Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry

Abstract

Liquid chromatography and tandem mass spectrometry (LC-MS/MS) has become the preferred method for conducting large-scale surveys of proteomes. Automated interpretation of tandem mass spectrometry (MS/MS) spectra can be problematic, however, for a variety of reasons. As most sequence search engines return results even for 'unmatchable' spectra, proteome researchers must devise ways to distinguish correct from incorrect peptide identifications. The target-decoy search strategy represents a straightforward and effective way to manage this effort. Despite the apparent simplicity of this method, some controversy surrounds its successful application. Here we clarify our preferred methodology by addressing four issues based on observed decoy hit frequencies: (i) the major assumptions made with this database search strategy are reasonable; (ii) concatenated target-decoy database searches are preferable to separate target and decoy database searches; (iii) the theoretical error associated with target-decoy false positive (FP) rate measurements can be estimated; and (iv) alternate methods for constructing decoy databases are similarly effective once certain considerations are taken into account.

Application of capillary isotachophoresis‐based multidimensional separations coupled with electrospray ionization‐tandem mass spectrometry for characterization of mouse brain mitochondrial proteome

“…Thus, many proteomic research laboratories routinely discard single peptide identifications to significantly reduce the FDR of distinct protein identifications. However, as pointed out by the work of Elias and Gygi [57] and our laboratories [47], these single-protein identifications after filtering are mostly correct and often represent 30-50% of a proteome dataset. The removal of these protein identifications greatly and negatively impacts the coverage and the sensitivity of overall proteome analysis.…”

“…The removal of these protein identifications greatly and negatively impacts the coverage and the sensitivity of overall proteome analysis. As advocated by Elias and Gygi [57] and also implemented in this study, the targetdecoy search strategy is employed as the routine practice to design more stringent criteria for single-peptide identifications.…”

The peptide-binding motif of HLA-DR8 shares important structural features with other type 1 diabetes-associated alleles

“…21 The target/decoy strategy is based on the principle that incorrect matches have an equal probability of being derived from either the target or the decoy database. 22,23 In contrast, spectra with relevant sequence information will match their corresponding sequence in the target database but not in the decoy database. Most peptides were present as 'peptide families' or 'nested sets', in which all the members share a common sequence with varying flanking residues along the C-and the N-termini, as is usual for MHC class II peptides.…”

Landscape of nuclear transport receptor cargo specificity

“…The reversed sequences of the target database were used as decoy database. Peptide and protein hits were filtered at a false discovery rate of 1% using a target‐decoy strategy (Elias & Gygi, 2007). Additionally, only protein groups identified by at least two unique peptides were retained.…”