Analysis abstract
Nature Methods 6, 423 - 430 (2009)
Published online: 17 May 2009 | Corrected online: 29 June 2009 | doi:10.1038/nmeth.1333
There is a Corrigendum (July 2009) associated with this Analysis.
There is a Corrigendum (July 2009) associated with this Analysis.
There is a Corrigendum (July 2009) associated with this Analysis.
A HUPO test sample study reveals common problems in mass spectrometry–based proteomics
Alexander W Bell1, Eric W Deutsch2, Catherine E Au1, Robert E Kearney3, Ron Beavis4, Salvatore Sechi5, Tommy Nilsson6, John J M Bergeron1 & HUPO Test Sample Working Group7
Abstract
We performed a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in liquid chromatography–mass spectrometry–based proteomics. We distributed an equimolar test sample, comprising 20 highly purified recombinant human proteins, to 27 laboratories. Each protein contained one or more unique tryptic peptides of 1,250 Da to test for ion selection and sampling in the mass spectrometer. Of the 27 labs, members of only 7 labs initially reported all 20 proteins correctly, and members of only 1 lab reported all tryptic peptides of 1,250 Da. Centralized analysis of the raw data, however, revealed that all 20 proteins and most of the 1,250 Da peptides had been detected in all 27 labs. Our centralized analysis determined missed identifications (false negatives), environmental contamination, database matching and curation of protein identifications as sources of problems. Improved search engines and databases are needed for mass spectrometry–based proteomics.
- Department of Anatomy and Cell Biology, McGill University, Montreal, Canada.
- The Institute for Systems Biology, Seattle, Washington, USA.
- Department of Biomedical Engineering, McGill University, Montreal, Canada.
- Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
- Division Diabetes, Endocrinology and Metabolic Diseases, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
- The Research Institute of the McGill University Health Centre and the Department of Medicine, McGill University, Montreal, Canada.
- A full list of authors appears at the end of this paper.
- Verdezyne, Inc., Carlsbad, California, USA.
- BioGrammatics Incorporated, Carlsbad, California, USA.
- Invitrogen Corporation, Carlsbad, California, USA.
- Allergan, Irvine, California, USA.
- Ambry Genetics, Aliso Viejo, California, USA.
- Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts, USA.
- State Key Laboratory of Proteomics, Beijing Proteome Research Center, Changping District, Beijing, China.
- Bochum University, Ruhr-Universitaet Bochum, Bochum, Germany.
- Proteomics, Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts, USA.
- Burnham Institute for Medical Research, La Jolla, California, USA.
- Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
- Georgetown University, Department of Oncology, Washington, DC, USA.
- Göteborg Proteomics Centre: The Proteomics Core Facility, Sahlgrenska Academy, University of Göteborg, Göteborg, Sweden.
- Harvard Partners Center for Genetics and Genomics, Cambridge, Massachusetts, USA.
- Thermo-Fisher BRIMS Center, Cambridge, Massachusetts, USA.
- Proteomics Platform, Quebec Genomic Center, Laval University Medical Research Center, Quebec, Canada.
- Health and Environment Unit, Laval University Medical Research Center, Quebec, Canada.
- Joint Proteomics Laboratory, Ludwig Institute for Cancer Research and The Walter & Eliza Hall Institute for Medical Research, Parkville, Australia.
- Genizon BioSciences Incorporated, Saint Laurent, Canada.
- McGill University and Genome Quebec Innovation Centre, Montréal, Canada.
- Ontario Cancer Biomarker Network, MaRS Centre, Toronto, Canada.
- Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada.
- The Scripps Research Institute, Department of Chemical Physiology, La Jolla, California, USA.
- Department of Biochemistry, University of Alberta, Edmonton, Canada.
- Departments of Physiology, Medicine and Division of Cardiology, David Geffen School of Medicine, University of California, Los Angeles, California, USA.
- Proteome Research Centre, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland.
- Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
- Clinical Proteomics Facility, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, USA.
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
- Department of Pathology, University of Pittsburgh School of Medicine, Magee-Womens Research Institute, Pittsburgh, Pennsylvania, USA.
- University of Victoria, Victoria, Canada.
- Department of Biochemistry, University of Western Ontario, London, Ontario, Canada.
- The Wistar Institute, Philadelphia, Pennsylvania, USA.
- Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
- Yonsei Proteome Research Center, Yonsei University, Sudaemoon-ku, Seoul, Korea.
- Agilent Technologies Incorporated, Santa Clara, California, USA.
- Applied Biosystems, Foster City, California, USA.
- Waters Corporation, Milford, Massachusetts, USA.
- EMBL Outstation, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.
Correspondence to: John J M Bergeron1 e-mail: john.bergeron@mcgill.ca
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