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.
Elias
Joshua E
JE
Department of Cell Biology, 240 Longwood Avenue, Harvard Medical School, Boston, Massachusetts 02115, USA.
Gygi
Steven P
SP
D000465:Algorithms; D000595:Amino Acid Sequence; D003628:Database Management Systems; D030562:Databases, Protein; D016247:Information Storage and Retrieval / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D020543:Proteome / Q000032:analysis / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*
D020543:Proteome