Profiling protein methylation with cofactor analog containing terminal alkyne functionality.
Profiling protein methylation with cofactor analog containing terminal alkyne functionality.
复制标题
DOI：
10.1002/9780470559277.ch120241
复制
发表时间：
2013-01-01
影响因子：
--
通讯作者：
Luo, Minkui
中科院分区：
文献类型：
--
作者：
Blum, Gil;Bothwell, Ian R;Luo, Minkui
从辅因子S-腺苷-L-蛋氨酸到生物分子的酶促转甲基化最近受到越来越多的关注，因为可能的底物的多样性以及在正常生物学和疾病中的意义。为了揭示蛋白质甲基转移酶(PMTs)的底物，本文重点介绍了一种含炔基的模拟蛋白甲基转移酶，即硒-腺苷-L-硒蛋氨酸(ProSeAM)和一种可切割的叠氮-偶氮-生物素探针，用于在细胞环境中描述内源性PMTs的靶标。本文描述了含有活性内源性PMTs的细胞裂解物的逐步制备以及随后用ProSeAM标记靶标的方法。本文继续用叠氮偶氮生物素探针作为下拉试剂对ProSeAM标记的蛋白质进行富集化，然后用二硫酸钠还原洗脱进行蛋白质组学分析。这里提供的方案是为ProSeAM配制的，作为一种剖析试剂，但也可以应用于其他含有末端炔的SAM模拟辅因子。
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.