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

PROTOCOLS

Labeling and Identification of Direct Kinase Substrates

Scott M. Carlson1,2* and Forest M. White1,2{dagger}

1 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
2 Koch Institute for Integrative Cancer Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.

* 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 {gamma}-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.

{dagger} Corresponding author. E-mail, fwhite{at}mit.edu

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

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