title: The stoichiometry of protein phosphorylation in adipocyte lipid droplets: analysis by N-terminal isotope tagging and enzymatic dephosphorylation.
abstract: Most phosphoproteomic studies to date have been limited to the identification of phosphoproteins and their phosphorylation sites, and have not assessed the stoichiometry of protein phosphorylation, a critical parameter reflecting the dynamic equilibrium between phosphorylated and non-phosphorylated pools of proteins. Here, we used a method for measuring phosphorylation stoichiometry through isotope tagging and enzymatic dephosphorylation of tryptic peptides. Using this method, protein digests are divided into two equal aliquots that are modified with either light or heavy isotope tags. One aliquot is dephosphorylated by alkaline phosphatase. Finally, the peptide mixtures are recombined and LC-MS/MS analysis is performed. With this method, we studied adipocytes of mice stimulated with CL316,243, a beta-3 adrenergic agonist known to induce lipolysis and marked phosphorylation changes in proteins of the lipid droplet surface. In lipid droplet preparations, CL316,243 administration increased phosphorylation of proteins related to regulation of signaling, metabolism and intracellular trafficking in white adipose tissue, including hormone-sensitive lipase which was 80% phosphorylated at the previously reported site, Ser-559, and the lipid surface protein perilipin, which was phosphorylated by approximately 60 and approximately 40% at previously unreported sites, Ser-410 and Ser-460.
authors.affiliation: Division of Medical Genetics, Department of Pediatrics, CHU Sainte-Justine, University of Montreal, 3175 Côte Ste-Catherine, Montreal, Quebec, Canada.
authors.forename: Evgeny
authors.initials: E
authors.lastname: Kanshin
authors.forename: Shupei
authors.initials: S
authors.lastname: Wang
authors.forename: Lyudmila
authors.initials: L
authors.lastname: Ashmarina
authors.forename: Michael
authors.initials: M
authors.lastname: Fedjaev
authors.forename: Ilya
authors.initials: I
authors.lastname: Nifant'ev
authors.forename: Grant A
authors.initials: GA
authors.lastname: Mitchell
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Pshezhetsky
mesh_terms: D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004149:Dioxoles / Q000494:pharmacology; D007098:Imino Acids / Q000737:chemistry; D007554:Isotopes / Q000737:chemistry; D008066:Lipolysis / Q000187:drug effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry; D010766:Phosphorylation / Q000187:drug effects; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D000318:Adrenergic beta-Agonists; D004149:Dioxoles; D007098:Imino Acids; D007554:Isotopes; D010748:Phosphopeptides; D011506:Proteins; C076126:disodium (R,R)-5-(2-((2-(3-chlorophenyl)-2-hydroxyethyl)-amino)propyl)-1,3-benzodioxole-2,3-dicarboxylate; C008109:iminodiacetic acid