Manual validation of peptide sequence and sites of tyrosine phosphorylation from MS/MS spectra

Mass spectrometry-based analysis of protein phosphorylation has become increasingly powerful over the past decade and has been applied to many different biological systems. One of the most significant concerns facing the phosphoproteomics community and the proteomics field as a whole is the quality and accuracy of the data generated in these large scale efforts. For protein identification in a given sample, the solution has been to require multiple peptides per protein, eliminating "one-hit-wonders" (proteins identified on the basis of a single peptide assignment) which may increase false positives in the data set. Unfortunately, most of the phosphoproteomics data fall into the latter category, as each phosphorylation site will most likely be represented by a single tryptic peptide. Here we provide a detailed protocol describing our manual validation efforts to assure accurate peptide and phosphorylation site assignment for individual MS/MS spectra. In this procedure we use a combination of tools to assign b-, y-, neutral loss, and internal fragment ions, with the goal of assigning all significant ions in the MS/MS spectrum. Confident peptide and phosphorylation site assignment requires good coverage of the peptide with minimal unassigned fragment ions. Using this approach it is possible to maximize the quality of the phospho-proteomics data while minimizing database contamination associated with false positive identifications.