on 05-01-201210:40 AM - edited on 11-09-202101:19 AM by usermigration2
Wielsch N, Thomas H, Surendranath V, Waridel P, Frank A, Pevzner P, Shevchenko A. J Proteome Res. 2006 Sep;5(9):2448-56. Protein identifications with the borderline statistical confidence are typically produced by matching a few marginal quality MS/MS spectra to database peptide sequences and represent a significant bottleneck in the reliable and reproducible characterization of proteomes. Here, we present a method for rapid validation of borderline hits that circumvents the need in, often biased, manual inspection of raw MS/MS spectra. The approach takes advantage of the independent interpretation of corresponding MS/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 and redundant candidate peptide sequences. In a case study involving the identification of more than 180 Caenorhabditis elegans proteins by nanoLC-MS/MS analysis on a linear ion trap LTQ mass spectrometer, the approach enabled rapid assignment (confirmation or rejection) of more than 70% of Mascot hits of borderline statistical confidence.