the centralized analysis. Using a series of the applied glucose limitation. LC-MS performance metrics, we benchmarked the performance of 8 LC-MS instruments (Orbitraps) and identified system components that 239 Evaluation of Two Separation vary the most across laboratories. Techniques, SCX and OFFGEL and of Two Fragmentation Methods, CAD and PQD, to Asses iTRAQ Quantitation Efficiency

G. Grigorean1, A. DiFonzo2, P. Soffientini2, T. Manousidou2 1European Institute of Oncology, IFOM-IEO Campus, Milan Italy; 2Cogentech, IFOM-IEO Campus, Milan, Italy

To evaluate various sample preparation and analysis platforms for isobaric tags for iTRAQ experiments, we compared two separation and two fragmentation techniques: Strong Cation Exchange (SCX) with

ABRF 2011 — Technologies to Enable Personalized Medicine • 99 isoelectric point separation. In addition, we compared two types of 241 Optimization of Nanospray Voltage mass analyzers: a quadrupole and a linear ion trap, the former within and Spray Stability: Impact on a quad-TOF hybrid mass spectrometer, and the later within an LTQ-FT hybrid MS. This was done by comparing two fragmentation techniques, Chromatographic Peak Area for collisionally activated dissociation (CAD) with pulsed Q dissociation Analyte Quantitation (PQD). Proteins from lysed HeLa cells were trypsinized and peptides 1, M. Lee2, A. Berg1 were iTRAQ labeled. Half of the labeled digest was separated via SCX G. Valaskovic into20 fractions. The other half of digested peptides was separated 1New Objective, Inc., Woburn, MA, United States; with an OFFGEL isoelectric point fractionator into 24 fractions. Each 2Milestone Development Services, Newton, PA, dried peptide fraction was desalted, and submitted to reverse-phase United States C-18 HPLC on-line with the mass spectrometer. Quantitative data analysis for the LTQ-FT data was performed using Proteome Discoverer State-of-the art liquid chromatography-tandem mass spectrometry (Thermo Electron) and for the QStar data, PEAKS Studio (Bioinformatic (LC-MS/MS) analysis uses a constant electrospray (ESI) voltage for Solution Inc.). R software was used for the statistical validation of data acquisition. Modern qualitative and quantitative LC-MS/MS results. The LTQ-FT data was also analyzed with the PEAKS software methods depend on highly efficient gradient elution chromatography. to asses the viability of PEAKS as a universally applicable data analysis The changing chemical composition of mobile phase during gradient package. Comparison between separation and dissociation was based elution results in an inherent disconnect with single point ESI on a) number of proteins quantified b) reproducibility of technical voltage optimization. A constant ESI voltage limits spray stability and replicates based on protein distribution c) standard deviation for data compromises chromatographic peak area quantification, limiting total obtained from the two dissociation techniques. Fractionation with peak area and increasing peak area relative standard deviation (RSD). OFFGEL increased the number of identified and quantified of proteins We have adapted a previously reported closed-loop “orthogonal” by approximately 20%. Number of mass spectra which can be used for feedback control system for qualitative proteomics† to one suitable quantitation is higher from the QStar instrument implying that the order for targeted peptide quantification. A digitally controlled nanospray of magnitude increased sensitivity of the LTQ-FT relative to the quad- source was modified to use the high-voltage output from an external, TOF is lost to the inefficiency of the PQD. Reproducibility of technical computer controlled power supply. A software program controlling replicates from PQD is lower than that obtained with data from CAD. the nanospray emitter stage position and the external power supply supported a programmable spray compensation voltage during 240 Quantification of Eicosanoid Pathway chromatograpghic analysis. One microliter of a standard peptide Proteins in Human Cerebrospinal (MRFA) was autosampler injected (CTC PAL) directly on column (New Fluid Using a Dual Pressure Linear Ion Objective PicoFrit, 75 um x 10 cm, C18) to the source equipped linear Trap Mass Spectrometer ion trap mass spectrometer (Thermo Scientific, LTQ). Repetitive on- column injections at different (fixed) target ESI voltage settings yields R. Biringer1, J. Horner1, A. Fonteh2, S. Kauffman2, a apparent compound dependant response curve in which total A. Huhmer1, M. Harrington2 chromatographic peak area for selected ion currents reaches a maximum value. Spray stability and chromatographic quality was directly 1Thermo Fisher Scientific, San Jose, CA, United correlated with the observed spray mode. Altering the ESI voltage from States; 2Molecular Neurology Program, Huntington 2 kV to 3 kV resulted in a peak area increase of 38%. Qualitative visual Medical Research Institutes, Pasadena, CA, United comparison of noise in the molecular ion signal (523.5 m/z) showed a States markedly higher level at 2.0kV. Fourier transform spectrum analysis of selected ion current, revealed a ten-fold increase in noise at 2 and 4 Objective: To develop a linear ion trap mass spectrometer-based Hz for the 2 kV acquisition. Spray instability results in increased noise protocol for quantifying eicosanoid pathway enzymes and to apply this of the reconstructed chromatogram, increasing the uncertainly of peak protocol to monitor enzyme changes in episodic migraine. Methods: area measurement. †Valaskovic, G.A, Murphy, J.P., Lee, M.S. “Automated Peptides diagnostic for eicosanoid pathway enzymes were determined Orthogonal Control System for Electrospray Ionization”, J. Am. Soc. from trypsin digests using standard LC/MSn methodologies. Mass Spectrom., 2004, 15, 1201-1215. Corresponding stable isotope peptide analogs (SI-peptides) were selected and synthesized. For each assay, a cocktail of all SI-peptides 242 Development of a High-Sensitivity is spiked into delipidated cerebrospinal fluid (CSF) from migraine Targeted MRM Assay for Peptide and study participants when well and sick. After trypsin digestion, each Protein Quantification Poster Abstracts of the SI-peptides and the corresponding endogenous peptides were simultaneously quantified using LC/stable isotope selected T. McKenna, A. Bartlett, C. Hughes, J. Langridge reaction monitoring (SI-SRM) using a dual pressure linear ion trap mass spectrometer. Results: Linear dynamic range for this assay spans nearly Water Corporation, Manchester, United Kingdom four orders of magnitude (0.5-4800 fmol). The LOQ is in the low to sub- fmol range depending on the peptide, and corresponds to eicosanoid Discovery phase proteomics has generated numerous candidate protein proteins at concentrations in the low ng/mL CSF range. Eicosanoid markers for a wide variety of biological processes and disease types. protein concentrations exhibit both preparation and migraine state To assess the viability of these protein expression changes requires the dependencies. Conclusions: We have developed a robust, targeted analysis of a larger number of samples, preferably in a targeted fashion, full scan MS/MS linear ion-trap based quantitative assay (LC/SI- and hence the use of MRM has become routine. Specific peptides from SRM) for eight enzymes of the eicosanoid pathway. Further, we have the proteins of interest are targeted as surrogate markers for that protein, demonstrated that this assay can be used to quantify changes in the in a screening assay using a triple quadrupole mass spectrometer expression of eicosanoid proteins during episodic migraine. operating in the multiple reaction monitoring (MRM) mode. The MRM method which is used to detect specific ions from target molecules has the capability to simultaneously quantify large numbers of proteins

100 • ABRF 2011 — Technologies to Enable Personalized Medicine with good limits of quantification (LOQ) and linear dynamic range. In Orbitrap XL. Following accurate mass and retention time alignment this mode of analysis the sensitivity and dynamic range are improved across all LC-MS runs performed within Rosetta Elucidator, a robust and providing sufficient data points across a chromatographic peak are mean normalization of precursor ion intensities was applied and area recorded then quantitation is accurate (CV 5-10%). This high sensitivity under the curve quantitative measurements were made for all precursor coupled with the specificity/selectivity afforded by MRM transitions ions. Qualitative identifications were assigned to each precursor ion allows extensive panels of peptide biomarkers to be monitored in following Mascot searches against a mus musculus database appended a single experiment from complex mixtures. We will describe the with reverse entries and scoring thresholds were adjusted to yield a development and implementation of novel high-sensitivity MRM assays peptide 1% FDR. Across all four treatment groups, a total of 778 for large scale peptide quantification. phosphopeptides were qualitatively identified. Analytical plus TiO2 enrichment variation of pre-digested bovine alpha-casein spiked into 243 High Sensitivity Protein Quantitation each sample prior to enrichment yielded a phosphopeptide intensity Using a Triple Quadrupole with a variation of < 20% RSD across approximately three orders of magnitude. Dual Ion Funnel In summary, these data demonstrate an effective and straight-forward methodology to study global changes in phosphorylation directly from C. Miller, Y. Yang, K. Waddell clinically relevant biological samples. Agilent Technologies, Palo Alto, CA, United States 245 Studying Whole-Body Protein Assays that are both specific and quantitative for target proteins are Turnover Using Stable-Isotope critical for preclinical validation of putative biomarkers. Such assays are Labeled Mice typically multiplexed, multiple reaction monitoring (MRM) analyses 1,2, A.A. Koller2, J. Relucio1, S. Nik1, which can provide the high-throughput required. Sensitivity is a key E. Chen 3 1 3 requirement for such assays as protein biomarker concentrations may B. Dost , H. Colognato , N. Bandeira be quite low in commonly used biofluids such as serum and plasma. 1Department of Pharmacological Sciences, Stony Improving the sensitivity of LC/MS can be achieved by using nanoflow Brook University, Stony Brook, NY, United States; LC, and by enhancing the sampling and transmission of ions in the 2Stony Brook University, Proteomics Center, School mass spectrometer. This study demonstrates the 5-10x sensitivity gain Of Medicine, NY, United States; 3Department of achieved for peptides using a triple quadrupole mass spectrometer modified with a dual ion funnel. The sensitivity achieved using a Computer Science and Engineering, University of microfluidic-based nanoflow LC system compared to a standard LC California, San Diego, CA, United States system will be shown discussed. Protein turnover is a neglected dimension in functional genomics studies delineating the dynamic changes of protein regulation and links 244 Development of a Label-Free transcriptome, proteome, and potentially metabolome. It is known that Quantitative Phosphoproteomics proteins are degraded and synthesized continuously at the cellular Platform Applicable to Non-Cell level. The balance of degradation and biosynthesis is tightly regulated Culture Biological Matrices and has great implications in normal physiology, cellular regulation, and human diseases. Recent technological advances in high-resolution mass 1 2 1 E. Soderblom , M.W. Foster , J.W. Thompson , spectrometry open up new opportunities to make proteome-wide M.A. Moseley1 prediction of protein turnover. However, studies of proteome turnover in live animals require high efficiency of labeling using labeled amino 1Duke Institue of Genome Science and Policy, 2 acids and bioinformatics to deal with highly complex mass spectra Durham, NC, United States; Duke University data. To unravel the dynamics of protein turnover in intact animals, School of Medicine, Durham, NC, United States we developed an in vivo pulse-chase strategy and bioinformatics tools (APCIE) to perform analysis of proteome turnover. We pulsed C57BL/6 The most commonly employed LC-MS based quantitative mice with 15N labeled amino acids through diet and chased 15N labeled phosphoproteomics experiments are performed using cultured cell- Poster Abstracts young (3 weeks old) and old (4 months old) mice with unlabeled diet lines with incorporated stable-isotope labeled amino acids. Although for 2 weeks. Lungs from these two groups of mice were harvested and these systems simplify experimental manipulation and provide analyzed by mass spectrometry-based shotgun proteomic techniques. relatively large amounts of soluble protein, they do not account for Using APCIE, we determined protein degradation (loss of 15N labeled the complexity of signal regulation from other factors or cell types amino acids) and protein synthesis (incorporation of 14N amino present in the cell’s native physiological environment. Here we acids) of over 300 proteins commonly found in young and old mice describe the general analytical and informatic methodologies used and observed aged-dependent differences in proteome turnover. As for the global quantitation of specific sites of phosphorylation from expected, most proteins are turned over faster in young mice than old clinically relevant matrices such as human tissue or biological fluids mice reflected by massive loss of 15N amino acids and incorporation of compatible from a variety of different experimental designs. As an 14N amino acids. Our newly developed in vivo pulse-chase technique exemplar application of the methodology, a quantitative analysis of and algorithm can be used for global profiling of protein turnover lipopolysaccharide challenged and ethyl nitrite protected mouse lung in intact animals to study complex mouse models mimicking human tissue was performed. Phosphopeptide enrichments from 600 ug diseases. Combining global profiling of protein turnover and protein aliquots of trypsin digested homogenized mouse lung tissue from four expression will make a tremendous impact towards mechanism-driven unique treatment groups was accomplished using an optimized TiO2 therapeutics based on dynamic regulation of proteins and protein spin-column protocol and were subjected to triplicate 1D-LC-MS/MS networks. analysis on a Waters NanoAcquity UPLC coupled to a Thermo LTQ

ABRF 2011 — Technologies to Enable Personalized Medicine • 101 246 Purification of Bacterial APOA-1 sample load between 50 % and 100 % of the total binding capacity. In and Characterization of Novel Anti- the second study screening for optimal solubilization and purification conditions for an integral membrane protein was performed, using Cancer Drug Delivery System seven detergents. The screening protocol involved a rapid purification 1 2 step before analyses, evaluation and final choice of detergent(s). T. Young , A. Lacko Obtained results from this screening gave reliable results that could also 1North Carolina State University, Raleigh, NC, be fed into further scale-up experiments. We will present results from United States; 2University of North Texas Health Western blotting, gel filtration and SDS-PAGE. The para-magnetic bead Science Cente, Fort Worth, TX, United States format simplified and shortened the handling procedures through-out both screening workflows. Although chemotherapy regimens have proved effective in attacking cancer cells and tumors, side effects and drug resistance remain a 248 Production of Recombinant Mouse major concern during cancer therapy. The use of reconstituted high Flower Protein in E. coli: Application density lipoprotein (rHDL) nanoparticles has been investigated as a of Mistic Fusion to Improving the drug delivery system, including the transporting of small interfering ribonucleic acid (siRNA). The use of rHDL nanoparticles has great Expression of Membrane Proteins potential, in this regard, due to their ability to specifically target 1 2 J.L. Martinez-Torrecuadrada , M. Marenchino , cancer cells via the HDL (SR-B1) receptor. The goal of these studies R. González1, J. López-Alonso2, was to improve the purification of Apolipoproteins A-1 (ApoA-1), a R. Campos-Olivas2, G. Roncador3, E. Moreno4 major component of rHDL, and the preliminary characterization of the siRNA carrying rHDL nanoparticles. E.coli cells transfected with 1Proteomics Core Unit, Spanish National Cancer the apo A-I gene was grown at 37OC, until an optical density of 0.6 Centre (CNIO), Madrid, Spain; 2Spectroscopy was reached. The cells were then induced with 0.5mM Isopropyl β-D- and Nuclear Magnetic Resonance Unit. Spanish 1-thiogalactopyranoside (IPTG) and centrifuged. Subsequently, the National Cancer Centre (CNIO), Madrid, Spain; pellets were suspended in the lysate solution and loaded onto a Nickel- 3Monoclonal Antibodies Core Unit, Spanish Sepharose column. Thereafter, rHDL nanoparticles using siRNA were prepared. 160 mg per liter of purified ApoA1 was obtained. Particle National Cancer Centre (CNIO), Madrid, Spain; 4 measurements and the chemical composition of the particles are being Cell Competition Group, Spanish National Cancer investigated. Further investigation regarding the efficiency of the Centre (CNIO), Madrid, Spain incorporation of siRNA, the physical and chemical properties as well as cytotoxicity of the particles will contribute to the assessment of the The structural and functional studies of membrane proteins have efficiency of these particles as a novel anti-cancer drug delivery system. been greatly hindered due to difficulties in their over-expression and production. It is also often difficult to generate effective antibodies to membrane proteins. In our laboratory, we have addressed the problem 247 Screening for Optimal Purification of producing high level amounts of membrane proteins in Escherichia Conditions for Histidine-Tagged coli by use of Mistic, a Bacillus subtilis protein, as a fusion partner. Flower Water-Soluble and Membrane (Fwe) is a membrane protein that is conserved in animals and proposed Proteins Using Magnetic Beads to be a Ca2+ channel in neurons. It has been recently reported that Methods in Drosophila Flower is a component of the cell competition response that is required and sufficient to label cells as “winners” or “losers”, H. Hedlund, E. Mascher, G. Risberg promoting the elimination of weaker cells from a growing population GE Healthcare Life Sciences, Uppsala, Sweden in order to optimize tissue fitness. This process may have biomedical implications because imbalances in cell fitness appear during aging, The development of methods for efficient expression screening of cancer formation and metastasis. In this work, we employed the multiple numbers of recombinant proteins or optimization of the membrane protein Mistic to assist in the production of this protein. purification conditions of proteins is essential to shorten the time from The construct 6xHis-Mistic – Flower carrying a TEV cleavage site was gene to drug targets. The combination of para- magnetic beads and efficiently overexpressed in E. coli. The IMAC-based purification and affinity purification using IMAC resins (pre-charged with Ni2+-ions) cleavage of the recombinant protein was achieved in the presence of for capture of histidine tagged proteins is an easy-to-use format and the detergent lauryldimethylamino oxide (LDAO). Circular dichroism showed a high content in helical structure as predicted from the amino Poster Abstracts a powerful tool allowing for rapid, small-scale purification. We will describe two studies for optimization of purification conditions of acid sequence by the program TMHMM. Also, the recombinant Mistic– histidine-tagged proteins. All experiments were done using Ni2+- Flower was used as antigen source to produce monoclonal antibodies in charged His Mag Sepharose™ Ni. In the first study optimal purification KO mice. The generated monoclonal antibodies were able to recognize conditions for a water-soluble histidine-tagged protein was investigated Flower protein by Western blot and immunohistochemistry, indicating at eight different buffer conditions and four different sample loads that this recombinant protein retained the antigenicity of the native (varying from 25 % to 100 % of the total binding capacity). The study form. These antibodies will facilitate importantly further functional was performed using a liquid handling workstation.A good balance studies on Flower protein. between yield and purity was obtained at 40 mM imidazole and at a

102 • ABRF 2011 — Technologies to Enable Personalized Medicine 249 Rapid Monoclonal Antibody Glycan electrochemical flow cell followed by online MS analysis. Based on the Profiling Using an Integrated intact protein mass and the resulting fragments and the MS/MS data unambiguous assignment of the disulfide bonds becomes possible. Microfluidic-Based mAb-Glyco Chip All these applications illustrate the tremendous power and broad and Quadrupole Tim-of-Flight Mass applicability of electrochemistry as a tool to mimic nature’s Redox Spectrometry reactions within a few seconds or minutes. C. Miller, N. Tang, K. Waddell 251 Protein Sequencing Research Group Agilent Technologies, Palo Alto, CA, United States (PSRG): Results of the PSRG 2011 Protein biologics now represent a significant share of pharmaceutical Study: Sensitivity Assessment of sales and future growth potential, particularly in an era of increasing Edman and Mass Spectrometric patent expirations. A range of analytical methods is required to Terminal Sequencing of an Unknown determine the purity, identity and integrity of protein biologics at Protein multiple points along the manufacturing process, from cell culture to 1, J.S. Smith2, W. Sandoval3, B. Xiang4, downstream purification, product characterization and lot release. H. Remmer 5 6 3 Characterization of glycans from antibodies is fundamentally important K. Mawuenyega , D. Suckau , V. Katta , in biotherapeutics design and disease progression and detection. The J.J. Walters7, P. Hunziker8 ability to characterize glycans rapidly has been limited by the sample 1University of Michigan, Ann Arbor, MI, United preparation steps and structural complexity of the glycoproteins. States; 2University of Texas Medical Branch, To address this problem, we have developed a microfluidic chip 3 that performs rapid on-line cleavage of glycans from monoclonal Galveston, TX, United States, Genentech, antibodies, captures the released glycans and then separates them Inc., South San Francisco, CA, United States, 4 prior to nanospray ionization in the mass spectrometer. The entire run Monsanto Company, St. Louis, MO, United States, time is 12 minutes. A glycan accurate mass database was established 5Washington University School of Medicine, allowing quick assignment and identification of the glycans. St. Louis, MO, United States, 6Bruker Daltonics, Bremen, Germany, 7Sigma-Aldrich, St. Louis, 250 Protein Cleavage, Disulfide Bonds MO, United States, 8University of Zurich, Zurich, Reduction, Metabolite Synthesis and Switzerland Much More Using Electrochemistry/ MS Establishing the N-terminal sequence of intact proteins plays a critical role in biochemistry and potential drug development. N-terminal J. Powers1, J. Purkerson1, A. Kraj2, M. Eysberg2, sequence analysis is necessary for quality control of protein biologics, J.P. Chervet2 for determining sites of signal peptide cleavage events, as a first step in elucidating the sequences of genes from uncommon species and 1Antec, Palm Bay, FL, United States; 2Antec, for the characterization of monoclonal antibodies. Automated Edman Zoeterwoude, The Netherlands degradation has been the method of choice for these analyses. However, alternate methods for N-terminal sequence analysis have emerged. The Recently, the scope of Electrochemistry (EC) upfront MS has been recent PSRG studies have established that Edman sequencing and mass extended from mimicking drug metabolism towards new applications spectrometry based techniques have varied strengths and weaknesses such as: protein/peptide cleavage, disulfide bonds reduction, covalent depending on several experimental factors and both play an important drug-protein binding, etc. In this presentation we will show the role in terminal sequencing. With this complimentary role realized, application of on-line EC/MS as a powerful tool to simulate various the 2011 PSRG study attempts to evaluate the sensitivity limits of the oxidation and reduction processes in life sciences. A specially designed various sequencing techniques. The PSRG distributed three sample µ-preparative electrochemical flow cell will be presented. The cell sets of 3 tubes each, varying by sample format (lyophilized, gel slice allows the synthesis of sufficient amounts of metabolites in a few minutes or membrane piece). Each set of three samples contains the same Poster Abstracts for subsequent use as reference material (e.g. NMR or MS). New recombinant protein with increasing amounts of material. The sequence scanning method was applied for oxidation of the highly concentrated of this protein is not listed in any database. Participants could request samples (mM range) to achieve high yield in the metabolites formation. any one, two, or all three sample sets. Including PSRG committee, a total Stable oxidation conditions were obtained without the need of any of 38 participants requested 74 sample sets. The participants were cell maintenance for a prolonged period of time. Electrochemistry asked to determine as many amino acids from both termini by their up front MS can be applied for protein and peptide cleavage (as a method of choice, and were encouraged to try multiple methods for promising new approach to enzymatic digestion). Electrochemical sequence elucidation. Study participants were directed to a website cleavage of proteins and peptides occurs very specifically at C-terminal to anonymously upload sequences and supporting data. Our analysis of the Tyrosine and Tryptophan peptide bonds. Examples of oxidative focuses on the length and accuracy of the sequence calls reported by cleavage will be presented. Disulfide bonds are one of the most the participants, and how that compares with decreasing amounts of important post-translational modifications for proteins. In this poster protein and the type of sample format analyzed. A comparison of the we present the structural analysis of biologically active peptides results obtained by Edman chemistry and by alternative technologies and proteins containing disulfide bonds (e.g., somatostatin, insulin, as well as information on the type of instruments and protocols is etc) using electrochemistry (EC) combined with mass spectrometry. reported. Therefore the sample undergoes electrolytic disulfide cleavage in the

ABRF 2011 — Technologies to Enable Personalized Medicine • 103 Author Index

Please note that only primary authors of poster presentations are listed in this index. A G K Adams, P. - 125 Gadush, M. - 173 Kain, S. - 136 Ahmed, A. - 103 Ganter, B. - 119 Kamberov, E. - 145 Albanese, J. - 207, 216 Garge, N.R. - 195 Khanna, A. - 150 Asperger, A. - 235 Gendeh, G. - 183, 184, 191, 198 Kinross, C. - 152 Giuffre, A. - 129 Koller, A. - 232 Grigorean, G. - 239 Küster-Schöck, E. - 176 B Grills, G. - 105, 113, 121 Barsch, A. - 222, 223 Gross, V. - 219 Berg, A. - 215 Grove, D. - 157 L Biringer, R. - 240 Langmore, J. - 188 Bishop, J. - 131 Lea, K. - 168 Bodi, K. - 156 H Liao, A. - 118 Boland, J. - 146 Hagen, A. - 102 Lin, S. - 108 Bolcato, C. - 233 Hamilton, J. - 107 Liu, C.S. - 134 Bramlett, K. - 163 Hawke, D.H. - 189 Liu, J. - 135 Hebert, N. - 210 Lopez, P. - 175 Hedlund, H. - 247 Lübbert, C. - 214 C Hemström, T. - 196 Luna, L. - 200 Campos, A. - 237 Hendrickson, W. - 115 Chamberlain, S. - 123 Heuermann, K. - 130 Chen, E. - 245 Hicks, B. - 104 M Chen, S.M. - 217 Hicks, L. - 177 Ma, B. - 194 Chuu, J. - 149 Hinerfeld, D. - 144 Magpiong, I. - 193 Curchoe, C. - 128 Holbrook, J. - 122, 165 Marshall-Waggett, C. - 212 Hughes, C. - 218 Martinez-Torrecuadrada, J.L. - 248 Masri, F. - 228 D McCary, A. - 141 Diaz, C. - 178 J McGuigan, J. - 133 Diaz Acosta, B. - 112 Jaggi, R. - 164 McKenna, T. - 242 Diaz-Candelas, P. - 142 Jen, J. - 161 McMillan, M. - 124 Donnelly, R. - 126 Jennings, S.F. - 114 Miller, C. - 221, 243, 249 Duhr, S. - 182, 201 Johnson, D. - 209 Müller, B. - 238 Jonscher, K. - 236 Murage, E. - 172 E Joshi, S. - 147 Murray, J. - 185 Jovanuvich, S. - 162 Myers, J. - 153 Eckart, S.P. - 179 Author Index F N Narayanan, A. - 197 Fadgen, K. - 227 Needleman, D. - 127 Fleming, B. - 106 Freeman, R. - 171, 208

104 • ABRF 2011 — Technologies to Enable Personalized Medicine O S W Ong, J. - 117 Salowsky, R. - 229 Wang, X. - 226 San Jose Hinahon, C. - 132 Whitley, P. - 138 Schweiger-Hufnagel, U. - 203 Williams, R. - 174 P Shan, P. - 190 Winkvist, M. - 186, 187 Patel, S. - 169 Smith, T. - 139 Wojtkiewicz, M. - 180 Peake, D. - 224 Snyder-Leiby, T. - 205 Person, M. - 109 Soderblom, E. - 244 Powers, J. - 160, 225, 250 Sondej, M. - 181 X Stapels, M. - 234 Xiang, F. - 206 Q Stewart, F.J. - 155 Supunpong Hernandez, N. - 158 Qu, L. - 170 Y T Yang, C. - 111 R Young, T. - 246 Thai, K. - 148 Ravi, H. - 151 Thompson, J.W. - 199 Z Remmer, H. - 251 Turner, M. - 137 Resemann, A. - 192, 204, 230 Turpen, P. - 110 Zavadil, J. - 120 Rezenom, Y. - 220 Zianni, M. - 154, 159 Rosato, C. - 101 Ruddat, V. - 202 V Vacek, G. - 140 Valaskovic, G. - 241 van Soest, R. - 211 Vincent, E. - 116 Vogel, C. - 143 Author Index

ABRF 2011 — Technologies to Enable Personalized Medicine • 105 ABRF 2011 Exhibits*

All ABRF 2011 Meeting attendees are invited to visit the Exhibit The purpose of the exhibition is to further the education of Hall featuring the leading experts in life sciences research and the scientists working in resource and research biotechnology biotechnology. laboratories. The exhibitors will showcase instruments, products or services for use in teaching, research, books or other Exhibit Hours publications in scientific fields of relevance. Sunday through Tuesday *Participation in the Exhibits Program, including attendance of Level 2, Grand Oaks Ballroom Exhibitor Presentations, does not constitute an endorsement by the Association of Biomolecular Resource Facilities (ABRF) of the Sunday, February 20 ...... 10:00 am – 6:30 pm claims, products, or services offered. Monday, February 21 ...... 10:00 am – 6:30 pm Tuesday, February 22 ...... 10:00 am – 2:00 pm

Exhibitor Directory in Alphabetical Order Exhibitors confirmed as of January 29, 2011.

AB SCIEX Booth 501 Advanced Analytical Booth 602 110 Marsh Drive 2711 South Loop Drive Foster City, CA 94404 Ames, IA 50010 United States United States P +1 508-383-7640 P +1 515-296-6600 F +1 650-627-2601 F +1 515-294-7141 www.absciex.com [email protected] www.aati-us.com AB SCIEX is a global leader in the development of life science analytical technologies that help answer complex scientific chal- Advanced Analytical is a leading innovator of analytical instru- lenges. The company provides mass spectrometry instrumenta- ments that improve laboratory throughput. The AdvanCETM tion, software and services for scientific analysis, including core FS96 and AdvanCETM FS12 are 96 and 12-channel parallel capil- and applied research. lary electrophoresis fluorescent instruments for analyzing dsD- NA fragments and RNA. A specialized reagent kit is available for ABRF Booth 317 automated, sensitive and rapid analysis of Next-Gen Sequencing 9650 Rockville Pike fragments. Bethesda, MD 20814 United States Agilent Technologies Booth 301 P +1 301-634-7306 2850 Centerville Road F +1 301-634-7455 Wilmington, DE 19808 [email protected] United States www.abrf.org P +1 800-227-9770 F +1 302-633-8944 The Association of Biomolecular Resource Facilities (ABRF) is a [email protected] unique membership association comprised of approximately www.agilent.com/chem 800 scientists working in resource and research biotechnology laboratories. Our members represent over 140 international Agilent is a leading supplier of life science instrumentation. LC, core laboratories in government, academia, research, industry GC & mass specs, DNA microarrays and scanners, thermal cy- and commercial settings. ABRF promotes the education and clers, automation instruments, reagents and software products career advancement of scientists through conferences, a quar- are used globally. Agilent has developed products and services terly journal, publication of ABRF Research Group studies, and utilized along the entire discovery value chain, from basic bio- conference travel awards. The ABRF Research Group studies are logical research through drug discovery and manufacturing. sponsored annually to help researchers assess and incorporate new biotechnologies into their laboratories, and are world-re- nowned for their practical and educational benefit. Exhibitor Directory

106 • ABRF 2011 — Technologies to Enable Personalized Medicine AnaSpec, Eurogentec Group Booth 417 Bio-Synthesis, Inc. Booth 104 34801 Campus Drive 612 East Main Street Fremont, CA 94555 Lewisville, TX 75057 United States United States P +1 510-791-9560 P +1 972-420-8505 F +1 510-791-9573 F +1 972-420-0442 [email protected] [email protected] www.anaspec.com www.biosyn.com

As a subsidiary of Eurogentec, AnaSpec offers expertise in Bio-Synthesis provides synthetic peptides including libraries and peptides, detection reagents, antibodies, assay kits, oligonucle- arrays, Antibodies, PNA, Bioconjugates, Custom DNA synthesis otides, and qPCRs. AnaSpec carries a broad product line of and its analogs, Gene synthesis, PCR based detection kits, Cell biochemicals and reagents for basic research, high-throughput line authentication, HLA typing and Analytical services. With screening and drug discovery. over two decades of experience producing custom products for both research and pharmaceutical use, BSI has developed a Bioinformatics Solutions reputation for both small and large scale synthesis using optimal Inc. (BSI) Booth 402 processes that meet your specifications. 470 Weber Street North, Suite 204 Waterloo, Ontario N2L6J2 Bruker Daltonics Inc. Booth 214 Canada 40 Manning Road P +1 519-885-8288 Billerica, MA 01821 F +1 519-885-9075 United States [email protected] P +1 978-663-3660 www.bioinfor.com F +1 978-667-5993 www.bdal.com BSI produces life science software to serve the needs of pharmaceutical, biotechnological and academic scientists; and to Bruker is a leading provider of Separation and Mass Spectrom- the progression of drug discovery research. Software solutions etry instruments for the Analytical Sciences. Our innovative and include: ZOOM for next-generation sequencing, PEAKS for easy to use product families (ESI-TOF, Ion Trap, FTMS, MALDI- proteomic mass spectrometry, RAPTOR for protein structure TOF, GC, GCMS, ICP-MS) provide the highest performance, prediction and PatternHunter for general purpose homology highest value systems for a wide range of small molecule and searching. protein analysis applications. Bioo Scientific Booth 718 Caliper Life Sciences Booth 304 3913 Todd Lane, Suite 312 68 Elm Street Austin, TX 78744 Hopkinton, MA 01748 United States United States P +1 888-208-2246 P +1 508-435-9500 [email protected] www.caliperLS.com www.biooscientific.com Caliper Life Sciences is a premier provider of cutting-edge tech- Bioo Scientific provides innovative, custom solutions to core nologies enabling researchers in the life sciences industry to facilities. These include DNA and RNA AIRTM Next-Generation create life-saving and enhancing medicines and diagnostic tests Sequencing library preparation kits, which offer increased sen- more quickly and efficiently. Caliper is aggressively innovating sitivity, flexibility and speed. We also offer the ExoMirTM kit for new technology to bridge the gap between in vitro assays and the isolation of RNA from exosomes and MaxDiscoveryTM kits for in vivo results and then translating those results into cures for toxicity and cytotoxicity analysis. human disease. Caliper’s portfolio of offerings includes state-of- the-art microfluidics, lab automation & liquid handling, optical imaging technologies, and discovery & development outsourcing solutions. Exhibitor Directory

ABRF 2011 — Technologies to Enable Personalized Medicine • 107 Exhibitor Directory — Continued

CEM Corporation Booth 303 DNAnexus Booth 404 P.O. Box 200, 3100 Smith Farm Road 420 Florence Street Matthews, NC 28106 Suite 210 United States Palo Alto, CA 94301 P +1 704-821-7015 United States F +1 704-821-8710 P +1 650-204-1938 [email protected] [email protected] www.cem.com www.DNAnexus.com

Make higher purity peptides faster with the Liberty™ Microwave DNAnexus provides solutions for both DNA sequencing centers Peptide Synthesizer. This system has the fastest cycle times avail- growing their next-gen capacity, and the researchers working able for routine and difficult peptides, including peptides you with next-gen sequence data. Our web-based platform solves can’t access under conventional conditions. The Discover® Sys- the data management and analysis challenges common to both tem provides improved sequence coverage and higher efficien- with a single, unified system. We support sequencing operations cies for your enzymatic digests in only 15 minutes. and research organizations of any size, with absolutely no upfront hardware investment needed. chemagen USA Booth 100 67 Millbrook Street eagle-i Consortium Booth 221 Suite 522 One Kendall Square, Suite B6303 Worcester, MA 01606 Cambridge, MA 02139 United States United States P +1 508-459-7727 P +1 617-384-8787 F +1 508-459-7548 [email protected] www.chemagen.com www.eagle-i.org

chemagic automation and reagents perform fast and reliable The eagle-i Consortium, made up of nine member institutions magnetic bead-based DNA and RNA extractions from sample across the United States, is building a prototype of a national volumes from 10 ul to 10 ml of blood, tissue, bacteria, food, PCR research resource discovery network. The eagle-i Network will and sequencing products, etc. Advantages of this unique system connect biomedical researchers with valuable resources, servic- are fast processing, unmatched sample volume range and robust es, and technologies in a faster and more cost-effective way than chemistry. previously possible. Dionex Corporation Booth 215 EdgeBio Booth 300 1228 Titan Way/P.O. Box 3603 201 Perry Parkway Sunnyvale, CA 94088-3603 Suite 5 United States Gaithersburg, MD 20877 P +1 408-737-0700 United States F +1 408-730-9403 P +1 800-326-2685 [email protected] F +1 301-990-0881 www.dionex.com [email protected] www.edgebio.com Dionex has developed a large portfolio of powerful instruments and chemistries for separations of biomolecules, such as EdgeBio offers next-generation sequencing services. From ex- proteins, peptides, monoclonal antibodies, nucleic acids, and perimental design to bioinformatics, EdgeBio is the perfect so- carbohydrates that serve in diverse applications ranging from lution for all your sequencing needs. EdgeBio also sells kits for biomarker discovery to drug R&D and QA/QC. Visit us at booth sequencing reaction cleanup, PCR purification, and plasmid pu- 215 to learn more about ground-breaking life science solutions rification, as well as competent cells. from Dionex. Exhibitor Directory

108 • ABRF 2011 — Technologies to Enable Personalized Medicine FASEB MARC Program Booth 416 Gene Codes Corporation Booth 220 9650 Rockville Pike 775 Technology Drive Bethesda, MD 20814 Suite 100A United States Ann Arbor, MI 48108 P +1 301-634-7020 United States F +1 301-634-7353 P +1 734-769-7249 [email protected] F +1 734-769-7074 www.faseb.org/marc [email protected] www.genecodes.com FASEB MARC Program provides a variety of activities to support the training of underrepresented minority students, postdoctor- Gene Codes is an international software firm. Our DNA se- ates, faculty and scientists in the biomedical sciences. We offer quence analysis software, SequencherTM is the industry leader. travel awards for scientific meetings, research conferences, and Customer-driven product development, combined with the lat- student summer research opportunity programs. We also spon- est programming techniques, and rigorous quality control to de- sor Career Development Programs including grantsmanship and velop products of the utmost usability and relevance has been leadership training seminars. the key to our success. Fluidigm Corporation Booth 614 Genetix Booth 616 7000 Shoreline Court, Suite 100 120 Baytech Drive South San Francisco, CA 94080 San Jose, CA 95134 United States United States P +1 866-358-4354 P +1 408-719-6400 F +1 650-871-7152 F +1 408-719-6401 www.fluidigm.com [email protected] www.genetix.com Fluidigm develops, manufactures and markets life-science systems based on integrated fluidic circuits (IFCs). This technology Genetix provides unrivalled solutions for imaging and image furthers research by minimizing costs and enhancing sensitivity analysis. The company’s imaging platforms offer solutions to for applications such as single-cell gene expression profiling, pick microbial colonies in genomic studies, to screen and select high-throughput SNP genotyping, and next-generation se- mammalian cell lines, monitor cell growth, evaluate cellular re- quencing. Fluidigm products are used for research only. sponse and quantify tissue biomarkers. GE Healthcare Booth 714 GENEWIZ, Inc. Booth 316 800 Centennial Avenue 115 Corporate Boulevard Piscataway, NJ 08854 South Plainfield, NJ 07080 United States United States P +1 800-526-3593 P +1 877-436-3949 F +1 877-295-8102 www.genewiz.com [email protected] www.gelifesciences.com GENEWIZ, Inc. provides award-winning DNA sequencing, molecular biology, and genomic services. We build codon-opti- GE Healthcare Life Sciences provides tools for drug discovery, mized genes through our powerful Gene Synthesis technologies, biopharmaceutical manufacturing and cellular technologies, so and provide high-quality plasmids from mini-to-giga scales using research scientists and specialists around the world can be more our Plasmid Preparation services. With over10 years experience productive, effective and creative. Our vision is to be the start- in DNA sequencing, we are well-equipped to handle sequenc- to-finish bioprocessing solution provider, the partner of choice ing projects of all complexities. in cell and protein research, and the leader in life sciences services. Exhibitor Directory

ABRF 2011 — Technologies to Enable Personalized Medicine • 109 Exhibitor Directory — Continued

Genomatix Software, Inc. Booth 218 iLab Solutions, LLC Booth 700 3025 Boardwalk, Suite 160 124 Lexington Avenue Ann Arbor, MI 48108 Cambridge, MA 02138 United States United States P +1 877-436-6628 P +1 617-297-2805 F +1 734-622-0477 F +1 877-812-6477 [email protected] [email protected] www.genomatix-software.com www.ilabsolutions.com

With a focus on translating NGS data from sequence files to Lab Solutions provides core facility management software to meaningful biological results, Genomatix leverages it’s 12+ year streamline requests, track usage, and automate billing. The web- history of software and data base development to bring you based solution supports the entire shared service work flow, a state-of-the-art, comprehensive, integrated platform for the from scheduling and request approvals through communication, analysis of your NGS data. billing and reporting. Integration available with financial system such as SAP, PeopleSoft, Banner, and Lawson. Geospiza Booth 519 100 West Harrison, North Tower, Suite 330 Illumina Booth 515 Seattle, WA 98119 9885 Towne Centre Drive United States San Diego, CA 92121 P +1 206-633-4403 United States F +1 206-633-4415 P +1 858-202-4500 [email protected] F +1 858-202-4766 www.geospiza.com [email protected] www.illumina.com Designed by biologists for biologists, Geospiza is the market leading developer of GeneSifter® software, providing integrat- Illumina enables discovery by providing comprehensive DNA ed solutions for Next Generation Sequencing analysis and LIMS. and RNA analysis solutions, including genotyping and copy For over 10 years, Geopsiza has set the industry standard for number analysis, gene expression, sequencing, and epigenetics: high value, low cost, “out of the box” solutions serving the life the cornerstones of genetic research. Our portfolio of tools in- sciences market. tegrates our sequencing and microarray technologies, as well as powerful assay protocols to make possible a new scale of bio- Hamilton Robotics Booth 223 logical research. 4970 Energy Way Reno, NV 89502 Intavis Booth 600 United States 945 West George Street P +1 800-548-5950 Suite 201 F +1 775-858-3024 Chicago, IL 60657 [email protected] United States www.hamiltonrobotics.com P +1 773-572-5799 www.intavis.com Hamilton Robotics is dedicated to the design and manufacture of automated liquid handling workstations. Key to our products Intavis is an industry leader in manufacturing robotic instru- is our air displacement pipetting and monitoring technology ments for in situ hybridization, immunohistochemistry, peptide and software controlling our systems. Our workstations and synthesis, protein digestion, and MALDI spotting. software serve as a common high precision and flexible base upon which to provide automated solutions. Exhibitor Directory

110 • ABRF 2011 — Technologies to Enable Personalized Medicine IntegenX, Inc. Booth 201 LC Sciences Booth 318 5720 Stoneridge Drive 2575 West Bellfort Street, #270 Suite 300 Houston, TX 77054 Pleasanton, CA 94588 United States United States P +1 713-664-7087 P +1 925-701-3400 F +1 713-664-8181 F +1 925-754-7373 [email protected] [email protected] www.lcsciences.com www.integenx.com LC Sciences is a genomics and proteomics company offer- IntegenX, Inc. produces automated sample processing systems ing innovative and quality products and services. We provide for life sciences and applied sciences markets. The company’s unique and customizable oligonucleotide and peptide micro- products: The new Apollo 324 automates Next-Gen library array products designed for nucleic acid and protein-profiling, preparation and the Apollo 100 automates sample processing biomarker-screening, drug screening, and development of di- for Sanger cycle sequencing. Our products dramatically reduce agnostic-devices. process time and reagent expenses. Leica Microsystems Booth 217 Integrated DNA Technologies Booth 305 2345 Waukegan Road 1710 Commercial Park Bannockburn, IL 60015 Coralville, IA 52241 United States United States P +1 800-248-0123 P +1 800-328-2661 F +1 847-236-3009 [email protected] [email protected] www.idtdna.com www.leica-microsystems.com

Serving the areas of academic research, biotechnology, clinical Leica Microsystems offers the latest imaging technology. With diagnostics and pharmaceutical development, IDT is the larg- the high sensitivity Leica TCS SP5, you can buy a basic confo- est manufacturer of custom oligo products in the United States. cal microscope today that is easily upgraded to keep pace with From oligos in plates to gene knockdown products, from fluo- your science. Other innovations: the Leica SCN400 slide scan- rescent DNA probes to gene synthesis, IDT offers the highest ner with dynamic focus tracking for fast scanning while reveal- quality, customer service and turnaround time. ing more details; and the Leica LMD6500 laser microdissection system that uses gravity to collect specimens in a contact-free Kapa Biosystems, Inc. Booth 619 manner. 600 West Cummings Park Suite 2250 Life Technologies Booth 601 Woburn, MA 01801 5791 Van Allen Way United States Carlsbad, California 92008 P +1 781-497-2933 United States F +1 781-497-2934 P +1 760-603-7200 [email protected] [email protected] www.kapabiosystems.com www.lifetechnologies.com

Kapa Biosystems, Inc. offers a portfolio of best-in-class PCR re- Life Technologies is a global biotechnology tools company agents engineered using our high-throughput molecular evolu- dedicated to improving the human condition. Our customers tion platform. The novel DNA polymerases contained within our do their work across the biological spectrum, working to ad- kits confer dramatic improvements to performance that enable vance personalized medicine, regenerative science, molecular results and applications not possible wtih wild-type enzymes. diagnostics, agricultural and environmental research, and 21st Kapa has engineered high performance reagents for real-time century forensics. PCR, high fidelity PCR, next-generation DNA sequencing, fast PCR, crude sample PCR, robust PCR, and molecular diagnostics. Exhibitor Directory

ABRF 2011 — Technologies to Enable Personalized Medicine • 111 Exhibitor Directory — Continued

MassTech, Inc. Booth 103 Morehouse School of 6992 Columbia Gateway Drive, #160 Medicine/RCMI Program Booth 204 Columbia, MD 21046 720 Westview Drive, SW United States Atlanta, GA 30310 P +1 443-539-1758 United States F +1 443-539-1759 P +1 404-752-1500 www.apmaldi.com www.msm.edu

MassTech, Inc. manufactures AP/MALDI ion sources and con- Morehouse School of Medicine (MSM) is a private, historically ducts on-going research and development in mass spectrometry black institution established in 1975. Major support for the bio- related technology. MassTech’s AP/MALDI ion source is compat- medical research infrastructure at MSM is received through the ible with mass spectrometers from manufacturers such as: Agi- NIH/NCRR funded RCMI Program. The MSM/RCMI supported lent, Applied Biosystems/MDS SCIEX, ThermoFinnigan, Waters/ core facilities are: Cell/Tissue Imaging, Gene Profiling, Analytical Micromass, Bruker and JEOL, and is available for OEMs and di- Chemistry/Protein Profiling and Biomedical Technology Service rect sales around the world. Laboratories. Matrix Science Booth 704 New England Biolabs, Inc. Booth 205 64 Baker Street 240 County Road London, W1U 7GB Ipswich, MA 01938 United Kingdom United States P +1 800-716-6702 P +1 800-632-5227 F +1 800-716-6704 [email protected] [email protected] www.neb.com www.matrixscience.com New England Biolabs, Inc. leads the industry in the discovery Take the guesswork out of protein identification with Mascot and production of enzymes for molecular biology applications Server. Get closer to your raw data with Mascot Distiller, giving including sample preparation for next-generation sequencing. direct access to all popular file formats for peak picking, de novo NEB’s global reputation for manufacturing products of the high- sequencing, quantitation, and more. Bring it all together with est quality coupled with best in class technical support makes Mascot Integra, a relational database system for proteomics. NEB a first choice for customers demanding optimized reagents for advanced technologies. Michrom Bioresources Booth 615 1945 Industrial Drive New Objective Booth 203 Auburn, California 95603 2 Constitution Way United States Woburn, MA 01801 P +1 530-888-6498 United States F +1 530-888-8295 P +1 781-933-9560 [email protected] F +1 781-933-9564 www.michrom.com [email protected] www.newobjective.com Michrom Bioresources is a premier supplier of LCMS instrumentation, accessories, and consumables for life science researchers New Objective brings state-of-the-art systems to nano- ESI-MS around the world. Michrom’s new Advance splitless nano-cap- and capillary LC/MS. In 2011, New Objective introduces the rev- illary UHPLC and revolutionary “Plug and Play” CaptiveSpray olutionary and rugged PicoFrit™ Chip. This integrated column/ ionization source interface to most MS instruments, providing high-voltage system provides high-throughput sample analysis optimum LCMS performance for qualitative and quantitative without compromising sensitivity or flexibility. Our industry- proteomics applications. proven PicoView® sources and PicoFrit columns continue to provide high-performance and sensitivity in LC-MS. Exhibitor Directory

112 • ABRF 2011 — Technologies to Enable Personalized Medicine Nonlinear Dynamics Booth 105 OriGene Technologies, Inc. Booth 400 2530 Meridian Parkway, 3rd Floor 9620 Medical Center Drive Durham, NC 27713 Suite 200 United States Rockville, MD 20850 P +1 866-GELS-USA United States [email protected] P +1 888 267 4436 www.nonlinear.com F +1 301-340-8606 [email protected] Nonlinear Dynamics’ range comprises analysis software for la- www.origene.com bel-free LC-MS, 2D electrophoresis gels, and MALDI data as well as tools for multivariate statistical analysis. Our unique analysis OriGene Technologies, Inc., is a gene centric life sciences com- approach has transformed proteomics research — it provides pany dedicated to support academic, pharmaceutical and bio- faster more accurate quantitation and has enabled cross-lab re- tech companies in their research of gene functions and drug producibility. discovery. OriGene develops proteins, antibodies, and other molecular tools to allow researchers to analyze their data on Omega Bio-Tek, Inc. Booth 605 a multiplex level. OriGene’s novel product line includes the 1850-E Beaver Ridge Circle world’s largest cDNA and shRNA clone collections, over 5,000 Norcross, GA 30071 purified human proteins, high quality monoclonal antibodies TM United States (TrueMAB ), 100,000 highly validated human tissues, and protein microarray products and services. OriGene also provides a P +1 770-931-8400 [email protected] broad range of antibody validation products including genome- www.omegabiotek.com wide tagged antigen standards and extensive IHC slides derived from our tissue collection. Omega Bio-Tek manufactures a complete line of DNA|RNA isolation kits utilizing magnetic beads and silica filter technology for Peak Scientific, Inc. Booth 101 both high throughput facilities and individual labs. Our novel no 19 Sterling Road spin, high-throughput magnetic bead based plasmid DNA pu- Boston, MA 01862 rification, PCR cleanup and DTR provides an affordability and United States selection that is unmatched. P +1 866-647-1649 F +1 978-608-9503 OpGen, Inc. Booth 603 [email protected] 708 Quince Orchard Road, #160 www.peakscientific.com Gaithersburg, MD 20878 United States Over the years our dynamic company has stimulated Peak design engineers into some remarkable developments in the man- P +1 301-869-9683 www.opgen.com ufacture of Laboratory Gas Generators. Smoother, quieter and more efficient than anything on the market, our Generators offer OpGen, Inc. provides the Argus™ Optical Mapping System for superb technical performance in the lab and our world-class af- generating high-resolution, ordered, whole-genome restriction ter sales service comes as standard. maps from single DNA molecules. Independent of sequence information, Optical Maps enable a comprehensive view of ge- Phenix Research Products Booth 518 nomic architecture. Use Optical Maps for comparative genom- 73 Ridgeway Road ics, strain typing and sequence assembly. Candler, NC 28715 United States P +1 800-767-0665 F +1 828-670-7020 [email protected] www.PhenixResearch.com

PHENIX Research was founded in 1990 and is recognized as a technical leader in providing outstanding laboratory products

at market beating prices. Our core competency and focus is to Exhibitor Directory provide value added solutions to life science researchers in Uni- versity, Biotech and Government settings. We offer outstanding customer service 8:00 am - 5:30 pm EST and a nationwide direct technical sales force.

ABRF 2011 — Technologies to Enable Personalized Medicine • 113 Exhibitor Directory — Continued

Photometrics Booth 516 Proteome Software Booth 514 3440 East Britannia Drive 1340 SW Bertha Boulevard Tucson, AZ 85706 Suite #10 United States Portland, OR 97219 P +1 520-889-9933 United States F +1 520-573-1944 P +1 800-944-6027 [email protected] F +1 928-244-6024 www.photometrics.com [email protected] www.proeomesoftware.com Developer of the new Evolve 128, Photometrics is the world’s premier designer and manufacturer of high-performance CCD Proteome Software produces industry standard products for and EMCCD cameras for life science research. Photometrics for MS/MS-based proteomics. Scaffold identifies biologically im- decades has led the industry with state-of-the-art imaging in- portant results by comparing data from Mascot, SEQUEST, Pro- strumentation for the scientific community. Researchers globally teome Discoverer, IdentityE, SpectrumMill, Phenyx, OMSSA and rely on Photometrics imaging solutions to meet their demanding X!Tandem. Scaffold Q+ adds iTRAQ and TMT quantification. requirements. MassQC monitors and troubleshoots LC-MS performance. Scaf- fold PTM determines sites of phosphorylation and other modi- PolyLC Inc. Booth 222 fications. 9151 Rumsey Road, Suite 175 Randox Pharma Services Booth 302 Columbia, MD 21045 55 Diamond Road United States Crumlin C. Antrim, BT29 4QY P +1 410-992-5400 United Kingdom F +1 410-730-8340 P +44 (0) 2894-422 413 [email protected] F +44 (0) 2894-452 912 www.polylc.com [email protected] www.randoxpharmaservices.com Unique HPLC columns for life sciences. Featuring ERLIC! 1) Se- lective isolation and separation of tryptic phosphopeptides and Randox Pharma Services offers the most comprehensive range glycopeptides; 2) Fractionation of tryptic peptides by isoelectric of biomarker solutions available to central labs, CRO’s and phar- point in volatile solvents. Products for proteomics: PolySULFOE- maceutical companies worldwide. Our award-winning, FDA ap- THYL A for 2-D SCX-RPC separations; mixed-bed ion-exchange proved, Biochip Array Technology is recognized as a ‘gold stan- and other columns for predigest fractionation of intact proteins, dard’ in biomarker multiplexing for the analysis of proteins, gene including histones and membrane proteins, metabolomics, and mutations, gene expression and SNP analysis. QC of biotech protein variants. Research Scientific Services Booth 216 Protea Biosciences, Inc. Booth 503 P.O. Box 9188 955 Hartman Run Road Gaithersburg, MD 20898 Morgantown, WV 26507 United States United States P +1 301-977-9344 P +1 877-776-8321 F +1 301-977-9572 F +1 304-292-7101 [email protected] www.proteabio.com www.resci.com

We are a diversified biotechnology company that applies novel Research Scientific Services (RSS) has provided professional bioanalytical technologies to the development of new pharma- service on Waters and AB Sciex mass spectrometers and selling ceuticals, products and services. Our products and services em- refurbished LC/MS/MS instruments for 20 years. Service is power Researchers with new capabilities in their own laboratory provided by a nationwide network of highly experienced former to improve the speed, quality and reproducibility of protein OEM engineers. Customer attentiveness and responsive service mass spectrometry data obtained from their biological samples. at a competitive price is why laboratories trust RSS service. Exhibitor Directory

114 • ABRF 2011 — Technologies to Enable Personalized Medicine Roche Applied Science Booth 500 SeqWright, Inc. Booth 702 9115 Hague Road 2575 West Bellfort, Suite 2001 Indianapolis, IN 46250 Houston, TX 77054 United States United States P +1 800-262-1640 P +1 800-720-4363 www.roche-applied-science.com F +1 713-528-6232 [email protected] Roche Applied Science’s new GS Junior system uses 454’s ultra- www.seqwright.com fast pyrosequencing technology to generate 400 to 500 bp sequencing read lengths for amplicon and transcriptome sequenc- SeqWright, a CLIA certified and GLP compliant facility, provides ing projects. Ask about the newest version of our Sequence FDA, Clinical, and Research-level Genomic services to the Phar- Capture sequence-enrichment technology, plus other new Nim- maceutical, Biotechnology and Academic communities. Special- bleGen microarray products. Investigate real-time PCR with the izing in Next Generation Genomics, Sequencing, Expression LightCycler® 480 and LightCycler® 1536 Instruments, plus the Analysis and Molecular Biology services, SeqWright has earned addition of rat and mouse targets to our preplated qPCR assays. a reputation for quality, technical expertise and a willingness to provide client-specific solutions. Sage Science Booth 617 500 Cummings Center Sigma Life Science Booth 505 Suite 3150 3050 Spruce Street Beverly, MA 1915 St. Louis, MO 63103 United States United States P +1 978-922-1832 P +1 314-771-5765 F +1 617-812-0540 [email protected] [email protected] www.wherebiobegins.com www.sagescience.com SIGMA® Life Science offers a wide portfolio of innovative tech- Sage Science manufactures the Pippin PrepTM DNA size selec- nologies, products and services spanning cell biology, protein as- tion system. The Pippin Prep automates size selection of DNA says, genomics, functional genomics, biomolecules, epigenetics, for next-gen sequencing library preparations, providing a labor- stem cell research and transgenic animal models. Through con- saving alternative to manual gel extraction and purification. In tinued investment in innovation and quality we are the leading addition to time savings, one can achieve higher yields and elim- destination for life science researchers to access deep biological inate lower molecular weight adapter contamination. information and market leading products that improve the quality of life. SeqGen, Inc. Booth 604 1725 Del Amo Boulevard SoftGenetics Booth 110 Torrance, CA 90501 100 Oakwood Ave United States Suite 350 P +1 877-377-3743 State College, PA 16803 F +1 800-790-4830 United States [email protected] P +1 814-237-9340 www.seqgen.net F +1 814-237-9343 [email protected] SeqGen is a full service provider of high quality, refurbished, www.softgenetics.com DNA Sequencers, PCR Instrumentation, and Service Contracts. We maintain a large inventory of equipment/parts and have Featuring NextGENe® software for analysis of all massively paral- many years of experience supporting Biotech laboratories. Our lel sequencer data with newly included RNA-Seq module; en- expert SeqGen Engineers conduct installations, warranty calls, hanced capabilities of functionalities added to GeneMarker® service contracts, and paid day service calls. Software include carbohydrate analysis, new large sizing algorithms, kinship analysis of wild populations, genotype merge tool and project comparison tool. SoftGenetics is providing demonstrations and 30-day trials. Exhibitor Directory

ABRF 2011 — Technologies to Enable Personalized Medicine • 115 Exhibitor Directory — Continued

Tecan Booth 419 VICI Valco Instruments Booth 102 4022 Stirrup Creek Drive P.O. Box 55603 Suite 310 Houston, TX 77255 Durham, NC 27703 United States United States P +1 800-347-8424 P +1 919-361-5200 F +1 713-956-3119 [email protected] [email protected] www.tecan.com www.vici.com

Tecan is a leading global provider of laboratory instruments and VICI Valco Instruments is a designer/ manufacturer of standard/ solutions. The company specializes in the development, produc- custom valves and fittings for precision analytical, biomedical, tion and distribution of instruments and automated workflow and biocompatible instrumentation. Product variety includes; solutions for laboratories in the life sciences sector. Its clients in- pneumatic and electric actuators, tubing, sampling loops, heat- clude pharmaceutical and biotechnology companies, university ed enclosures, valve sequence and temperature controllers, gas research departments, forensic and diagnostic laboratories. purifiers, GC detectors, and digital interfaces. Devices and instrumentation for generation of calibration gas standards, con- Thermo Scientific Booth 200 tainment traps and gas specific purifiers. 355 River Oaks Parkway San Jose, CA 95134 Waters Corporation Booth 108 United States 34 Maple Street P +1 800-532-4752 Milford, MA 01757 [email protected] United States www.thermoscientific.com P +1 508-478-2000 F +1 508-872-1990 Thermo Scientific brand solutions leverage the market-leading [email protected] sample preparation products and ion trap mass spectrometers www.waters.com for definitive quantitation of protein mixtures, greatly improving protein characterization, accelerating protein database searches Waters helps laboratory-dependent organizations by provid- and reducing false identification rates. ing breakthrough technologies and solutions. Pioneering a con- nected portfolio of separation and analytical science, laboratory Vanderbilt University informatics and mass spectrometry, Waters provides the tools to Medical Center Booth 319 improve the quality of today’s science and explore the infinite 3401 West End Building possibilities of tomorrow. Waters, The Science of What’s Pos- Suite 300 sible. Nashville, TN 37203-8375 United States P +1 615-875-3363 F +1 615-936-6530 [email protected] www.mc.vanderbilt.edu/CORES

Vanderbilt University Medical Center (VUMC) is a comprehensive healthcare facility. Vanderbilt’s mission is to advance health and wellness through preeminent programs in patient care, education, and research. CORES (Core Ordering & Reporting Enter- prise System) is a web-based system developed by VUMC used to process core billing and produce usage reporting. Exhibitor Directory

116 • ABRF 2011 — Technologies to Enable Personalized Medicine Exhibit Hall Floorplan Grand Oaks Ballroom, Level 2 Level Ballroom, Oaks Grand Exhibit Hall Floorplan

ABRF 2011 — Technologies to Enable Personalized Medicine • 117 Exhibitor List in Alphabetical Order

Exhibitors confirmed as of January 29, 2011.

M = ABRF Annual Meeting Sponsor C = ABRF Corporate Sponsor

Company Booth Company Booth AB SCIEX ...... 501 Leica Microsystems ...... 217 ABRF ...... 317 Life Technologies ...... 601 Advanced Analytical ...... 602 MassTech, Inc...... 103

Agilent Technologies M C ...... 301 Matrix Science M ...... 704

AnaSpec, Eurogentec Group M C ...... 417 Michrom Bioresources ...... 615 Bioinformatics Solutions Inc. (BSI) ...... 402 Morehouse School of Medicine/RCMI Program ...... 204 Bioo Scientific ...... 718 New England Biolabs, Inc...... 205 Bio-Synthesis, Inc...... 104 New O bjective ...... 203 Bruker Daltonics Inc...... 214 Nonlinear Dynamics ...... 105

Caliper Life Sciences C ...... 304 Omega Bio-Tek ...... 605 CEM C orporation ...... 303 OpGen, In c...... 603 chemagen U SA ...... 100 OriGene Technologies, Inc...... 400 Dionex Corporation ...... 215 Peak Scientific, Inc...... 101 DNAnexus ...... 404 Phenix Research Products ...... 518 eagle-i Consortium ...... 221 Photometrics ...... 516

EdgeBio C ...... 300 PolyLC Inc...... 222 FASEB MARC Program ...... 416 Protea Biosciences, Inc...... 503 Fluidigm Corporation ...... 614 Proteome S oftware ...... 514 GE H ealthcare ...... 714 Randox Pharma Services ...... 302 Gene Codes Corporation ...... 220 Research Scientific Services...... 216

Genetix ...... 616 Roche Applied Science M C ...... 500 GENEWIZ, I nc...... 316 Sage Science ...... 617 Genomatix Software, Inc ...... 218 SeqGen, Inc...... 604 Geospiza ...... 519 SeqWright, In c...... 702 Hamilton Robotics ...... 223 Sigma Life Science ...... 505 iLab Solutions, LLC ...... 700 SoftGenetics...... 110

Illumina M C ...... 515 Tecan ...... 419 Intavis ...... 600 Thermo Scientific ...... 200

IntegenX, Inc. C ...... 201 Vanderbilt University Medical Center ...... 319 Integrated DNA Technologies ...... 305 VICI Valco Instruments ...... 102

Kapa Biosystems, Inc...... 619 Waters Corporation M C ...... 108 LC S ciences ...... 318 Exhibitor List – Alpha

118 • ABRF 2011 — Technologies to Enable Personalized Medicine Exhibitor List in Booth Order

Exhibitors confirmed as of January 29, 2011.

M = ABRF Annual Meeting Sponsor C = ABRF Corporate Sponsor

Booth Company Booth Company 100 ...... chemagen USA 400 ...... OriGene Technologies, Inc. 101 ...... Peak Scientific, Inc. 402 ...... Bioinformatics Solutions Inc. (BSI) 102 ...... VICI Valco Instruments 404 ...... DNAnexus 103 ...... MassTech, In c. 416 ...... FASEB MARC Program

104 ...... Bio-Synthesis, I nc. 417 ...... AnaSpec, Eurogentec Group M C 105 ...... Nonlinear D ynamics 419 ...... Tecan

108 ...... Waters C orporation M C 500 ...... Roche Applied Science M C 110 ...... SoftGenetics 501 ...... AB S CIEX 200 ...... Thermo S cientific 503 ...... Protea Biosciences, Inc.

201 ...... IntegenX, In c. C 505 ...... Sigma Life Science 203 ...... New O bjective 514 ...... Proteome S oftware

204 ...... Morehouse School of Medicine/RCMI Program 515 ...... Illumina M C 205 ...... New England Biolabs, Inc. 516 ...... Photometrics 214 ...... Bruker Daltonics Inc. 518 ...... Phenix Research Products 215 ...... Dionex C orporation 519 ...... Geospiza 216 ...... Research Scientific Services 600 ...... Intavis 217 ...... Leica M icrosystems 601 ...... Life Technologies 218 ...... Genomatix Software, Inc. 602 ...... Advanced A nalytical 220 ...... Gene Codes Corporation 603 ...... OpGen, In c. 221 ...... eagle-i C onsortium 604 ...... SeqGen, In c. 222 ...... PolyLC Inc. 605 ...... Omega B io-Tek 223 ...... Hamilton Robotics 614 ...... Fluidigm C orporation

300 ...... EdgeBio C 615 ...... Michrom B ioresources

301 ...... Agilent Technologies M C 616 ...... Genetix 302 ...... Randox Pharma Services 617 ...... Sage Science 303 ...... CEM C orporation 619 ...... Kapa Biosystems, Inc.

304 ...... Caliper Life Sciences C 700 ...... iLab Solutions, LLC 305 ...... Integrated DNA Technologies 702 ...... SeqWright, Inc.

316 ...... GENEWIZ, I nc. 704 ...... Matrix S cience M 317 ...... ABRF 714 ...... GE H ealthcare 318 ...... LC S ciences 718 ...... Bioo Scientific 319 ...... Vanderbilt University Medical Center Exhibitor List – Booth

ABRF 2011 — Technologies to Enable Personalized Medicine • 119 Vendor Presentations

Sunday, February 20 — 12:00 pm – 1:30 pm

Alyssum Room, Level 3 Grand Oaks Ballroom, Rooms N&O, Level 2 Exhibit Booth: 214 Exhibit Booth: 714

Moving Beyond Proteomics 12:00 pm – 12:30 pm Speaker: Detlev Suckau, Bruker Daltonik GmbH In current proteomics studies as well as biopharmaceutical de- Advances in Sample Preparation of Low velopment and characterization, post-translational modifica- Abundant Proteins using Magnetic Beads tions obtain increasing attention. Amongst these, glycosylation is Speaker: Helena Hedlund, prominent but the lack of bioinformatics solutions slowed down Global Product Manager, GE the analysis of glycoproteins in both, the biopharmaceutical in- Healthcare Life Sciences dustry as well as in proteomics studies. Low abundant proteins are often biologically important but difficult to detect. Some proteins are present in a few copies In this seminar, we introduce new solutions that include a new whereas others have a short transient presence. To be able to glycoprotein and glycoproteomics enabling bioinformatics plat- increase the detection and identification rate, efficient sample form and show examples from selected glycoprotein character- preparation methods are necessary. We have combined estab- izations including glycan and direct glycopeptide analysis using lished affinity chromatography methods with magnetic bead Brukers leading ESI-MSn and MALDI platforms. technology for the enrichment of such challenging proteins. In the first study changes in tyrosine phoshorylation in cancer cells Dedicated solutions for the development and quality control of upon drug treatment was investigated. Immmonoprecipitation Biologicals and Biosimilars will be described that show you how using Protein G Mag SepharoseTM was used for enrichment of to rapidly characterize therapeutic proteins with the confidence phosphorylated proteins prior to a 2-D DIGE experiment. The that is derived from the high specificity of Brukers ultra-high effect of the treatment will be presented as well as protein iden- resolution QTOF maXis. tification from MS analysis.

As a further extension of classical protein characterization and In the second study screening for optimal purification conditions validation workflows, we describe recent advances in Top-Down for a number of integral membrane proteins was performed. Vi- protein characterization tools that will make your C- and N-ter- tal parameters as choice of detergent wash and elution buffer minal definitions specific and rapid. The ESI-ETD and MALDI concentrations were evaluated. All experiments were done us- Top-Down Sequencing approaches will also forward your pro- ing Ni2+-charged His Mag Sepharose Ni. Results from screening teomics research as they provide simultaneous access to PTM for several detergents gave reliable results that could be fed into analysis, protein isoform differentiation and proteolytic regula- further scale-up experiments. The magnetic bead format simpli- tion events that are not addressed with current technologies. fied and shortened the handling procedures through-out these two different workflows.

12:30 pm – 1:00 pm Quantitative Western Blotting with AmershamTM ECLTM Prime Speaker: Maria Winkvist, Scientist, GE Healthcare Life Sciences Western blotting is a well established technique in life science research. Traditionally the technique has been restricted to qualitative protein analysis. However, development of refined detection methods and reagents has opened up the possibility to use the technique in quantitative analysis, for accurate monitoring of small changes in protein abundance and for detection of posttranslational modifications. Vendor Presentations Vendor

120 • ABRF 2011 — Technologies to Enable Personalized Medicine This workshop will present ways to achieve quantitative West- Grand Oaks Ballroom, ern blotting by optimizing the imaging system and detection re- Rooms P&Q, Level 2 agent. The impact of normalization, signal-to-noise, background noise and other experimental factors will also be discussed. Exhibit Booth: 515

1:00 pm – 1:30 pm Illumina’s New 2011 Portfolio and Announcements 2-D DIGE Analysis of Multicellular Tumor Spheroids in Evaluation of Breast Cancer Illumina’s broad portfolio of sequencing and array solutions Treatment empowers scientists in labs of all sizes to take their research Speaker: Viola Ruddat, Ph.D., Senior Applications Scientist, further, faster. Please join us to hear the details about the recent GE Healthcare Life Sciences announcements of the terabase sequencing milestone, our ability to enable our customers to reach 600 Gb/run this spring, Many cancers can be diagnosed using Positron Emission Tomog- and our newest Platform, MiSeq. raphy (PET) to visualize tumors. PET can also be used to monitor how effective various treatments are in individual patients We’ll also describe our progressive research path for GWAS, by observing the decrease in tumor tissue. Tumor spheroids are our sequencing service without compromise and Eco, our ultra- cancer cells grown on agar coated dishes forming a 3D structure. accessible qPCR system. They are widely used in preclinical cancer research, where the multicellular tumor spheroid model is considered biologically and physiologically similar to in vivo grown tumors.

In this workshop, we will present how we have used Two-dimensional Difference Gel Electrophoresis (2-D DIGE) analysis to gain more insight in changes of protein expression as a result of drug treatment of multicellular tumor spheroids.

Monday, February 21 — 12:00 pm – 1:30 pm

Grand Oaks Ballroom, Rooms R&S, Level 2 and quantify the responses of cells to different treatments. For rapid determination of cell growth in microwell plates, Clone- Exhibit Booth: 616 Select ImagerTM provides objective, quantitative and consistent characterization of cell growth prior to investigation of cellular Advanced Technologies for Antibody responses to different treatments such as siRNA, small molecule Discovery and Cell Screening or antibody. To determine the responses of cells, CellReport- erTM is used for fluorescent cell- and bead-based assays. Intuitive Speakers: Mark Truesdale, Gabe Longoria and Alison workflows provide flexibility to optimize and standardize assays Glaser from image acquisition through to data analysis. Typical applica- The use of antibodies in investigating and treating diseases is tions include cell-based assays for cell cycle analysis, apoptosis universal, however traditional methods for antibody develop- or monitoring protein translocation and bead-based homoge- ment are time consuming and costly. The Genetix ClonePixTM FL neous assays for quantifying cellular protein production. system improves operational efficiency by reducing timelines, minimizing resource requirement and allows higher throughput This seminar will present the latest technologies and applica- of projects. tions for screening cells, identification and isolation of clones, and data-rich cell based assays. Understanding disease mechanisms through the culture and study of relevant cell lines requires multiple technologies. Imag- ing systems from Genetix are used to characterize cell growth Vendor Presentations

ABRF 2011 — Technologies to Enable Personalized Medicine • 121 Vendor Presentations — Continued

Monday, February 21 — 12:00 pm – 1:30 pm (Continued)

Grand Oaks Ballroom, Next Generation qPCR solutions from Life Technologies Rooms N&O, Level 2 In 2010, Life TechnologiesTM released the Applied BiosystemsTM ViiaTM 7 Real-Time PCR System. This seventh generation system Exhibit Booth: 601 combines advances in optical and thermal cycling technology to provide one the most powerful and flexible Real-Time PCR From Discovery to Translational instruments available on the market today. After a brief review Genomics with Life Technologies’ of the ViiaTM 7 Real-Time PCR System, we will explore the next Comprehensive Sequencing Solutions generation of qPCR systems. The OpenArray® Real-Time PCR platform employs 3072 well Life Technologies offers the most comprehensive and comple- reaction plates to increase throughput and to lower costs for mentary portfolio of research sequencing solutions — from qPCR. The throughput and cost efficiencies of the OpenArray® the market leading Capillary Electrophoresis solutions, through Real-Time PCR platform are enabling applications such as Next- the recently launched Ion Torrent Personal Genome Machine, Gen Sequencing library quantitation by digital PCR, gene ex- (PGM) to SOLiDTM systems — with the 5500 series SOLiDTM sys- pression pathway analysis, high-throughput genotyping studies, tems becoming available soon. The re-engineered 5500 series and miRNA profiling. The OpenArray® Real-Time PCR platform SOLiDTM system generates up to 30 gigabases of mapped data delivers TaqMan® sensitivity and specificity in miRNA profiling per day and can be used in a myriad of research applications while achieving cost savings, comparable to microarray systems. — including complete cancer genome re-sequencing, single cell The throughput for miRNA profiling is up to 9 samples com- transcriptome analysis, interrogation of methylation status, and pleted in a 2.5 hour run, and up to 36 samples per day. de novo sequencing. The increasing throughput of the evolv- TM ing SOLiD platform and other next generation sequencing The next generation of Life Technologies’TM qPCR systems also technologies now makes it possible to sequence a whole human includes a novel technology platform based upon a continuous genome. As additional human genomes are sequenced, new flow liquid-bridge manifold. This will enable high throughput biological insights are obtained, and we begin to truly appreci- Genotyping and Gene Expression applications. The Stokes Bio ate the critical need for sequencing accuracy. Consensus accu- nanodroplet qPCR technology will be reviewed during this talk. racy can overcome some inherent error rates, however a floor is ultimately reached, thereby limiting identification of low frequency somatic mutations or SNV’s when using low coverage Alyssum Room, Level 3 techniques for GWAS based projects. A new chemistry incorporating error-correction codes has been developed that enables Exhibit Booth: 500 the SOLiDTM system to achieve 99.99% accuracy within a single sequencing read. This, and other technological improvements, will be discussed in the context of the emerging “Translational Amplicon Sequencing with GS FLX and Genomics” era that we have recently entered. GS Junior Systems PostLight Sequencing with Semiconductor Chips Ion Torrent has invented the first device—a new semiconduc- Access ArrayTM System High-Throughput Resequencing tor chip—capable of directly translating chemical signals into Speaker: Steven Hoffman, Fluidigm Corporation digital information. The first application of this technology is se- Flexible Next-Generation Sequencing Approaches Enabled quencing DNA. The device leverages decades of semiconduc- by the GS FLX and GS Junior Systems tor technology advances, and in just a few years has brought the Speaker: Bruce Taillon, Ph.D., 454 Life Sciences entire design, fabrication and supply chain infrastructure of that Featuring GS FLX Titanium series chemistry, the Genome Se- industry—a trillion dollar investment—to bear on the challenge quencer FLX and GS Junior Systems offer a powerful combina- of sequencing. The result is Ion semiconductor sequencing, the tion of long sequencing reads (400 to 500 base pairs) and dedi- first commercial sequencing technologythat does not use light, cated GS Amplicon Variant Analyzer (AVA) software, enabling and as a result delivers unprecedented speed, scalability and the sequencing, alignment, and analysis of amplicons against a low cost. reference sequence. Vendor Presentations Vendor

122 • ABRF 2011 — Technologies to Enable Personalized Medicine Grand Oaks Ballroom, Begonia Room, Level 3 Rooms P&Q, Level 2 Exhibit Booth: 108 Exhibit Booth: 505

Controlling Experimental Variables Recent Developments in Waters Speaker: Jim Walters, Ph.D., Principal Investigator, Analytical Proteomics Technology Research Department, Sigma Life Science Speaker: Martha Stapels, Principle Research Scientist, The enormous amount of data generated from complex pro- Waters teomic analyses with mass spectrometry is often overwhelming; We will be discussing advances in ETD, Ion Mobility and requiring iterative analysis and optimization of a number of vari- separations. Also featured will be a presentation by J. Will ables. To counteract the effects of potentially false data, a num- Thompson from Duke University: “Absolute Quantitation Using ber of defined protein and peptide standards have been de- 2DLC-MSE and Species-Specific Correction to Investigate veloped to allow for universal performance comparisons across Chlamydia trachomatis Developmental Forms.” platforms and between labs. We will highlight our efforts in the development, commercialization, and utilization of proteomics standards. We will discuss our newly developed standards which include both phosphopeptide standards as well as a synthetic protein designed to fully assess standard LC-MS platforms.

Monday, February 21 — 7:30 pm – 9:00 pm

Alyssum Room, Level 3 Exhibit Booth: 500

Reviews of SeqCap EZ Exome v2.0 Vendor Presentations

ABRF 2011 — Technologies to Enable Personalized Medicine • 123 Vendor Presentations — Continued

Tuesday, February 22 — 12:00 pm – 1:30 pm

Alyssum Room, Level 3 Grand Oaks Ballroom, Rooms N&O, Level 2 Exhibit Booth: 214 Exhibit Booth: 501

The Latest Solutions for Metabolomics Breakthrough Solutions for Proteomics, and Small Molecule Research Biomarker, and Lipidomics Research Speaker: Aiko Barsch, Bruker Daltonik GmbH Speakers: Doug Simmons and Jenny Albanese, AB SCIEX Metabolomics nowadays has several analytical challenges to Join us for lunch and learn about powerful new breakthrough in- overcome. The identification of metabolites is often defined as novations that can really boost your productivity in proteomics, the major bottleneck in metabolomics applications as well as in biomarker, and lipidomics research! general small molecule research. Begonia Room, Level 3 The confident generation of molecular formulae is the first step in the identification of unknowns. Especially for m/z values above Exhibit Booth: 108 500, the number of ambiguous assignments increases exponentially. maXisTM Ultra high resolution Q-TOF accurate mass and Recent Developments in Waters isotopic pattern data from MS and MS/MS spectra significantly Metabolomics and Lipidomics Biomarker extends the m/z range for reliable formula suggestions. The mo- Technologies lecular formula can then be queried against public databases to quickly match already known compounds. Hyphenating gas Speaker: Henry Shion, Principal Applications Scientist, chromatography with soft atmospheric pressure ionisation Waters (APCI) to high resolution Q-TOF-MS technology can preserve We will be discussing innovations in metabolomic/lipidomic the molecular ion information which enables the identification separations with UPLC, comprehensive data collection with MSE of unknowns in GC-MS data. and QuanTOF technology, and advanced data processing and database searching. One crucial advantage of applying maXis UHR-TOF in untargeted metabolomic studies is the speed of analysis by coupling U-HPLC systems without compromising the performance in terms of mass accuracy or resolution even at high acquisition rates up to 20Hz. Overall, the sample throughput is increased, thus addressing the necessity of handling large sample numbers needed for statistically relevant data. Vendor Presentations Vendor

124 • ABRF 2011 — Technologies to Enable Personalized Medicine Notes Notes

ABRF 2011 — Technologies to Enable Personalized Medicine • 125 Demos (as of January 29, 2011)

Monday, February 21 — 12:00 pm – 12:15 pm Monday, February 21 — 12:30 pm – 12:45 pm

Exhibit Hall Demo Stage Exhibit Hall Demo Stage Exhibit Booth: 503

Comprehensive Genomic Tools from Innovations in protein mass Clinical Research to Translational spectrometry from Protea Biosciences: Medicine: Paving the Way for New bioanalytical tools for protein Personalized Medicine analysis and characterization Speaker: Jessica Parra, Ph.D., Marketing Manager, Global Speakers: Christopher A. Bolcato, Technical Services Field Marketing Manager, and Haddon E. Goodman, Product Marketing Clinical researchers are making personalized medicine a real- Manager ity by translating biomarker discoveries into clinical diagnostic Presentation will include discussion around innovative technolo- tests. Although many biomarker signatures have been identified gies and techniques in gel protein separation and recovery, high in research labs, only a few microarray-based clinical diagnostic abundant protein depletion, and new surfactant technology tests have received clearance for commercialization. Clinical re- developed by Protea Biosciences. Several new products will be searchers face many challenges from the discovery process to discussed that aid in the preparation of protein samples for mass validation and routine testing. Using a single platform from clini- spectrometry. cal research discovery to clinical applications helps researchers to overcome the operational hurdles of commercialization. Af- Monday, February 21 — 12:15 pm – 12:30 pm fymetrix is paving the path to market with a toolkit that includes instrumentation, arrays, and reagents for use in developing clinical diagnostic tests. The convenience of a cGMP-manufactured Exhibit Hall Demo Stage kit helps to reduce rework and risk during the development process to fast-track commercialization Exhibit Booth: 515

Monday, February 21 — 1:00 pm – 1:15 pm Illumina’s Genotyping Portfolio Featuring a New FFPE Solution Exhibit Hall Demo Stage The revolutionary Infinium HD FFPE Restore kit has expanded Exhibit Booth: 110 Illumina’s broad portfolio of array solutions to enable genotyping of formalin-fixed, paraffin-embedded (FFPE) samples on the Infinium platform. Join us to hear more about this exciting option available for the CyotSNP and OmniExpress arrays. We’ll also NextGENe’s RNA-Seq Analysis tool describe the latest evolution of iSelect, the most flexible custom Speaker: Megan R. Manion genotyping option available today, and give a brief update on NextGENe’s RNA-Seq Analysis tool, which includes a proprie- the Omni GWAS Roadmap. tary alignment algorithm specifically designed for transcriptome data, will be presented. Project set-up and results will be shown. Results, which include mutation detected, expression analysis and alternative splicing analysis, are displayed in the interactive NextGENe Viewer which includes detailed annotation. Next- GENe is a standalone Windows-based program for the analysis of Next Generation Sequencing data and is compatible with data from Illumina Genome Analyzers, Roche/454 GS FLX, FLX Titanium and Junior, the SOLiD System and Ion Torrent’s Ion Personal Genome Machine. In addition to RNA-Seq analysis,

Demos 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 pm

Exhibit Hall Demo Stage Exhibit Booth: 515

My samples. My study. MiSeqTM. Introducing Illumina’s personal sequencing system.

The MiSeq personal sequencing system delivers the fastest time to answer, a revolutionary workflow, and the widest breadth of sequencing applications, all in a compact and economical instrument. This session will provide an introduction to the instrument and showcase the revolutionary workflow — come experience it for yourself. Demos

ABRF 2011 — Technologies to Enable Personalized Medicine • 127 Membership Application

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128 • ABRF 2011 — Technologies to Enable Personalized Medicine DO, RY RESORT • ORLAN FLORIDA TEMPORA ’S CON SNEY • DI 012 0, 2 6-2 H 1 RC MA

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