Sidoli Simone, Bhanu Natarajan V, Karch Kelly R, Wang Xiaoshi, Garcia Benjamin A
Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania.
Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania;
J Vis Exp. 2016 May 17(111):54112. doi: 10.3791/54112.
Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.
核小体是染色质的最小结构单位，由缠绕在组蛋白八聚体上的147个碱基对的DNA组成。组蛋白的功能是由多种核蛋白进行广泛的翻译后修饰介导的。这些修饰对于核完整性至关重要，因为它们调节染色质结构并招募参与基因调控、DNA修复和染色体凝聚的酶。尽管科学界的很大一部分采用基于抗体的技术来表征组蛋白PTM丰度，但这些方法通量低且对超修饰蛋白有偏差，因为表位可能会被附近的修饰所阻碍。本方案描述了使用纳升液相色谱（nLC）和质谱（MS）对组蛋白修饰进行准确定量。该方法旨在在单次分析中表征多种组蛋白PTM以及几种组蛋白变体的相对丰度。在本方案中，组蛋白先用丙酸酐衍生化，然后用胰蛋白酶消化，以产生长度为5 - 20个氨基酸的肽段。消化后，组蛋白肽段新暴露的N末端进行衍生化，以改善nLC-MS期间的色谱保留。该方法允许对跨越四个数量级的组蛋白PTM进行相对定量。
