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.

phosphoproteomics, PRM, targeted mass spectrometry, quantitative proteomics and phosphorylation ratio
dx.doi.org/10.1021/acs.jproteome.7b00911, hdl.handle.net/1765/105684
Journal of Proteome Research
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Erasmus MC: University Medical Center Rotterdam

Dekker, L.J.M, Zeneyedpour, L, Snoeijers, S, Joore, J, Leenstra, S, & Luider, T.M. (2018). Determination of Site-Specific Phosphorylation Ratios in Proteins with Targeted Mass Spectrometry. Journal of Proteome Research, 17(4), 1654–1663. doi:10.1021/acs.jproteome.7b00911