Quantitative proteomic analysis of posttranslational modifications of human histones
Quantitative proteomic analysis of posttranslational modifications of human histones
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DOI：
10.1074/mcp.m600007-mcp200
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发表时间：
2006-07-01
影响因子：
7
通讯作者：
Jensen, Ole Norregaard
中科院分区：
文献类型：
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作者：
Beck, Hans Christian;Nielsen, Eva C.;Jensen, Ole Norregaard
在活细胞中，组蛋白受到一系列转录后修饰的影响。这些修饰的组合性质构成了“组蛋白密码”，它决定了细胞发育、生长、分化和内稳态过程中的染色质结构和功能。由于缺乏分析方法来监测组蛋白的可逆多位点修饰的组合复杂性，包括乙酰化和甲基化，阻碍了组蛋白密码的破译。为了解决这个问题，我们使用LC-MSMS技术和虚拟专家质谱仪软件对从人小细胞肺癌细胞中提取的组蛋白进行了定性和定量的蛋白质组学分析。人组蛋白H_2A、H_2B、H_3和H_4共有32个乙酰化、甲基化和泛素化，其中包括7个新的修饰。应用LC-MSMS定量蛋白质组学方法研究了组蛋白去乙酰化酶抑制剂(HDACi)PXD101对人细胞培养中组蛋白乙酰化的量效关系。三重LC-MSMS在6种不同的HDACi浓度下运行表明，PXD101以位置特异性和剂量依赖的方式影响组蛋白H_2A、H_2B、H_3和H_4的乙酰化。这一无偏见的分析表明，组蛋白变异体H_2A、H_2B和H_4的乙酰化肽的相对增加伴随着组蛋白H_2B的二甲基化赖氨酸(57)的相对减少。从HDACi处理的细胞组蛋白定量蛋白质组学获得的剂量-反应结果与组蛋白乙酰化、细胞毒性以及p21和细胞周期蛋白A2的剂量依赖性表达谱的Western印迹分析一致。这表明基于质谱学的翻译后修饰的定量蛋白质组学分析是进行HDAC抑制剂等候选药物功能分析的一种可行的方法。
Histone proteins are subject to a range of post-transcriptional modifications in living cells. The combinatorial nature of these modifications constitutes the "histone code" that dictates chromatin structure and function during development, growth, differentiation, and homeostasis of cells. Deciphering of the histone code is hampered by the lack of analytical methods for monitoring the combinatorial complexity of reversible multisite modifications of histones, including acetylation and methylation. To address this problem, we used LC-MSMS technology and Virtual Expert Mass Spectrometrist software for qualitative and quantitative proteomic analysis of histones extracted from human small cell lung cancer cells. A total of 32 acetylations, methylations, and ubiquitinations were located in the human histones H2A, H2B, H3, and H4, including seven novel modifications. An LC-MSMS-based method was applied in a quantitative proteomic study of the dose-response effect of the histone deacetylase inhibitor (HDACi) PXD101 on histone acetylation in human cell cultures. Triplicate LC-MSMS runs at six different HDACi concentrations demonstrated that PXD101 affects acetylation of histones H2A, H2B, H3, and H4 in a site-specific and dose-dependent manner. This unbiased analysis revealed that a relative increase in acetylated peptide from the histone variants H2A, H2B, and H4 was accompanied by a relative decrease of dimethylated Lys(57) from histone H2B. The dose-response results obtained by quantitative proteomics of histones from HDACi-treated cells were consistent with Western blot analysis of histone acetylation, cytotoxicity, and dose-dependent expression profiles of p21 and cyclin A2. This demonstrates that mass spectrometry-based quantitative proteomic analysis of post-translational modifications is a viable approach for functional analysis of candidate drugs, such as HDAC inhibitors.