Labeling and Identification of Direct Kinase Substrates
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White_Labeling and.pdf
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1.93 MB
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Adobe PDF
Checksum (MD5)
e6947aa1f9a4a4b50adf207f4d206803
Author(s)
Carlson, Scott M.
White, Forest M.
Date Issued
May 2012
Journal
Science Signaling
Publisher
American Association for the Advancement of Science (AAAS)
Citation
Carlson, S. M., and F. M. White. “Labeling and Identification of Direct Kinase Substrates.” Science Signaling 5, no. 227 (May 29, 2012): pl3–pl3.
Version
Author's final manuscript
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.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Koch Institute for Integrative Cancer Research at MIT
Terms of Use
Creative Commons Attribution-Noncommercial-Share Alike
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DOI of Published Version
https://doi.org/10.1126/scisignal.2002568
