Mapping of Phosphorylation Sites by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 134

dramatic influence on the search output. For a phosphopeptide
search, variable (or dynamic) modifications have to be set for the
residues researched. Typically, these modifications are set only for
serine (S), threonine (T), and tyrosine (Y), but phosphorylations
have also been reported on aspartic acid (D) ( 15 ) , histidine (H)
( 16 ) , cysteine (C) ( 17 ) , and arginine (R) ( 18 ) , albeit less com-
monly.
Although identifying phosphopeptides using database search
engines is rather straightforward with high accuracy mass spec-
trometers, the assignment of the phosphorylation site is in most
cases not trivial. This problem occurs when there are multiple
residues that can be phosphorylated in the peptides sequence
assigned. For this ambiguity to be resolved, fragment ions unique
to that phosphosite must be detected. Manual interpretation of
tandem mass spectra is often biased and not feasible for large
data sets per definition. An eloquent way to remove this bias is
to automatically calculate the so-called ambiguity score (Ascore)
( 19 ) . This score is a measure for the likelihood of matching a
difference of at least the number of matched phosphopeptide-
specific fragment ions by chance from the top two candidate
sites as determined by the search algorithm. An Ascore of 20
(P

0.01) should then result in the site being localized with a 99%
certainty.

=

4. Notes

1. In order to preserve the endogenous phosphorylation state
of the biological system, it is recommended to add phos-
phatase inhibitors to the lysis buffer ( 17 ) .
2. Water in this text is referred to as HPLC-grade water.
3. Underloading of the gel bead results in the co-purification
of non-phosphorylated peptides, therefore use the correct
peptide to resin ratio.
4. If you want to perform quantification, use D 0 -D 3 -methanol
for the methyl esterification.
5. Drying under vacuum, lyophilizing under centrifugation,
vacuum concentration, and Speedvac are used throughout
the text and refer to the same technique.
6. Phosphate, phospholipids, DNA, RNA, high salt, and EDTA
disturb the phosphopeptide isolation by IMAC and TiO 2 .
7. Important : Make sure that the pH is between 2.5 and 3. If
necessary, adjust the pH.