title: Validation of peptide MS/MS spectra using metabolic isotope labeling for spectral matching-based shotgun proteome analysis.
abstract: We report an isotope labeling shotgun proteome analysis strategy to validate the spectrum-to-sequence assignments generated by using sequence-database searching for the construction of a more reliable MS/MS spectral library. This strategy is demonstrated in the analysis of the E. coli K12 proteome. In the workflow, E. coli cells were cultured in normal and (15)N-enriched media. The differentially labeled proteins from the cell extracts were subjected to trypsin digestion and two-dimensional liquid chromatography quadrupole time-of-flight tandem mass spectrometry (2D-LC QTOF MS/MS) analysis. The MS/MS spectra of the two samples were individually searched using Mascot against the E. coli proteome database to generate lists of peptide sequence matches. The two data sets were compared by overlaying the spectra of unlabeled and labeled matches of the same peptide sequence for validation. Two cutoff filters, one based on the number of common fragment ions and another one on the similarity of intensity patterns among the common ions, were developed and applied to the overlaid spectral pairs to reject the low quality or incorrectly assigned spectra. By examining 257,907 and 245,156 spectra acquired from the unlabeled and (15)N-labeled samples, respectively, an experimentally validated MS/MS spectral library of tryptic peptides was constructed for E. coli K12 that consisted of 9,302 unique spectra with unique sequence and charge state, representing 7,763 unique peptide sequences. This E. coli spectral library could be readily expanded, and the overall strategy should be applicable to other organisms. Even with this relatively small library, it was shown that more peptides could be identified with higher confidence using the spectral search method than by sequence-database searching.
authors: Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors: Mingguo
authors: M
authors: Xu
authors: Liang
authors: L
authors: Li
mesh_terms: D002853:Chromatography, Liquid; D029968:Escherichia coli Proteins / Q000378:metabolism*; D020543:Proteome; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D020543:Proteome