Unit

Profiling Protein Methylation with Cofactor Analog Containing Terminal Alkyne Functionality

  1. Gil Blum1,2,
  2. Ian R. Bothwell1,2,
  3. Kabirul Islam1,
  4. Minkui Luo1

Published Online: 1 MAR 2013

DOI: 10.1002/9780470559277.ch120241

Current Protocols in Chemical Biology

Current Protocols in Chemical Biology

How to Cite

Blum, G., Bothwell, I. R., Islam, K. and Luo, M. 2013. Profiling Protein Methylation with Cofactor Analog Containing Terminal Alkyne Functionality. Current Protocols in Chemical Biology. 5:67–88.

Author Information

  1. 1

    Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York

  2. 2

    Tri-Institutional Training Program in Chemical Biology, Memorial Sloan-Kettering Cancer Center, New York, New York

Publication History

  1. Published Online: 1 MAR 2013

Abstract

Enzymatic transmethylation from the cofactor S-adenosyl-l-methionine (SAM) to biological molecules has recently garnered increased attention because of the diversity of possible substrates and implications in normal biology and diseases. To reveal the substrates of protein methyltransferases (PMTs), the present article focuses on an alkyne-containing SAM mimic, Se-adenosyl-l-selenomethionine (ProSeAM), and a cleavable azido-azo-biotin probe to profile the targets of endogenous PMTs in cellular contexts. This article describes the stepwise preparation of cell lysates containing active, endogenous PMTs and subsequent target labeling with ProSeAM. The article continues with the enrichment of the ProSeAM-labeled proteins with the azido-azo biotin probe as a pulldown reagent and the subsequent reductive elution with sodium dithionate for proteomic analysis. The protocols provided here were formulated for ProSeAM as a profiling reagent but can be applied to other terminal-alkyne-containing SAM analog cofactors. Curr. Protoc. Chem. Biol. 5:67-88 © 2013 by John Wiley & Sons, Inc.

Keywords:

  • Se-adenosyl-l-selenomethionine;
  • ProSeAM;
  • azido-azo biotin probe;
  • click chemistry