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
∼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.