Analysis of Protein Phosphorylation by Hypothesis-Driven Multiple-Stage Mass Spectrometry

We describe a strategy, which we term hypothesis-driven
multiple-stage mass spectrometry (HMS-MS), for the
sensitive detection and identification of phosphopeptides
derived from enzymatic digests of phosphoproteins. In
this strategy, we postulate that any or all of the potential
sites of phosphorylation in a given protein may be phosphorylated. Using this assumption, we calculate the m/z
values of all the corresponding singly charged phosphopeptide ions that could, in theory, be produced by the
enzyme employed for proteolysis. We test ions at these
m/z values for the presence of phosphoserine or phosphothreonine residues using tandem mass spectrometry
(MS2) in a vacuum MALDI ion trap mass spectrometer,
where the neutral loss of the elements of H3PO4 (98 Da)
provides a sensitive assay for the presence of phosphopeptides. Subsequent MS3 analysis of the (M + H − 98)+
peaks allows us to confirm or reject the hypotheses that
the putative phosphopeptides are present in the sample.
HMS-MS was successfully applied to the detection and
identification of phosphopeptides from substrates of the
Saccharomyces cerevisiae cyclin-dependent kinase (Cdk)
Cdc28, phosphorylated in vitro (Ipl1) and in vivo (Orc6),
basing hypothesis formation on the minimal Cdk consensus phosphorylation motif Ser/Thr-Pro. The method was
also used to find in vitro phosphopeptides from a domain
of the Drosophila melanogaster protein PERIOD, hypothesizing possible phosphorylations of all Ser/Thr
residues without assuming a consensus motif. Our results
demonstrate that HMS-MS is a sensitive, highly specific
tool for systematically surveying proteins for Ser/Thr
phosphorylation, and represents a significant step toward
our goal of comprehensive phosphorylation mapping.