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A HUPO test sample study reveals common problems in mass spectrometry–based proteomics

Nature Methods volume 6, pages 423–430 (2009)Cite this article

A Corrigendum to this article was published on 01 July 2009

This article has been updated

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.

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Figure 1: Number of tandem mass spectra assigned to tryptic peptides.
Figure 2: Discrepancies between reported data and centralized analysis identify erroneous reporting.

Change history

  • 29 June 2009

    NOTE: In the version of this article initially published, the author name Steven A Carr was spelled incorrectly, and the name of an organization described in the text, the HUPO Proteomics Standards Initiative (PSI), was given incorrectly. These errors have been corrected in the PDF and HTML versions of this article.

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Acknowledgements

Supported in part by Canadian Institutes of Health Research to the HUPO Head Quarters (S. Ouellette) for coordination of this HUPO test sample initiative. A.W.B. and C.E.A. were supported by Genome Quebec and McGill University. We thank D. Juncker, G. Temple, J. van Oostrum, G. Omenn, K. Colwill, J. Langridge and M. Hallett for their comments on the manuscript, and D.M. Desiderio for helpful comments on the manuscript. This test sample effort builds on pioneering efforts from several other groups and especially Association of Biomolecular Resource Facilities. This study is a HUPO test sample initiative and HUPO welcomes collaborative efforts to benefit proteomics. We acknowledge the following sources of grant support: E.W.D. is supported by the National Heart, Lung and Blood Institute, National Institutes of Health (NIH), under contract N01-HV-28179; the University of California, Los Angeles Burnham Institute for Medical Research NIH grant number RR020843; University of California, Los Angeles (National Heart, Lung and Blood Institute P01-008111); University of Michigan, NIH P41RR018627; Beijing Proteome Research Center, affiliated with The Beijing Institute of Radiation Medicine for National Key Programs for Basic Research grant 2006CB910801 and Hi-Tech Research grant 2006AA02A308. We acknowledge access and use of The University College Dublin Conway Mass Spectrometry Resource instrumentation, supported by Science Foundation, Ireland grant 04/RPI/B499. PRIDE, J.A.V. is a postdoctoral fellow of the “Especialización en Organismos Internacionales” program from the Spanish Ministry of Education and Science. L.M. is supported by the “ProDaC” grant LSHG-CT-2006-036814 of the EU. Samuel Lunenfeld Research Institute, Mount Sinai, Toronto is supported by Genome Canada through Ontario Genomics Institute. J.A.V. and L.M. thank H. Hermjakob and R. Apweiler for their support. A.W.B. thanks L. Roy and Z. Bencsath-Makkai for help in data submission and analysis.

Author information

Authors and Affiliations

  1. Department of Anatomy and Cell Biology, McGill University, Montreal, Canada

    Alexander W Bell, Catherine E Au & John J M Bergeron

  2. The Institute for Systems Biology, Seattle, Washington, USA

    Eric W Deutsch

  3. Department of Biomedical Engineering, McGill University, Montreal, Canada

    Robert E Kearney

  4. Biomedical Research Centre, University of British Columbia, Vancouver, Canada

    Ron Beavis

  5. Division Diabetes, Endocrinology and Metabolic Diseases, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Salvatore Sechi

  6. The Research Institute of the McGill University Health Centre and the Department of Medicine, McGill University, Montreal, Canada

    Tommy Nilsson

  7. Verdezyne, Inc., Carlsbad, California, USA

    Thomas A Beardslee

  8. BioGrammatics Incorporated, Carlsbad, California, USA

    Thomas Chappell

  9. Invitrogen Corporation, Carlsbad, California, USA

    Gavin Meredith, Mahbod Hajivandi, Marshall Pope & Paul Predki

  10. Allergan, Irvine, California, USA

    Peter Sheffield

  11. Ambry Genetics, Aliso Viejo, California, USA

    Phillip Gray

  12. Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts, USA

    Majlinda Kullolli, Marina Hincapie & William S Hancock

  13. State Key Laboratory of Proteomics, Beijing Proteome Research Center, Changping District, Beijing, China

    Wei Jia, Lina Song, Lei Li, Junying Wei, Bing Yang, Jinglan Wang, Wantao Ying, Yangjun Zhang, Yun Cai, Xiaohong Qian & Fuchu He

  14. Bochum University, Ruhr-Universitaet Bochum, Bochum, Germany

    Helmut E Meyer, Christian Stephan, Martin Eisenacher, Katrin Marcus, Elmar Langenfeld & Caroline May

  15. Proteomics, Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts, USA

    Steven A Carr & Rushdy Ahmad

  16. Burnham Institute for Medical Research, La Jolla, California, USA

    Wenhong Zhu & Jeffrey W Smith

  17. Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

    Samir M Hanash, Jason J Struthers, Hong Wang & Qing Zhang

  18. Department of Oncology, Georgetown University, Washington, DC, USA

    Yanming An & Radoslav Goldman

  19. Göteborg Proteomics Centre: The Proteomics Core Facility, Sahlgrenska Academy, University of Göteborg, Göteborg, Sweden

    Elisabet Carlsohn & Sjoerd van der Post

  20. Harvard Partners Center for Genetics and Genomics, Cambridge, Massachusetts, USA

    Kenneth E Hung, Kenneth Parker & Raju Kucherlapati

  21. Thermo-Fisher BRIMS Center, Cambridge, Massachusetts, USA

    David A Sarracino & Bryan Krastins

  22. Proteomics Platform, Quebec Genomic Center, Laval University Medical Research Center, Quebec, Canada

    Sylvie Bourassa

  23. Health and Environment Unit, Laval University Medical Research Center, Quebec, Canada

    Guy G Poirier

  24. Joint Proteomics Laboratory, Ludwig Institute for Cancer Research and The Walter & Eliza Hall Institute for Medical Research, Parkville, Australia

    Eugene Kapp, Heather Patsiouras, Robert Moritz & Richard Simpson

  25. Genizon BioSciences Incorporated, Saint Laurent, Canada

    Benoit Houle

  26. McGill University and Genome Quebec Innovation Centre, Montréal, Canada

    Sylvie LaBoissiere

  27. Ontario Cancer Biomarker Network, MaRS Centre, Toronto, Canada

    Pavel Metalnikov

  28. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada

    Vivian Nguyen & Tony Pawson

  29. Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA

    Catherine C L Wong, Daniel Cociorva & John R Yates III

  30. Department of Biochemistry, University of Alberta, Edmonton, Canada

    Michael J Ellison, Ana Lopez-Campistrous & Paul Semchuk

  31. Departments of Physiology, Medicine and Division of Cardiology, David Geffen School of Medicine, University of California, Los Angeles, California, USA

    Yueju Wang & Peipei Ping

  32. Proteome Research Centre, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland

    Giuliano Elia, Michael J Dunn & Kieran Wynne

  33. Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA

    Angela K Walker, John R Strahler, Philip C Andrews & Jayson A Falkner

  34. Clinical Proteomics Facility, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, USA

    Brian L Hood, William L Bigbee & Thomas P Conrads

  35. Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Brian L Hood & Thomas P Conrads

  36. Department of Pathology, University of Pittsburgh School of Medicine, Magee-Womens Research Institute, Pittsburgh, Pennsylvania, USA

    William L Bigbee

  37. University of Victoria, Victoria, Canada

    Derek Smith & Christoph H Borchers

  38. Department of Biochemistry, University of Western Ontario, London, Ontario, Canada

    Gilles A Lajoie & Sean C Bendall

  39. The Wistar Institute, Philadelphia, Pennsylvania, USA

    Kaye D Speicher & David W Speicher

  40. Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan

    Masanori Fujimoto & Kazuyuki Nakamura

  41. Yonsei Proteome Research Center, Yonsei University, Sudaemoon-ku, Seoul, Korea

    Young-Ki Paik, Sang Yun Cho, Min-Seok Kwon, Hyoung-Joo Lee, Seul-Ki Jeong & An Sung Chung

  42. Agilent Technologies Incorporated, Santa Clara, California, USA

    Christine A Miller & Rudolf Grimm

  43. Applied Biosystems, Foster City, California, USA

    Katy Williams

  44. Waters Corporation, Milford, Massachusetts, USA

    Craig Dorschel

  45. EMBL Outstation, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK

    Lennart Martens & Juan Antonio Vizcaíno

Authors
  1. Alexander W Bell
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  2. Eric W Deutsch
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  3. Catherine E Au
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  4. Robert E Kearney
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  5. Ron Beavis
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  6. Salvatore Sechi
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  7. Tommy Nilsson
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  8. John J M Bergeron
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Consortia

HUPO Test Sample Working Group

  • Thomas A Beardslee
  • , Thomas Chappell
  • , Gavin Meredith
  • , Peter Sheffield
  • , Phillip Gray
  • , Mahbod Hajivandi
  • , Marshall Pope
  • , Paul Predki
  • , Majlinda Kullolli
  • , Marina Hincapie
  • , William S Hancock
  • , Wei Jia
  • , Lina Song
  • , Lei Li
  • , Junying Wei
  • , Bing Yang
  • , Jinglan Wang
  • , Wantao Ying
  • , Yangjun Zhang
  • , Yun Cai
  • , Xiaohong Qian
  • , Fuchu He
  • , Helmut E Meyer
  • , Christian Stephan
  • , Martin Eisenacher
  • , Katrin Marcus
  • , Elmar Langenfeld
  • , Caroline May
  • , Steven A Carr
  • , Rushdy Ahmad
  • , Wenhong Zhu
  • , Jeffrey W Smith
  • , Samir M Hanash
  • , Jason J Struthers
  • , Hong Wang
  • , Qing Zhang
  • , Yanming An
  • , Radoslav Goldman
  • , Elisabet Carlsohn
  • , Sjoerd van der Post
  • , Kenneth E Hung
  • , David A Sarracino
  • , Kenneth Parker
  • , Bryan Krastins
  • , Raju Kucherlapati
  • , Sylvie Bourassa
  • , Guy G Poirier
  • , Eugene Kapp
  • , Heather Patsiouras
  • , Robert Moritz
  • , Richard Simpson
  • , Benoit Houle
  • , Sylvie LaBoissiere
  • , Pavel Metalnikov
  • , Vivian Nguyen
  • , Tony Pawson
  • , Catherine C L Wong
  • , Daniel Cociorva
  • , John R Yates III
  • , Michael J Ellison
  • , Ana Lopez-Campistrous
  • , Paul Semchuk
  • , Yueju Wang
  • , Peipei Ping
  • , Giuliano Elia
  • , Michael J Dunn
  • , Kieran Wynne
  • , Angela K Walker
  • , John R Strahler
  • , Philip C Andrews
  • , Brian L Hood
  • , William L Bigbee
  • , Thomas P Conrads
  • , Derek Smith
  • , Christoph H Borchers
  • , Gilles A Lajoie
  • , Sean C Bendall
  • , Kaye D Speicher
  • , David W Speicher
  • , Masanori Fujimoto
  • , Kazuyuki Nakamura
  • , Young-Ki Paik
  • , Sang Yun Cho
  • , Min-Seok Kwon
  • , Hyoung-Joo Lee
  • , Seul-Ki Jeong
  • , An Sung Chung
  • , Christine A Miller
  • , Rudolf Grimm
  • , Katy Williams
  • , Craig Dorschel
  • , Jayson A Falkner
  • , Lennart Martens
  •  & Juan Antonio Vizcaíno

Contributions

A.W.B. coordinated all steps of the study. C.E.A., T.N. and J.J.M.B. coordinated data analysis and the final manuscript. E.W.D., R.B. and R.K. did the centralized analysis of the collective data retrieved from the raw data supplied from each lab to Tranche. S.A.C., P.P., L.M., E.K., C.D., S.S., X.Q., K.W., T.P.C., K.P. and T.A.B. provided comments. Invitrogen prepared, designed and distributed the test sample proteins.

Corresponding author

Correspondence to John J M Bergeron.

Ethics declarations

Competing interests

There is a potential to market the test samples used in this study.

Additional information

A full list of authors appears at the end of this paper.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 , Supplementary Tables 1–5 and 7–11, Supplementary Note, Supplementary Methods (PDF 2922 kb)

Supplementary Table 6

Initial results as submitted by 24 academic laboratories and 3 vendors. (XLS 110 kb)

Supplementary Table 12

Final results and repeat analyses as submitted by 24 academic laboratories and 3 vendors. (PDF 136 kb)

Supplementary Table 13

Peptides identified by centralized analysis of the data. (XLS 607 kb)

Supplementary Table 14

Tranche hash and passphrase codes. (XLS 25 kb)

Supplementary Table 15

Proteins identified by centralized analysis of the data. (XLS 177 kb)

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Bell, A., Deutsch, E., Au, C. et al. A HUPO test sample study reveals common problems in mass spectrometry–based proteomics. Nat Methods 6, 423–430 (2009). https://doi.org/10.1038/nmeth.1333

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