Anal Chem. 2018 Mar 6;90(5):3083-3090. doi: 10.1021/acs.analchem.7b03948. Epub 2018 Feb 15.
Dynamic post-translational modifications of histones regulate transcriptional gene expression in eukaryotes. Unique combinations of modifications, almost exclusively displayed at the flexible N-terminal tails on histones, create distributions of proteoforms that need to be characterized in order to understand the complexity of gene regulation and how aberrant modification patterns influence disease. Although mass spectrometry is a preferred method for the analysis of histone modifications, information is lost when using conventional trypsin-based histone methods. Newer "middle-down" protocols may retain a greater fraction of the full proteoform distribution. We describe a strategy for the simultaneous characterization of histones H3 and H4 with near-complete retention of proteoform distributions, using a conventional proteomics liquid chromatography-tandem mass spectrometry (LC-MS/MS) configuration. The selective prolyl endoprotease neprosin generates convenient peptide lengths for retention and dispersion of modified H3 and H4 peptides on reversed-phase chromatography, offering an alternative to the hydrophilic interaction liquid chromatography typically used in middle-down methods. No chemical derivatizations are required, presenting a significant advantage over the trypsin-based protocol. Over 200 proteoforms can be readily profiled in a single analysis of histones from HeLa S3 cells. An in-gel digestion protocol provides additional options for effective histone analysis.
组蛋白的动态翻译后修饰调节真核生物的转录基因表达。修饰的独特组合，几乎只在组蛋白的灵活 N 端尾部显示，形成了需要加以表征的蛋白形式分布，以便了解基因调控的复杂性以及异常修饰模式如何影响疾病。尽管质谱是分析组蛋白修饰的首选方法，但传统的基于胰蛋白酶的组蛋白方法会丢失信息。更新的“中向下”方案可能会保留更大比例的完整蛋白形式分布。我们描述了一种策略，用于使用常规蛋白质组学液相色谱-串联质谱 (LC-MS/MS) 配置同时对组蛋白 H3 和 H4 进行特征分析，几乎完全保留蛋白形式分布。选择性脯氨酰内切蛋白酶 neprosin 生成方便的肽长度，用于保留和分散反相色谱上修饰的 H3 和 H4 肽，为中向下方法中通常使用的亲水相互作用液相色谱提供了替代方案。不需要化学衍生化，这与基于胰蛋白酶的方案相比具有显著优势。在单个 HeLa S3 细胞组蛋白分析中，可轻松分析超过 200 种蛋白形式。胶内消化方案为有效的组蛋白分析提供了更多选择。
