Confident Site Localization Using a Simulated Phosphopeptide Spectral Library

We have investigated if phosphopeptide
identification and simultaneous
site localization can be achieved by spectral library searching. This
allows taking advantage of comparison of specific spectral features,
which would lead to improved discrimination of differential localizations.
For building a library, we propose a spectral simulation strategy
where all possible single phosphorylations can be simply and accurately
(re)­constructed on enzymatically dephosphorylated peptides, by predicting
the diagnostic fragmentation events produced in beam-type CID. To
demonstrate the performance of our approach, enriched HeLa phosphopeptides
were dephosphorylated with alkaline phosphatase and analyzed with
higher energy collisional dissociation (HCD), which were then used
for creating a spectral library of simulated phosphopeptides. Spectral
library searching using SpectraST was performed on data sets of synthetic
phosphopeptides and the HeLa phosphopeptides, and subsequently compared
to Mascot and Sequest database searching followed by phosphoRS and
Ascore afforded localization, respectively. Our approach successfully
led to accurate localization, and it outperformed other methods, when
phosphopeptides were covered by the library. These results suggest
that the searching with simulated spectral libraries serves as a crucial
approach for both supplementing and validating the phosphorylation
sites obtained by database searching and localization tools. For future
development, simulation of multiply phosphorylated peptides remains
to be implemented.