Nuccio A G, Bui M, Dalal Y, Nita-Lazar A
Cellular Networks Proteomics Unit, Laboratory of Systems Biology, NIAID, NIH, Bethesda, MD, United States.
Chromatin Structure and Epigenetic Mechanisms Unit, Laboratory of Receptor Biology and Gene Expression, CCR, NCI, NIH, Bethesda, MD, United States.
Methods Enzymol. 2017;586:275-290. doi: 10.1016/bs.mie.2016.09.035. Epub 2016 Nov 2.
Histone posttranslational modifications (PTMs) are key epigenetic marks involved in gene silencing or activation. Histone modifications impact chromatin organization and transcriptional processes through the changes in charge density between histones and DNA. They also serve as recognition and binding sites for specific binding proteins. Histone tails and globular cores contain many basic amino acid residues, which are subject to various dynamic modifications, making the modification repertoire extremely diverse. Consequently, determination of histone PTM identity and quantity has been a challenging task. In recent years, mass spectrometry-based methods have proven useful in histone PTM characterization. This chapter provides a brief overview of these methods and describes the approach to analyze the PTMs of the histone variant CENP-A, essential for the cell cycle progression, when present in minute amounts from tumor and mammalian tissues. Because this method does not rely on antibody-based immunopurification, we anticipate that these tools could be readily adaptable to the investigation to other histone variants in a range of mammalian tissues and solid tumors.
组蛋白翻译后修饰（PTMs）是参与基因沉默或激活的关键表观遗传标记。组蛋白修饰通过组蛋白与DNA之间电荷密度的变化影响染色质组织和转录过程。它们还作为特定结合蛋白的识别和结合位点。组蛋白尾巴和球状核心包含许多碱性氨基酸残基，这些残基会发生各种动态修饰，使得修饰种类极其多样。因此，确定组蛋白PTM的种类和数量一直是一项具有挑战性的任务。近年来，基于质谱的方法已被证明在组蛋白PTM表征中很有用。本章简要概述了这些方法，并描述了分析组蛋白变体CENP-A的PTM的方法，CENP-A对细胞周期进程至关重要，当它以微量存在于肿瘤和哺乳动物组织中时。由于该方法不依赖基于抗体的免疫纯化，我们预计这些工具可轻松适用于一系列哺乳动物组织和实体瘤中其他组蛋白变体的研究。
