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A HUPO test sample study reveals common problems in mass spectrometrybased proteomics
2009 Nature America, Inc. All rights reserved.
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
We performed a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in liquid chromatographymass spectrometrybased proteomics. We distributed an equimolar test sample, comprising 20 highly puried 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 ofonly 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 identications (false negatives), environmental contamination, database matching and curation of protein identications as sources of problems. Improved search engines and databases are needed for mass spectrometrybased proteomics.
Liquid chromatographymass spectrometry (LC-MS) has become the most popular technique for proteomics analysis. In this strategy, proteins of a sample are typically separated by PAGE and then digested with trypsin. After extraction from the gel, peptides are separated by liquid chromatography and upon elution are ionized via electrospray into the mass spectrometer for characterization by mass analysis. The mass spectrometer subsequently selects peptides for fragmentation to yield mass values that are then used to identify the peptide and the corresponding protein by searching sequence databases. This technique, termed tandem mass spectrometry (MS), is repeated to continuously select ionized peptides from the liquid chromatography column. Depending on protein abundance and complexity, the mass spectrometer type and its setup, up to about 15,000 peptides and up to about 4,000 proteins can be identied in a single experiment1.
Despite the high mass accuracy of modern mass spectrometers, the general perception of the reliability of MS-based proteomics is that it is low. Previous test sample studies have demonstrated
that there is both a lack of reproducibility between different laboratories as well as...