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Molecular & Cellular Proteomics 4:1614-1625, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Differential Multisite Phosphorylation of the Trehalose-6-phosphate Synthase Gene Family in Arabidopsis thaliana

A Mass Spectrometry-based Process for Multiparallel Peptide Library Phosphorylation Analysis^*

Mirko Glinski and Wolfram Weckwerth

From the Max Planck Institute of Molecular Plant Physiology, 14476 Potsdam-Golm, Germany

Multisite protein phosphorylation plays a fundamental role in metabolic regulation. To detect and quantify in vitro kinase phosphorylation activities, we developed a highly selective LC-MS/MS-based method using high resolution multiple reaction monitoring on a triple quadrupole mass spectrometer. This method eliminates the need for stable isotope labeling and enables multiparallel kinase target assays. Using these assays, we made the first observation of in vitro phosphorylation of different trehalose-6-phosphate synthase (TPS) isozymes. TPSs possess putative Ca²⁺-independent, sucrose non-fermenting 1-related protein kinase 1 (SnRK1) phosphorylation sites. Sixteen synthetic peptides from six different Arabidopsis thaliana TPS isozymes containing the SnRK1 consensus recognition motif were phosphorylated simultaneously in vitro, and their phosphorylation dynamics were determined. We achieved absolute quantification of TPS peptide phosphorylation by tuning the mass spectrometer to the corresponding synthetic standard phosphopeptides. The selectivity of the mass spectrometer in the multiple reaction monitoring mode compensates for the low ionization efficiency of phosphopeptides in the presence of a complex matrix. Results are in close agreement with recent in vivo studies of TPS phosphorylation and regulation and reveal significant differences in the phosphorylation levels of different TPS members within the TPS gene family ranging over 3 orders of magnitude. Substituting EGTA for CaCl₂ in the reaction mixture reduced the formation of some of the phospho-TPS peptides drastically, indicating that Ca²⁺-dependent kinases are active in the presence of Ca²⁺-independent SnRKs. This agrees with the proposed overlap of the consensus motifs of these kinases and enables delineation between Ca²⁺-independent and Ca²⁺-dependent phosphorylation. Results demonstrate that multiparallel kinase target assays are sensitive enough to provide evidence for differential multisite phosphorylation of homologous TPS proteins and their highly conserved putative phosphorylation sites.

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