title: Bioorthogonal profiling of protein methylation (BPPM) using an azido analog of S-adenosyl-L-methionine.
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.
authors.affiliation: Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
authors.forename: Gil
authors.initials: G
authors.lastname: Blum
authors.forename: Kabirul
authors.initials: K
authors.lastname: Islam
authors.forename: Minkui
authors.initials: M
authors.lastname: Luo
mesh_terms: D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry*; D006649:Histocompatibility Antigens / Q000235:genetics / Q000378:metabolism; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008745:Methylation; D008969:Molecular Sequence Data; D009154:Mutation; D011506:Proteins / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives* / Q000737:chemistry*; D013194:Staining and Labeling
chemical_list: D001386:Azides; D006649:Histocompatibility Antigens; D011506:Proteins; D020543:Proteome; D012436:S-Adenosylmethionine; C445405:EHMT2 protein, human; D011495:Histone-Lysine N-Methyltransferase