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.
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Acknowledgements
We thank Kathleen C. A. Garber for helpful discussions. This work was supported by NIH DP2OD008592, a Pew Biomedical Scholar Award (E.E.C.), the Research Corporation for Science Advancement (Cottrell Scholar Award) (E.E.C.), a Sloan Research Fellowship (E.E.C), and an Indiana University Quantitative and Chemical Biology Training Fellowship (K.E.W.).
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Wilke, K.E., Carlson, E.E. (2016). Thiol-ene-Enabled Detection of Thiophosphorylation as a Labeling Strategy for Phosphoproteins. In: von Stechow, L. (eds) Phospho-Proteomics. Methods in Molecular Biology, vol 1355. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-3049-4_1
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DOI: https://doi.org/10.1007/978-1-4939-3049-4_1
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