Technology for Identification of Epiproteomes in Tissue Samples

 描述(由申请人提供)：这项提案的总体目标是开发一种用于识别组织样本中的表观蛋白质组的尖端技术，这将提供一种方法，重新定义NIDA研究人员如何研究药物滥用/成瘾的表观遗传反应。我们将表观蛋白质组定义为位于~1kb染色体位置的蛋白质、组蛋白和组蛋白翻译后修饰。在之前的工作中，我们开发了一个突破性的技术平台，称为CHAP-MS(染色质亲和纯化与质谱学)，它允许我们以生物化学的方式分离~1kb的染色体片段，并使用高分辨率蛋白质组学来鉴定表观蛋白质组。我们的CHAP-MS平台首次为蛋白质组分析提供了特定的、天然的染色质基因座的分离。第一代和第二代CHAP-MS技术使用异位表达的亲和探针分离染色质，并设计用于细胞培养研究；因此，排除了对动物模型的分析。正如这项提议所概述的，我们将在酵母中开发一种名为重组rCRISPR-CHAP-MS的第三代方法，并将其翻译成脑组织样本。第三代方法将绕过亲和探针的异位表达，从而使组织分析成为可能。我们展望了rCRISPR-CHAP-MS方法在定义控制药物滥用/成瘾的表观遗传机制方面的长期应用。具体目标1将侧重于开发应用于组织样本的rCRISPR-CHAP-MS。以酿酒酵母为模型系统，在目标1中，我们将通过分析转录活跃和抑制条件下GAL1启动子的表观蛋白质组来建立rCRISPR-CHAP-MS。具体目标2将专注于将rCRISPR-CHAP-MS翻译到脑组织。在目标2中，我们将针对长期暴露于甲基苯丙胺的大鼠纹状体内AMPA受体亚单位GluA1基因启动子的优化方法。

 DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop a cutting-edge technology for identifying epiproteomes in tissue samples, which will provide an approach that will redefine how NIDA researchers study epigenetic responses to drug abuse/addiction. We define an epiproteome as the proteins, histones and histone posttranslational modifications at a defined ~1 kb chromosomal location. In previous work, we have developed a groundbreaking technology platform called ChAP-MS (Chromatin Affinity Purification with Mass Spectrometry) that allows us to biochemically isolate a ~1 kb section of a chromosome and identify the epiproteome using high resolution proteomics. Our ChAP-MS platform provided for the first ever isolation of a specific, native chromatin locus for proteomic analysis. The first and second generation ChAP-MS technologies used ectopic expression of affinity probes for chromatin isolation and were designed for cell culture studies; thus, precluding the analysis of animal models. As outlined in this proposal, we will develop a third generation approach called recombinant rCRISPR-ChAP-MS in yeast and translate it to brain tissue samples. The third generation approach will circumvent ectopic expression of affinity probes; thus, enabling the analysis of tissue. We envision the long term application of the rCRISPR-ChAP-MS approach in defining epigenetic mechanisms regulating drug abuse/addiction. Specific Aim 1 will focus on developing rCRISPR-ChAP-MS for application to tissue samples. Using S. cerevisiae as a model system in Aim 1, we will develop rCRISPR-ChAP-MS by analysis of epiproteomes at the GAL1 promoter under transcriptionally active and repressed conditions. Specific Aim 2 will focus on translating rCRISPR-ChAP-MS to brain tissue. In Aim 2, we will target our optimized approach to the gene promoter of AMPA receptor subunit GluA1 in striatum from rats chronically exposed to methamphetamine.