The stoichiometry of protein phosphorylation in adipocyte lipid droplets: analysis by N-terminal isotope tagging and enzymatic dephosphorylation

Proteomics
Evgeny KanshinAlexey V Pshezhetsky

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 ...Continue Reading

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Citations

May 5, 2012·Purinergic Signalling·Herbert ZimmermannNorbert Sträter
May 1, 2013·Molecular BioSystems·Johan Renes, Edwin Mariman
Jan 1, 2012·Adipocyte·David Brockman, Xiaoli Chen
Dec 17, 2014·Journal of Proteome Research·Pieter GlibertDieter Deforce
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Sep 15, 2018·Proteomics·Congyan Zhang, Pingsheng Liu

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