A new strategy to filter out false positive identifications of peptides in SEQUEST database search results

Based on the randomized database method and a linear discriminant function (LDF) model, a new strategy to filter out false positive matches in SEQUEST database search results is proposed. Given an experiment MS/MS dataset and a protein sequence database, a randomized database is constructed and merged with the original database. Then, all MS/MS spectra are searched against the combined database. For each expected false positive rate (FPR), LDFs are constructed for different charge states and used to filter out the false positive matches from the normal database. In order to investigate the error of FPR estimation, the new strategy was applied to a reference dataset. As a result, the estimated FPR was very close to the actual FPR. While applied to a human K562 cell line dataset, which is a complicated dataset from real sample, more matches could be confirmed than the traditional cutoff-based methods at the same estimated FPR. Also, though most of the results confirmed by the LDF model were consistent with those of PeptideProphet, the LDF model could still provide complementary information. These results indicate that the new method can reliably control the FPR of peptide identifications and is more sensitive than traditional cutoff-based methods.