Rapid validation of protein identifications with the borderline statistical confidence via de novo sequencing and MS BLAST searches |
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Authors: | Wielsch Natalie Thomas Henrik Surendranath Vineeth Waridel Patrice Frank Ari Pevzner Pavel Shevchenko Andrej |
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Institution: | Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany. |
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Abstract: | 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. |
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