Dual protease type XIII/pepsin digestion offers superior resolution and overlap for the analysis of histone tails by HX-MS
Dual protease type XIII/pepsin digestion offers superior resolution and overlap for the analysis of histone tails by HX-MS
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DOI：
10.1016/j.ymeth.2020.01.016
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发表时间：
2020-12-01
期刊：
影响因子：
4.8
通讯作者：
Papanastasiou, Malvina
中科院分区：
文献类型：
--
作者：
Mullahoo, James;Zhang, Terry;Papanastasiou, Malvina
组蛋白的N-末端区域（尾部）是从核小体突出的动态元件，并参与染色质组织的许多方面。它们的表观遗传作用是公认的，并且这些区域上存在的翻译后修饰（PTM）有助于转录调控。虽然氢/氘交换质谱法（HX-MS）非常适合于动态结构的分析，但是由于使用胃蛋白酶获得的差的N-末端覆盖，它很少用于分析组蛋白。在这里，我们测试了双蛋白酶类型XIII/胃蛋白酶消化柱对这类蛋白质的适用性。我们使用H4单体优化在线消化条件，并将该方法扩展到单体状态和核小体核心颗粒（NCPs）的组蛋白分析。我们表明，双蛋白酶柱产生许多短的和重叠的N-末端肽。我们通过对不同时间点的NCP进行氢交换实验来评估我们的方法，并以优异的分辨率对尾部进行全覆盖。我们进一步采用电子转移解离，并展示了前所未有的重叠程度在多个肽，是几倍高于以前报道的方法。我们在这里报告的方法可以很容易地应用于组蛋白动力学和组蛋白结合蛋白的足迹核小体的HX-MS调查。
The N-terminal regions of histone proteins (tails) are dynamic elements that protrude from the nucleosome and are involved in many aspects of chromatin organization. Their epigenetic role is well-established, and post-translational modifications (PTMs) present on these regions contribute to transcriptional regulation. While hydrogen/deuterium exchange mass spectrometry (HX-MS) is well-suited for the analysis of dynamic structures, it has seldom been employed to analyze histones due to the poor N-terminal coverage obtained using pepsin. Here, we test the applicability of a dual protease type XIII/pepsin digestion column to this class of proteins. We optimize online digestion conditions using the H4 monomer, and extend the method to the analysis of histones in monomeric states and nucleosome core particles (NCPs). We show that the dual protease column generates many short and overlapping N-terminal peptides. We evaluate our method by performing hydrogen exchange experiments of NCPs for different time points and present full coverage of the tails at excellent resolution. We further employ electron transfer dissociation and showcase an unprecedented degree of overlap across multiple peptides that is several fold higher than previously reported methods. The method we report here may be readily applied to the HX-MS investigation of histone dynamics and to the footprints of histone binding proteins on nucleosomes.