Restellini Camilla, Cuomo Alessandro, Lupia Michela, Giordano Marco, Bonaldi Tiziana, Noberini Roberta
Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy.
Unit of Gynecological Oncology Research, IEO, European Institute of Oncology IRCCS, Milan, Italy.
Proteomics Clin Appl. 2019 Jan;13(1):e1700166. doi: 10.1002/prca.201700166. Epub 2018 Dec 6.
Profiling histone posttranslational modifications (PTMs) in clinical samples holds great potential for the identification of epigenetic biomarkers and the discovery of novel epigenetic targets. MS-based approaches to analyze histone PTMs in clinical samples usually rely on SDS-PAGE separation following histone enrichment in order to eliminate detergents and further isolate histones. However, this limits the digestions options and hence the modification coverage.
The aim of this study is the implementation of a procedure involving acetone protein precipitation followed by histone enrichment through a C18 StageTip column to obtain histone preparations suitable for various in-solution digestion protocols. Among them, the Arg-C digestion, which allows profiling histone H4 modifications, and the Prop-PIC method, which improves the detection of short and hydrophilic peptides, are tested. This approach is validated on different types of samples, including formalin-fixed paraffin-embedded pathology tissues, and employed to profile histone H4 modifications in cancer samples and normal tissues, identifying previously reported differences, as well as novel ones.
This protocol widens the number of applications available in the toolbox of clinical epigenomics, allowing the investigation of a larger spectrum of histone marks in patient samples.
分析临床样本中的组蛋白翻译后修饰（PTM）对于鉴定表观遗传生物标志物和发现新的表观遗传靶点具有巨大潜力。基于质谱的分析临床样本中组蛋白PTM的方法通常依赖于在组蛋白富集后进行SDS-PAGE分离，以去除去污剂并进一步分离组蛋白。然而，这限制了酶切选择，从而限制了修饰覆盖范围。
本研究的目的是实施一种方法，该方法包括丙酮蛋白沉淀，然后通过C18 StageTip柱进行组蛋白富集，以获得适用于各种溶液内酶切方案的组蛋白制剂。其中，测试了可分析组蛋白H4修饰的精氨酸酶C（Arg-C）酶切以及可改善短肽和亲水肽检测的丙氨酸-脯氨酸-异亮氨酸-半胱氨酸（Prop-PIC）方法。该方法在包括福尔马林固定石蜡包埋病理组织在内的不同类型样本上得到验证，并用于分析癌症样本和正常组织中的组蛋白H4修饰，识别出先前报道的差异以及新的差异。
该方案拓宽了临床表观基因组学工具箱中可用的应用数量，使得能够在患者样本中研究更大范围的组蛋白标记。
