Protein sequence database searching of tandem mass spectrometry data is commonly employed to identify post-translational modifications (PTMs) to peptides in global proteomic studies. In these studies, the accurate identification of these modified peptides relies on strategies to ensure high-confidence results from sequence database searching in which differential mass shift parameters are employed to identify PTMs to specific amino acids. Using lysine acetylation as an example PTM, we have observed that the inclusion of differential modification information in sequence database searching dramatically increases the potential for false-positive sequence matches to modified peptides, making the confident identification of true sequence matches difficult. In a proof-of-principle study of whole cell yeast lysates, we demonstrate the combination of preparative isoelectric focusing using free-flow electrophoresis, and an adjusted peptide isoelectric point prediction algorithm, as an effective means to increase the confidence of lysine-acetylated peptide identification. These results demonstrate the potential utility of this general strategy for improving the identification of PTMs which cause a shift to the intrinsic isoelectric point of peptides.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|