Improving Peptide-Level Mass Spectrometry Analysis via Double Competition

Lin, Andy; Short, Temana; Noble, William Stafford; Keich, Uri

doi:10.1021/acs.jproteome.2c00282

Improving Peptide-Level Mass Spectrometry Analysis via Double Competition

Journal Article · Tue Sep 27 00:00:00 EDT 2022 · Journal of Proteome Research

DOI:https://doi.org/10.1021/acs.jproteome.2c00282· OSTI ID:1895576

^[1]; Short, Temana ^[2]; ^[3]; ^[2]

Pacific Northwest National Lab. (PNNL), Seattle, WA (United States)
Univ. of Sydney, NSW (Australia)
Univ. of Washington, Seattle, WA (United States)

A critical statistical task in the analysis of shotgun proteomics data involves controlling the false discovery rate (FDR) among the reported set of discoveries. This task is most commonly solved at the peptide-spectrum match (PSM) level by using target-decoy competition (TDC), in which a set of observed spectra are searched against a database containing a mixture of real (target) and decoy peptides. The PSM-level procedure can be adapted to the peptide level by selecting the top-scoring PSM per peptide prior to FDR estimation. Here, we investigate both PSM-level and peptide-level FDR control methods and come to two conclusions. First, although the TDC procedure is provably correct under certain assumptions, we observe that one of these assumptions - that incorrect PSMs are independent of one another - is frequently violated. Hence, we empirically demonstrate that TDC-based PSM-level FDR estimates can be liberally biased. Further, we propose that researchers avoid PSM-level results and instead focus on peptide-level analysis. Second, we investigate three ways to carry out peptide level TDC and show that the most common method ("PSM-only") offers the lowest statistical power in practice. The most powerful method, peptide-level FDR with PSM competition ("PSM-and-peptide"), carries out competition first at the PSM level and then again at the peptide level. In our experiments, this approach yields an average increase of 17% more discovered peptides at a1% FDR threshold relative to the PSM-only method.

View Accepted Manuscript (DOE)

Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Organization:: National Institutes of Health (NIH); USDOE

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 1895576

Report Number(s):: PNNL-SA-173059

Journal Information:: Journal of Proteome Research, Journal Name: Journal of Proteome Research Journal Issue: 10 Vol. 21; ISSN 1535-3893

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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