A probability-based approach for high-throughput protein phosphorylation analysis and site localization

• Published in: Nature biotechnology Vol. 24; no. 10; pp. 1285 - 1292
• Main Authors: Beausoleil, Sean A, Villén, Judit, Gerber, Scott A, Rush, John, Gygi, Steven P
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.10.2006; Nature; Nature Publishing Group
• Subjects: Agriculture; Algorithms; Analytical, structural and metabolic biochemistry; Biochemistry; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; HeLa Cells - drug effects; Humans; Image Processing, Computer-Assisted; Ions; letter; Life Sciences; Liquid chromatography; Mass spectrometry; Mass Spectrometry - methods; Methods; Nocodazole - pharmacology; Peptide Mapping - methods; Phosphorylation; Probability; Proteins; Scientific imaging; Validation; Data analysis; Phosphorylation; Liquid chromatography; Method; Localization; Hela cell line; Mass spectrometry; Protein
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/nbt1240

Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) ( Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.