Two-Dimensional Target Decoy Strategy for Shotgun Proteomics

Abstract

The target-decoy approach to estimating and controlling false discovery rate (FDR) has become a de facto standard in shotgun proteomics, and it has been applied at both the peptide-to-spectrum match (PSM) and protein levels. Current bioinformatics methods control either the PSM- or the protein-level FDR, but not both. In order to obtain the most reliable information from their data, users must employ one method when the number of tandem mass spectra exceeds the number of proteins in the database and another method when the reverse is true. Here we propose a simple variation of the standard target-decoy strategy that estimates and controls PSM and protein FDRs simultaneously, regardless of the relative numbers of spectra and proteins. We demonstrate that even if the final goal is a list of PSMs with a fixed low FDR and not a list of protein identifications, the proposed two-dimensional strategy offers advantages over a pure PSM-level strategy.

Protein Analysis by Shotgun/Bottom-up Proteomics

“…609 Considering both PSM and protein false discovery simultaneously in a two-dimensional target decoy strategy yielded a more versatile and accurate representation of peptides and proteins within a sample. 610 A strategy has been presented to infer the proteins present within a complex sample from hundreds of LC-MS runs where protein false discovery rates are difficult to control using conventional PSM false discovery rates. 611 Similarly, protein database size and quality directly influence the size and quality of the identified protein and peptide candidates.…”

Determining the calibration of confidence estimation procedures for unique peptides in shotgun proteomics

“…Such procedures include commonly used algorithms such as ProteinProphet [15], MaxQuant [16] and Fido [17]. It should be noted, however, that protein confidence is sometimes estimated directly from PSMs [18, 19]. …”

ProteinInferencer: Confident protein identification and multiple experiment comparison for large scale proteomics projects

“…Decoy protein hits usually contain a limited number of PSMs and therefore will be scored lower at the protein level. As demonstrated previously,[33] (Figure 2) to exclude the false proteins Y and Z with DTASelect, extremely stringent criteria are required which may also deplete many true PSMs and peptides. With ProteinInferencer, the proteins Y and Z are discarded because of their low protein scores.…”