Labeling and identification of direct kinase substrates.
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.
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Scott M
SM
Carlson
Forest M
FM
White
D016475:3T3 Cells; D000818:Animals; D002846:Chromatography, Affinity; D051379:Mice; D010770:Phosphotransferases / Q000378:metabolism*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
D010770:Phosphotransferases