Stransky Stephanie, Saskya-Joseph Jazmine, Sun Yan, Sidoli Simone
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA.
Methods Mol Biol. 2025;2919:251-266. doi: 10.1007/978-1-0716-4486-7_14.
Histone proteins regulate essential cellular processes by being decorated with a myriad of posttranslational modifications (PTMs). These processes are mostly led by the ability of these PTMs to recruit protein readers involved in gene transcription, DNA replication, DNA damage, chromatin remodeling, and other functions. Identifying histone readers is critical for the understanding of mechanisms leading to these functions and potentially predict targets for treatment in anomalous phenotypes. For this reason, histone reader identification has been performed for several years using strategies aiming to increase depth, resolution, and accuracy, e.g., ChIP-MS, proximity biotinylation, photo-crosslinking, and array-based technologies. In this chapter, we describe a protocol for identifying histone readers in a straightforward and unbiased manner: peptide pull-down combined with high-resolution mass spectrometry. Synthesized and immobilized modified histone peptides are incubated with nuclear extracts, and the PTMs' interactors are analyzed by mass spectrometry, which allows the identification of thousands of proteins with high confidence. We also describe the steps for a proteomic data analysis and present tools for a comprehensive data integration.
组蛋白通过多种翻译后修饰（PTM）进行修饰，从而调节细胞的基本过程。这些过程主要是由这些PTM招募参与基因转录、DNA复制、DNA损伤、染色质重塑和其他功能的蛋白质识别蛋白的能力所主导。识别组蛋白识别蛋白对于理解导致这些功能的机制以及潜在预测异常表型的治疗靶点至关重要。因此，多年来一直使用旨在提高深度、分辨率和准确性的策略进行组蛋白识别蛋白的鉴定，例如染色质免疫沉淀-质谱联用（ChIP-MS）、邻近生物素化、光交联和基于阵列的技术。在本章中，我们描述了一种以直接且无偏倚的方式识别组蛋白识别蛋白的方案：肽段下拉结合高分辨率质谱。将合成并固定的修饰组蛋白肽与核提取物一起孵育，然后通过质谱分析PTM的相互作用蛋白，这使得能够高可信度地鉴定数千种蛋白质。我们还描述了蛋白质组数据分析的步骤，并介绍了用于全面数据整合的工具。
