title: 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.
authors.affiliation: Max-Planck-Institute for Molecular Plant Physiology, 14424 Potsdam, Germany.
authors.forename: Mirko
authors.initials: M
authors.lastname: Glinski
authors.forename: Tina
authors.initials: T
authors.lastname: Romeis
authors.forename: Claus-Peter
authors.initials: CP
authors.lastname: Witte
authors.forename: Stefanie
authors.initials: S
authors.lastname: Wienkoop
authors.forename: Wolfram
authors.initials: W
authors.lastname: Weckwerth
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry*; D010936:Plant Extracts / Q000032:analysis; D018515:Plant Leaves / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin
chemical_list: D010748:Phosphopeptides; D010936:Plant Extracts; D010940:Plant Proteins; D010770:Phosphotransferases; D014357:Trypsin