Biology Reference
In-Depth Information
3. Upload the database to be searched, adjust default settings as
desired. Add Phospho (STY) to the tolerated protein modifi -
cations, relax Peptide and Protein FDRs if desired. We recom-
mend keeping the Site FDR at 0.01 for high quality mapping
of phosphosites. Check label-free quantifi cation if applicable.
4. Run MaxQuant. If suffi cient computing power is available
increase the number of threads. On 64 bit machines with 4 or
8 Gb of RAM most small to medium scale jobs should be
completed within a day. If problems arise uncheck protein
quantifi cation options. MaxQuant will no longer quantify
phsophopeptide intensities but will still map phosphosites.
5. Open Viewer to analyze search results. The modifi cations tab
contains information on all identifi ed phosphopeptides includ-
ing q-values and posterior error probabilities (PEPs) as well as
likelihood estimates of phosphorylated residues (phosphoryla-
tion sites) and intensities. The peptides tab contains informa-
tion on all peptide spectral matches (PSMs) so redundant
PSMs for each phosphorylated peptide and possible unphos-
phorylated counterparts can be found here.
Another good option for phosphopeptide identifi cation and
probabilistic phosphosite mapping is phosphoRS, integrated into
Proteome Discoverer versions 1.3 and higher. Results similar to
MaxQuant are achieved.
4
Notes
1. Liquid chromatography (LC) on-line with high resolution
accurate mass (HR/AM) mass spectrometry (MS) is the pre-
ferred method for large scale analysis of phosphopeptides. The
precise analytical strategy in the framework of LC-MS how-
ever is dependent on the instrumental capabilities at hand, the
focus of the study, the researcher's expertise and experience,
and other parameters, so a wide range of applications are pos-
sible downstream of tandem MOAC enrichment of phospho-
peptides. In our research we have performed large scale
identifi cation and quantifi cation of site-specifi c phosphoryla-
tion using a shotgun proteomics approach modifi ed especially
for phosphopeptide identifi cation but targeted strategies
aimed at specifi c phosphopeptides or phosphorylation sites are
also conceivable. Keeping this in mind we describe our
approach commenting on possible alterations.
2. Mass spectrometry is a powerful application for the large scale
mapping of phosphorylation sites to protein primary structure
that also allows quantifi cation of site specifi c phosphorylation
stoichiometries.
