title: Thiol-ene-Enabled Detection of Thiophosphorylation as a Labeling Strategy for Phosphoproteins.
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.
authors.affiliation: Department of Chemistry, Indiana University, 800 E. Kirkwood Ave., Bloomington, IN, 47405, USA.
authors.forename: Kaelyn E
authors.initials: KE
authors.lastname: Wilke
authors.affiliation: Department of Chemistry, Indiana University, 800 E. Kirkwood Ave., Bloomington, IN, 47405, USA. carlsone@umn.edu.|Department of Molecular and Cellular Biochemistry, Indiana University, 212 S. Hawthorne St., Bloomington, IN, 47405, USA. carlsone@umn.edu.|Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN, 55455, USA. carlsone@umn.edu.
authors.forename: Erin E
authors.initials: EE
authors.lastname: Carlson
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008163:Luminescent Measurements; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D057188:Workflow
chemical_list: D010750:Phosphoproteins; D013438:Sulfhydryl Compounds; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D011494:Protein Kinases
keywords: ATPγS; Kinase; Phosphoprotein; Substrate; Thiol capping; Thiol-ene; Thiophosphorylation