The analysis of histone post-translational modifications (PTMs) by mass spectrometry (MS) has been critical to the advancement of the field of epigenetics. The most sensitive and accurate workflow is similar to the canonical proteomics analysis workflow (bottom-up MS), where histones are digested into short peptides (4-20 aa) and quantitated in extracted ion chromatograms. However, this limits the ability to detect even very common co-occurrences of modifications on histone proteins, preventing biological interpretation of PTM crosstalk. By digesting with GluC rather than trypsin, it is possible to produce long polypeptides corresponding to intact histone N-terminal tails (50-60 aa), where most modifications reside. This middle-down MS approach is used to study distant PTM co-existence. However, the most sensitive middle-down workflow uses weak cation exchange-hydrophilic interaction chromatography (WCX-HILIC), which is less robust than conventional reversed-phase chromatography. Additionally, since the buffer systems for middle-down and bottom-up proteomics differ substantially, it is cumbersome to toggle back and forth between both experimental setups on the same LC system. Here, we present a new workflow using porous graphitic carbon (PGC) as a stationary phase for histone analysis where bottom-up and middle-down sized histone peptides can be analyzed simultaneously using the same reversed-phase buffer setup. By using this protocol for middle-down sized peptides, we identified 406 uniquely modified intact histone tails and achieved a correlation of 0.85 between PGC and WCX-HILIC LC methods. Together, our method facilitates the analysis of single and combinatorial histone PTMs with much simpler applicability for conventional proteomics labs than the state-of-the-art middle-down MS.

中文翻译：

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通过自下而上肽和中-下多肽尾巴的集成色谱定量单个和组合组蛋白修饰。

通过质谱（MS）分析组蛋白翻译后修饰（PTM）对表观遗传学领域的发展至关重要。最灵敏，最准确的工作流程类似于标准蛋白质组学分析工作流程（自下而上的MS），在该流程中，组蛋白被消化成短肽（4-20aa）并在提取的离子色谱图中进行定量。但是，这限制了检测甚至非常常见的组蛋白修饰共存的能力，从而阻止了PTM串扰的生物学解释。通过用GluC而不是胰蛋白酶消化，有可能产生与完整的组蛋白N末端尾巴相对应的长多肽（50-60个氨基酸），其中大部分修饰都存在于此。这种从中到下的MS方法用于研究遥远的PTM共存。然而，最敏感的中下工作流程使用弱阳离子交换-亲水相互作用色谱（WCX-HILIC），它不如传统的反相色谱稳定。此外，由于中下蛋白质组学和自下而上蛋白质组学的缓冲系统有很大不同，因此在同一LC系统上的两个实验设置之间来回切换很麻烦。在这里，我们介绍了一种使用多孔石墨碳（PGC）作为固定相进行组蛋白分析的新工作流程，其中可以使用相同的反相缓冲液设置同时分析自下而上和中下大小的组蛋白肽。通过将此协议用于中等大小的肽段，我们鉴定了406个独特修饰的完整组蛋白尾巴，并且在PGC和WCX-HILIC LC方法之间实现了0.85的相关性。一起，