Stable isotope labeling of phosphopeptides for multiparallel kinase target analysis and identification of phosphorylation sites.

Abstract

An approach for multiparallel target identification and relative quantification of in vitro kinase activities in two different biological samples, using liquid chromatography/mass spectrometry (LC/MS), is described. Synthetic target peptides, containing the putative regulatory phosphorylation sites of sucrose-phosphate synthase (SPS) isoenzymes from Arabidopsis thaliana, were simultaneously in vitro phosphorylated and their phosphorylation states determined. Quantification was achieved by stable isotope labeling of the phosphoserine moiety with ethanethiol and [(2)D(5)]-ethanethiol. This revealed different kinase activities in extracts of wild-type (WT) plants and mutant plants lacking plastidic phosphoglucomutase (PGM). The multiparallel assay allowed the determination of favored substrate specificities among the putative phosphorylation sites in SPS. Additionally, we extended the method to unambiguously identify phosphorylation sites in peptides via differential labeling.