Blum Gil, Bothwell Ian R, Islam Kabirul, Luo Minkui
Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
Curr Protoc Chem Biol. 2013;5(1):67-88. doi: 10.1002/9780470559277.ch120241.
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.
由于可能的底物种类多样以及在正常生物学和疾病中的意义，从辅因子S-腺苷-L-甲硫氨酸（SAM）到生物分子的酶促转甲基作用最近受到了越来越多的关注。为了揭示蛋白质甲基转移酶（PMT）的底物，本文重点研究了一种含炔基的SAM类似物，即硒代腺苷-L-硒代甲硫氨酸（ProSeAM），以及一种可裂解的叠氮基-偶氮-生物素探针，用于在细胞环境中分析内源性PMT的靶点。本文描述了含有活性内源性PMT的细胞裂解物的逐步制备过程，以及随后用ProSeAM进行靶点标记的过程。本文接着介绍了用叠氮基-偶氮生物素探针作为下拉试剂富集ProSeAM标记的蛋白质，以及随后用连二亚硫酸钠进行还原洗脱以进行蛋白质组学分析的过程。这里提供的方案是为将ProSeAM用作分析试剂而制定的，但也可应用于其他含末端炔基的SAM类似辅因子。
