Generation of acetyllysine antibodies and affinity enrichment of acetylated peptides.
Generation of acetyllysine antibodies and affinity enrichment of acetylated peptides.
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DOI：
10.1038/nprot.2010.117
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发表时间：
2010-09
期刊：
影响因子：
14.8
通讯作者：
Zhao S
中科院分区：
文献类型：
--
作者：
Guan KL;Yu W;Lin Y;Xiong Y;Zhao S
赖氨酸乙酰化已成为主要的翻译后修饰之一，其在染色质重塑、转录因子激活以及最近的代谢酶调节中的作用表明了这一点。蛋白质中乙酰化位点的鉴定是生理调节中乙酰化功能表征的第一个重要步骤。然而，由于乙酰基缺乏合适的物理和生化特性以及细胞中存在高丰度乙酰化组蛋白，乙酰组的研究受到阻碍，需要一种稳健的方法来克服这些问题。在这里，我们提出以下方案：（i）使用化学乙酰化卵清蛋白和合成乙酰化肽分别生成泛乙酰赖氨酸抗体和 Lys288-乙酰化精氨琥珀酸裂合酶的位点特异性抗体； (ii) 使用亚细胞分级分离来减少高丰度的乙酰化组蛋白； (iii) 使用乙酰赖氨酸抗体亲和纯化和质谱分析来表征人肝组织的乙酰组。整个表征过程需要约 2-3 天才能完成。
Lysine acetylation has emerged as one of the major post-translational modifications, as indicated by its roles in chromatin remodeling, activation of transcription factors and, most recently, regulation of metabolic enzymes. Identification of acetylation sites in a protein is the first essential step for functional characterization of acetylation in physiological regulation. However, the study of the acetylome is hindered by the lack of suitable physical and biochemical properties of the acetyl group and existence of high-abundance acetylated histones in the cell, and needs a robust method to overcome these problems. Here we present protocols for (i) using chemically acetylated ovalbumin and synthetic acetylated peptide to generate a pan-acetyllysine antibody and a site-specific antibody to Lys288-acetylated argininosuccinate lyase, respectively; (ii) using subcellular fractionation to reduce highly abundant acetylated histones; and (iii) using acetyllysine antibody affinity purification and mass spectrometry to characterize acetylome of human liver tissue. The entire characterization procedure takes ~2–3 d to complete.