Labeling and Identification of Direct Kinase Substrates

Sci. Signal., 5 June 2012
Vol. 5, Issue 227, p. pl3
DOI: 10.1126/scisignal.2002568

Labeling and Identification of Direct Kinase Substrates

  1. Scott M. Carlson1,2,* and
  2. Forest M. White1,2,
  1. 1Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
  2. 2Koch Institute for Integrative Cancer Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
  1. Corresponding author. E-mail, fwhite{at}mit.edu
  • * Present address: Department of Biology, Stanford University, Stanford, CA, USA.

Abstract

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.

Citation:

S. M. Carlson and F. M. White, Labeling and Identification of Direct Kinase Substrates. Sci. Signal. 5, pl3 (2012).

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