Name: HUPO test samples - A HUPO test sample study reveals common problems in mass spectrometry-based proteomics
Keywords: Not available

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

ABSTRACT: Not available

INSTRUMENT(S): LTQ Orbitrap, Q-Tof Premier, Bruker Daltonics micrOTOF series, Bruker Daltonics esquire series, LTQ, 4800 Proteomics Analyzer, Q-Tof ultima, LTQ FT, instrument model, LCQ Deca XP Plus, QSTAR Pulsar, LTQ XL, QSTAR

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: A BELL

PROVIDER: PRD000069 | Pride | 2009-05-15

REPOSITORIES: Pride

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Publications

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

Bell Alexander W AW Deutsch Eric W EW Au Catherine E CE Kearney Robert E RE Beavis Ron R Sechi Salvatore S Nilsson Tommy T Bergeron John J M JJ

Nature methods 20090601 6

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 ...[more]

PMID: 19448641

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

Project description: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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A proteomic study of the HUPO Plasma Proteome Project's pilot samples using an accurate mass and time tag strategy.

Project description:Characterization of the human blood plasma proteome is critical to the discovery of routinely useful clinical biomarkers. We used an accurate mass and time (AMT) tag strategy with high-resolution mass accuracy cLC-FT-ICR MS to perform a global proteomic analysis of pilot study samples as part of the HUPO Plasma Proteome Project. HUPO reference serum and citrated plasma samples from African Americans, Asian Americans, and Caucasian Americans were analyzed, in addition to a Pacific Northwest National Laboratory reference serum and plasma. The AMT tag strategy allowed us to leverage two previously published "shotgun" proteomics experiments to perform global analyses on these samples in triplicate in less than 4 days total analysis time. A total of 722 (22% with multiple peptide identifications) International Protein Index redundant proteins, or 377 protein families by ProteinProphet, were identified over the six individual HUPO serum and plasma samples. The samples yielded a similar number of identified redundant proteins in the plasma samples (average 446 +/- 23) as found in the serum samples (average 440 +/- 20). These proteins were identified by an average of 956 +/- 35 unique peptides in plasma and 930 +/- 11 unique peptides in serum. In addition to this high-throughput analysis, the AMT tag approach was used with a Z-score normalization to compare relative protein abundances. This analysis highlighted both known differences in serum and citrated plasma such as fibrinogens, and reproducible differences in peptide abundances from proteins such as soluble activin receptor-like kinase 7b and glycoprotein m6b. The AMT tag strategy not only improved our sample throughput but also provided a basis for estimated quantitation.

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