title: Profiling protein methylation with cofactor analog containing terminal alkyne functionality.
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.
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: Ian R
authors.initials: IR
authors.lastname: Bothwell
authors.forename: Kabirul
authors.initials: K
authors.lastname: Islam
authors.forename: Minkui
authors.initials: M
authors.lastname: Luo
mesh_terms: D001386:Azides / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002460:Cell Line; D002725:Chloroform / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D000432:Methanol / Q000737:chemistry; D008745:Methylation; D011496:Protein Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D012645:Selenomethionine / Q000031:analogs & derivatives* / Q000737:chemistry; D012997:Solvents; D013194:Staining and Labeling; D014867:Water / Q000737:chemistry
chemical_list: D001386:Azides; D005456:Fluorescent Dyes; D011506:Proteins; D020543:Proteome; D012997:Solvents; C577600:propargylic Se-adenosyl-L-selenomethionine; D014867:Water; D001710:Biotin; D002725:Chloroform; D012645:Selenomethionine; D011496:Protein Methyltransferases; D000432:Methanol
keywords: ProSeAM; Se‐adenosyl‐L‐selenomethionine; azido‐azo biotin probe; click chemistry