Comprehensive phosphorylation site analysis of individual phosphoproteins applying scoring schemes for MS/MS data

We have developed novel scoring schemes for the identification of (phospho)peptides (PeptideScore) and for pinpointing phosphorylation sites (PhosphoSiteScore) using MS/MS data. These scoring schemes have been developed for the in-depth analysis of individual phosphoproteins, not for large-scale phosphoproteomic-type data. The scoring schemes are implemented into the new software tool Phosm, which provides a concise and comprehensive presentation of the results. For development and evaluation of these schemes, we have analyzed approximately 500 phosphopeptide MS/MS spectra, most of them nontryptic peptides. The novel scoring schemes turned out to be very powerful, even with CID MS/MS spectra of very low quality. Many phosphopeptides and phosphorylation sites that remained unassigned in our LC-MS/MS data sets with Mascot could be identified with Phosm. Especially the number of identified multiply phosphorylated peptides could be significantly increased. The applied scoring parameters are described, and the scoring for several selected examples of phosphopeptides is discussed in detail. Furthermore, a new and simple nomenclature for all types of phosphorylated fragment ions is introduced in this publication.