Abstract
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.
Highlights
Multisite protein phosphorylation plays a fundamental role in metabolic regulation
Kinase Target Analysis via Multiple Reaction Monitoring sucrose-phosphate synthase (SPS) and nitrate reductase, whose activity states are rapidly altered by protein kinase action in response to environmental changes and/or genetic perturbations present in mutant plants (14 –17), the trehalose-6-phosphate synthase (TPS) enzyme is thought to be regulated by reversible protein phosphorylation/dephosphorylation [18]
Mass spectrometric fullscan detection of the reaction products formed in the presence and absence of ATP unambiguously revealed the exclusive formation of singly phosphorylated TPS peptides
Summary
We have observed clear differences in the phosphorylation states of Arabidopsis wild-type and mutant plant SPS [42] It is a very homologous system using substrates similar to those used in sucrose biosynthesis, no investigations on the regulation of TPS isoforms via post-translational phosphorylation have been reported. Based method allows a significant enhancement of signal-tonoise (S/N) ratios in phosphopeptide analysis and compensates for the low ionization efficiency of phosphopeptides in positive ionization mode by the high selectivity of the mass spectrometer using MRM. This enabled us to look at TPS isoform phosphorylation states in Arabidopsis for the first time
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