Thiol-ene-Enabled Detection of Thiophosphorylation as a Labeling Strategy for Phosphoproteins

Kaelyn E. Wilke, Erin E Carlson

Research output: Contribution to journalArticlepeer-review

Abstract

The adenosine triphosphate (ATP) analogue adenosine 5'-O-(3-thiotriphosphate) (ATPγS) has been applied as a tool to study kinase-substrate phosphorylation. Not only does the transfer of a thiophosphate group represent a unique modification amid the phosphoproteome, but it can also be stable to phosphatase activity. However, detection of this species is complicated due to the similar chemical reactivity of thiophosphate and proteinaceous thiols. Here, we describe a novel method for detection of protein thiophosphorylation utilizing the thiol-ene reaction. By first chemoselectively capping protein thiols through radical chemistry, kinase-catalyzed thiophosphorylation can be visualized specifically.

Original languageEnglish (US)
Pages (from-to)3-15
Number of pages13
JournalMethods in molecular biology (Clifton, N.J.)
Volume1355
DOIs
StatePublished - Jan 1 2016

Keywords

  • ATPγS
  • Kinase
  • Phosphoprotein
  • Substrate
  • Thiol capping
  • Thiol-ene
  • Thiophosphorylation

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