Open AccessBioorthogonal Profiling of Protein Methylation (BPPM) Using an Azido Analog of S ‐Adenosyl‐ L ‐Methionine
Author(s) -
Blum Gil,
Islam Kabirul,
Luo Minkui
Publication year - 2013
Publication title -
current protocols in chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.503
H-Index - 14
ISSN - 2160-4762
DOI - 10.1002/9780470559277.ch120240
Subject(s) - bioorthogonal chemistry , methyltransferase , proteome , methylation , chemistry , protein methylation , biochemistry , click chemistry , methionine , computational biology , combinatorial chemistry , biology , dna , amino acid
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.