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

Alexander W. Bell, Eric W. Deutsch, Catherine E. Au, Robert E. Kearney, Ron Beavis, Salvatore Sechi, Tommy Nilsson, John J.M. Bergeron*, 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 JiaLina 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, Steve 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, 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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

293 Scopus citations


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.

Original languageEnglish
Pages (from-to)423-430
Number of pages8
JournalNature Methods
Issue number6
StatePublished - 2009
Externally publishedYes


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