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

Abstract

We carried out a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in LC-MS-based proteomics. We distributed a test sample consisting of an equimolar mix of 20 highly purified recombinant human proteins, to 27 laboratories for identification. Each protein contained one or more unique tryptic peptides of 1250 Da to also test for ion selection and sampling in the mass spectrometer. Of the 27 labs, initially only 7 labs reported all 20 proteins correctly, and only 1 lab reported all the tryptic peptides of 1250 Da. Nevertheless, a subsequent centralized analysis of the raw data revealed that all 20 proteins and most of the 1250 Da peptides had in fact been detected by all 27 labs. The centralized analysis allowed us to determine sources of problems encountered in the study, which include missed identifications (false negatives), environmental contamination, database matching, and curation of protein identifications. Improved search engines and databases are likely to increase the fidelity of mass spectrometry-based proteomics.

Cell Biology of the Endoplasmic Reticulum and the Golgi Apparatus through Proteomics

“…Indeed, of the 27 labs, only seven were able to do this correctly (Bell et al 2009). This was not because the quality of the tandem mass spectra generated was poor.…”

The transition of the European Proteomics Association into the future

“…ABRF, HUPO and ProteoRed [1][2][3][4][5] have developed test standards to evaluate end-to-end quality control for proteomics. A detailed effort towards systematic QC has been undertaken by the National Cancer Institute (NCI) via its large-scale Clinical Proteomic Technology Assessment for Cancer (CPTAC) project [6,7]. Clearly these efforts underscore the importance of excellence in proteomics standards and by virtue of their findings have defined actions for improving the quality of data and evaluating the robustness, repeatability and reproducibility of defined proteomics workflows [8].…”

Role of proteomics in nutrigenomics and nutrigenetics

“…Editorial Kussman range) of current mass spectrometers, but the dynamic range of protein concentrations (e.g., estimated to be 10 12 in human blood) [14], which still leads to under-sampling, redundancy and irreproducibility in protein identifications and quantification [15,16]. Current mass spectrometry (MS)-based proteomic platforms can deliver a dynamic range of 10 4 .…”