Labeling and Identification of Direct Kinase Substrates
Author(s)Carlson, Scott M.; White, Forest M.
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Identifying kinase substrates is an important step in mapping signal transduction pathways, but it remains a difficult and time-consuming process. Analog-sensitive (AS) kinases have been used to selectively tag and identify direct kinase substrates in lysates from whole cells. In this approach, a γ-thiol adenosine triphosphate analog and an AS kinase are used to selectively thiophosphorylate target proteins. Thiophosphate is used as a chemical handle to purify peptides from a tryptic digest, and target proteins are identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Here, we describe an updated strategy for labeling AS kinase substrates, solid-phase capture of thiophosphorylated peptides, incorporation of stable isotope labeling in cell culture for filtering nonspecific background peptides, enrichment of phosphorylated target peptides to identify low-abundance targets, and analysis by LC-MS/MS.
DepartmentMassachusetts Institute of Technology. Department of Biological Engineering; Koch Institute for Integrative Cancer Research at MIT
American Association for the Advancement of Science (AAAS)
Carlson, S. M., and F. M. White. “Labeling and Identification of Direct Kinase Substrates.” Science Signaling 5, no. 227 (May 29, 2012): pl3–pl3.
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