title: Rapid validation of protein identifications with the borderline statistical confidence via de novo sequencing and MS BLAST searches.
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.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
authors.forename: Natalie
authors.initials: N
authors.lastname: Wielsch
authors.forename: Henrik
authors.initials: H
authors.lastname: Thomas
authors.forename: Vineeth
authors.initials: V
authors.lastname: Surendranath
authors.forename: Patrice
authors.initials: P
authors.lastname: Waridel
authors.forename: Ari
authors.initials: A
authors.lastname: Frank
authors.forename: Pavel
authors.initials: P
authors.lastname: Pevzner
authors.forename: Andrej
authors.initials: A
authors.lastname: Shevchenko
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000032:analysis* / Q000302:isolation & purification; D019295:Computational Biology / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*
chemical_list: D029742:Caenorhabditis elegans Proteins