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

Nature Methods volume 4, pages 207214 (2007) | Download Citation



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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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.

Author information


  1. Department of Cell Biology, 240 Longwood Avenue, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Joshua E Elias
    •  & Steven P Gygi
  2. Taplin Biological Mass Spectrometry Facility, 240 Longwood Avenue, Harvard Medical School, Boston, Massachusetts 02115, USA.

    • Steven P Gygi


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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Steven P Gygi.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    False positive identifications can be estimated by doubling decoy hits from a search against a concatenated target/decoy database.

  2. 2.

    Supplementary Fig. 2

    The distributions of potential peptide matches is consistent between target and decoy databases.

  3. 3.

    Supplementary Fig. 3

    Example supporting the necessity for target/decoy competition.

  4. 4.

    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.

  5. 5.

    Supplementary Fig. 5

    Using decoy hits to guide selection of appropriate selection criteria.

  6. 6.

    Supplementary Table 1

    Slopes of best-fit lines for precision values shown in Figure 5b.

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  1. 1.

    Supplementary Methods

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