Unit

Bioorthogonal Profiling of Protein Methylation (BPPM) Using an Azido Analog of S-Adenosyl-L-Methionine

  1. Gil Blum1,2,
  2. Kabirul Islam1,
  3. Minkui Luo1

Published Online: 1 MAR 2013

DOI: 10.1002/9780470559277.ch120240

Current Protocols in Chemical Biology

Current Protocols in Chemical Biology

How to Cite

Blum, G., Islam, K. and Luo, M. 2013. Bioorthogonal Profiling of Protein Methylation (BPPM) Using an Azido Analog of S-Adenosyl-L-Methionine. Current Protocols in Chemical Biology. 5:45–66.

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

Protein methyltransferases (PMTs) utilize S-adenosyl-l-methionine (SAM) as a cofactor and transfer its sulfonium methyl moiety to diverse substrates. These methylation events can lead to meaningful biological outcomes, from transcriptional activation/silencing to cell cycle regulation. This article describes recently developed technology based on protein engineering in tandem with SAM analog cofactors and bioorthogonal click chemistry to unambiguously profile the substrates of a specific PMT. The protocols encapsulate the logic and methods of selectively profiling the substrates of a candidate PMT by (1) engineering the selected PMT to accommodate a bulky SAM analog; (2) generating a proteome containing the engineered PMT; (3) visualizing the proteome-wide substrates of the designated PMT via bioorthogonal labeling with a fluorescent tag; and finally (4) pulling down the proteome-wide substrates for mass spectrometric analysis. Curr. Protoc. Chem. Biol. 5:45-66 © 2013 by John Wiley & Sons, Inc.

Keywords:

  • G9a;
  • protein engineering;
  • synthetic cofactor;
  • click chemistry