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


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.

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Figure 1: Overlap between target (forward) and decoy (reversed) sequences is negligible.
Figure 2: The distributions of potential peptide matches is consistent between target and decoy databases at several mass tolerances.
Figure 3: Incorrect identifications are equally selected from target and decoy (reversed) sequences.
Figure 4: Separate searching overestimates FP rates by underestimating low-scoring correct identifications.
Figure 5: Estimating the theoretical error associated with target-decoy estimations.
Figure 6: Evaluation of alternate decoy databases.

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This work was supported in part by US National Institutes of Health (GM67945 and HG00041 to S.P.G.). We thank S. Beausoleil, P. Everley, S. Gerber and W. Haas for continuing and insightful discussions, and Sage-N for implementing our idea of the pseudo-reversed searches on their SEQUEST platform.

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Correspondence to Steven P Gygi.

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Supplementary information

Supplementary Fig. 1

False positive identifications can be estimated by doubling decoy hits from a search against a concatenated target/decoy database. (PDF 416 kb)

Supplementary Fig. 2

The distributions of potential peptide matches is consistent between target and decoy databases. (PDF 22 kb)

Supplementary Fig. 3

Example supporting the necessity for target/decoy competition. (PDF 47 kb)

Supplementary Fig. 4

Relative scores shift to smaller values for less than half of peptide hits when searched against composite target-decoy databases as opposed to separate databases. (PDF 41 kb)

Supplementary Fig. 5

Using decoy hits to guide selection of appropriate selection criteria. (PDF 160 kb)

Supplementary Table 1

Slopes of best-fit lines for precision values shown in Figure 5b. (PDF 14 kb)

Supplementary Methods (DOC 43 kb)

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Elias, J., Gygi, S. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4, 207–214 (2007).

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