Mass spectrometric quantification of histone post-translational modifications by a hybrid chemical labeling method.
Mass spectrometric quantification of histone post-translational modifications by a hybrid chemical labeling method.
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DOI：
10.1074/mcp.o114.046573
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发表时间：
2015-04
期刊：
影响因子：
--
通讯作者：
Arnott D
中科院分区：
文献类型：
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作者：
Maile TM;Izrael-Tomasevic A;Cheung T;Guler GD;Tindell C;Masselot A;Liang J;Zhao F;Trojer P;Classon M;Arnott D
质谱法是一个强大的替代抗体为基础的方法分析组蛋白翻译后修饰（标记）。这种方法的一个关键发展是组蛋白的故意丙酰化，以提高富含赖氨酸和亲水性尾部的序列覆盖率，这些尾部具有大多数修饰。然而，一些标记仍然是有问题的，特别是组蛋白H3的二甲基化和三甲基化赖氨酸4，我们发现其在样品制备和液相色谱-质谱法期间遭受大量和选择性损失。我们开发了一种新的方法，采用“一锅”混合化学衍生的组蛋白，其中一个初步的自由赖氨酸转化为他们的丙酰化形式在温和的水性条件下，随后由胰蛋白酶消化和标记的新肽N末端与异氰酸苯酯。使用高分辨率质谱法收集定性和定量数据，并开发了一种新的基于网络的软件应用程序（Fishtones），用于查看和量化所得数据集中的组蛋白标记。组蛋白H3.1上的53个甲基、乙酰基和磷酰基标记的回收率总体上平均提高了3倍，H3 K4二甲基和三甲基标记的回收率提高了50倍以上。通过测量赖氨酸脱甲基酶的小分子抑制剂诱导的H3 K4三甲基化的定量变化和表观遗传修饰剂ASH 2L和WDR 5的siRNA敲低，证明了这种工作流程在表观遗传研究和药物发现中的作用。
Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a “one-pot” hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5.