Determination of Site-Specific Phosphorylation Ratios in Proteins with Targeted Mass Spectrometry

We show that parallel reaction monitoring
(PRM) can be used for
exact quantification of phosphorylation ratios of proteins using stable-isotope-labeled
peptides. We have compared two different PRM approaches on a digest
of a U87 cell culture, namely, direct-PRM (tryptic digest measured
by PRM without any further sample preparation) and TiO2-PRM (tryptic digest enriched with TiO2 cartridges, followed
by PRM measurement); these approaches are compared for the following
phosphorylation sites: neuroblast differentiation-associated protein
(AHNAK S5480-p), calcium/calmodulin-dependent protein kinase type
II subunit delta (CAMK2D T337-p), and epidermal growth factor receptor
(EGFR S1166-p). A reproducible percentage of phosphorylation could
be determined (CV 6–13%) using direct-PRM or TiO2-PRM. In addition, we tested the approaches in a cell culture experiment
in which U87 cells were deprived of serum. As a “gold standard”
we included immune precipitation of EGFR followed by PRM (IP-PRM).
For EGFR (S1166) and AHNAK (S5480) a statistical significant change
in the percentage of phosphorylation could be observed as a result
of serum deprivation; for EGFR (S1166) this change was observed for
both TiO2-PRM and IP-PRM. The presented approach has the
potential to multiplex and to quantify the ratio of phosphorylation
in a single analysis.