Identification and Significance of Protein Adducts

长期以来，蛋白质一直被认为是环境化学品的关键目标， 健康影响。质谱（MS）电离方法和仪器的最新发展， 使蛋白质的快速、高通量分析成为可能。该应用程序的目标是开发和 完善目前最先进的技术，用于全球低丰度蛋白质翻译后检测 环境化学物质引起的修饰（PTM）。我们假设拓扑学、化学和 物理特征联合收割机确定哪些蛋白质是化学加合的目标， 加合引起蛋白质结构/功能的变化，这随后有助于蛋白质的结构/功能的改变。 对化学品暴露的毒理学反应。这些PTM以低丰度存在，并且不 可通过常规协议检测到。具体分析低丰度PTM的MS方法是 目前正在我们的实验室里开发。一系列分离和富集步骤， MS和光谱处理算法的选择性检测将用于全球识别 PTM。将通过免疫沉淀分离加合蛋白，通过2D凝胶电泳分离， 通过MALDI和多维I-IPLC-ESI-MS/MS进行分析。SALSA（光谱分析的评分 分析）将用于表征蛋白质修饰的位点。有了明确的内收部位， 将决定蛋白质加合的结果。该应用程序的目标是（i）开发 MS方法识别环境化学物质的蛋白质靶点，（ii）确定那些特征， 使某些蛋白质或蛋白质内的基序（“亲电体结合基序”）易于化学加合， 和（iii）确定合理选择的加合物的潜在生物学/毒理学后果 “亲电蛋白”。对于后一个目标，我们将利用一个良好的表征模型的化学诱导毒性 为了确定化学诱导的PTM对（a）核肌动蛋白的功能和细胞核内肌动蛋白的功能的特异性影响， PTM-肌动蛋白对染色质重塑的影响，和（B）细胞色素c的功能，以及PTM-肌动蛋白对染色质重塑的影响。 PTM-细胞色素c对细胞凋亡的启动和进展。

Proteins have long been appreciated as critical targets of environmental chemicals that produce adverse health effects. Recent developments in mass spectrometry (MS) ionization methods and instrumentation now make possible the rapid, high throughput analysis of proteins. The goal of this application is to develop and refine current state-of-the-art technology for the global detection of low abundance protein post-translational modifications (PTMs) induced by environmental chemicals. We hypothesize that topological, chemical and physical features combine to determine which proteins are targets for chemical adduction, and such adduction causes a change in structure/function of the protein which subsequently contributes to the toxicological response to chemical exposure. These PTMs are present at low abundance and are not detectable by conventional protocols. MS approaches to specifically analyze low abundance PTMs are currently being developed in our laboratory. A series of isolation and enrichment steps combined with selective detection with MS and spectral processing algorithms will be used for global identification of PTMs. Adducted proteins will be isolated by immune-precipitation, separated by 2D gel electrophoresis and analyzed by MALDI and multidimensional I-IPLC-ESI-MS/MS. SALSA (Scoring ALgorithm for Spectral Analysis) will be used to characterize sites of protein modification. With well defined sites of adduction we will then determine the consequences of protein adduction. The goals of this application are to (i) develop MS methods to identify protein targets of environmental chemicals, (ii) ascertain those features that predispose certain proteins, or motifs within proteins ("electrophile binding motifs") to chemical adduction, and (iii) to determine the potential biological/toxicological consequences of adduction to rationally selected "electrophilins". For the latter aim, we will utilize a well characterized model of chemical-induced toxicity to determine the specific effects of chemical-induced PTMs on (a) the function of nuclear actin and the effects of PTM-actin on chromatin remodeling, and (b) the function of cytochrome c, and the effects of PTM-cytochrome c on the initiation and progression of apoptosis.