Labeling and Identification of Direct Kinase Substrates

• Main Authors: Carlson, Scott M. (Contributor), White, Forest M. (Contributor)
• Other Authors: Massachusetts Institute of Technology. Department of Biological Engineering (Contributor), Koch Institute for Integrative Cancer Research at MIT (Contributor)
• Format: Article
• Language: English
• Published: American Association for the Advancement of Science (AAAS), 2014-08-26T16:17:46Z.
• Subjects: Article
• Online Access: Get fulltext

 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.