21st Century Analysis of Histone Modifications (pp 589-634)

组蛋白负责DNA的有效包装，更重要的是，对 几乎所有以染色质为模板的活动。组蛋白“密码”假说指出，一组 组蛋白中的PTMS可能会引发特定的转录程序。在这三个目标中，我们将制定、验证和 将基于两种口味的质谱学的综合方法应用于组蛋白的分析 对几个疾病和模型系统进行了修改。 具体目标1.建立和验证一种定量三重四极(QQQ)方法来监测100 使用自下而上的方法从1,000个细胞进行组蛋白修饰。 具体目标2.开发和验证一种自顶向下的定量质谱学方法来监测 来自10,000个细胞的1000种组蛋白蛋白质形式。 具体目标3.将组蛋白修饰的定量测量与遗传 信息。-开发和验证用于临床标本的坚固管道。 利用在特定目标1和2中开发的技术，特定目标3集成了这些质谱学 利用基因数据对来自癌症患者的临床样本提供洞察的方法 表观遗传修饰物的靶向突变。在许多情况下，这些突变被认为是背后的“驱动因素” 肿瘤发生学。虽然研发1、2和3侧重于蛋白质组学分析中的具体技术障碍，但研发4 寻求协调多个、密集、多样化的数据集。这个研发项目将推动我们这一新兴领域的发展 通过标准化方法和最大限度地提高信息的质量和数量而被称为“表观蛋白质组学” 从有限和不同种类的临床标本中获得，以深入了解复杂的疾病。

Histone proteins are responsible for efficient packaging of DNA and, more importantly, the modulation of almost every chromatin-templated activity. The histone ‘code’ hypothesis states that the combination of a set of PTMs in histone may elicit a specific transcriptional program. In these three aims, we will develop, validate and apply a comprehensive approach, based on two flavors of mass spectrometry, to the analysis of histone modifications in several disease and model systems. Specific Aim 1. To Develop and Validate a Quantitative Triple Quadrupole (QqQ) Method to Monitor 100 Histone Modifications from 1,000 Cells using a Bottom-Up Approach. Specific Aim 2. To Develop and Validate a Quantitative Top-Down Mass Spectrometry Method to Monitor 1000 Histone Proteoforms from 10,000 Cells. Specific Aim 3. To Integrate Quantitative Measurements of Histone Modifications with Genetic Information. – Development and Validation of a Robust Pipeline for Clinical Specimens. Using technology developed in Specific Aims 1 and 2, Specific Aim 3 integrates those mass spectrometry approaches with genetic data to provide insight into clinical samples coming from cancer patients carrying targeted mutations in epigenetic modifiers. In many cases, these mutations are thought to be the “drivers” behind tumorigenesis. While TR&Ds 1, 2 and 3 focus on specific technical hurdles in proteomics analyses, TR&D 4 seeks to harmonize multiple, dense, diverse datasets. This TR&D project will advance the burgeoning field we refer to as “epiproteomics” by standardizing methods and maximizing the quality and quantity of information acquired from limited and heterogeneous clinical specimens to gain insight into complex diseases.