文摘
Protein identifications with the borderline statistical confidence are typically produced by matching afew marginal quality MS/MS spectra to database peptide sequences and represent a significantbottleneck in the reliable and reproducible characterization of proteomes. Here, we present a methodfor rapid validation of borderline hits that circumvents the need in, often biased, manual inspection ofraw MS/MS spectra. The approach takes advantage of the independent interpretation of correspondingMS/MS spectra by PepNovo de novo sequencing software followed by mass spectrometry-driven BLAST(MS BLAST) sequence-similarity database searches that utilize all partially inaccurate, degenerate andredundant candidate peptide sequences. In a case study involving the identification of more than 180Caenorhabditis elegans proteins by nanoLC -MS/MS analysis on a linear ion trap LTQ massspectrometer, the approach enabled rapid assignment (confirmation or rejection) of more than 70% ofMascot hits of borderline statistical confidence.