Identification and Significance of Protein Adducts

描述(由申请人提供)：长期以来，蛋白质一直被认为是产生有害健康影响的环境化学品的关键目标。质谱仪(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。这项应用的目标是开发和完善目前最先进的技术，用于全球检测由环境化学品引起的低丰度蛋白质翻译后修饰(PTM)。我们假设，拓扑、化学和物理特征结合在一起决定了哪些蛋白质是化学加合物的靶标，这种加合物导致蛋白质结构/功能的变化，从而促进了对化学物质暴露的毒理学反应。这些PTM以低丰度存在，并且不能被传统的方案检测到。我们实验室目前正在开发专门分析低丰度PTM的质谱学方法。将使用一系列分离和浓缩步骤，结合MS选择性检测和光谱处理算法，对PTMS进行全球鉴定。采用免疫沉淀法分离加合物蛋白，经双向凝胶电泳法分离，用MALDI和多维I-IPLC-ESI-MS/MS分析，用SALSA(计分光谱分析算法)表征蛋白质修饰位点。有了明确的内收部位，我们将确定蛋白质内收的后果。这项应用的目标是(I)开发MS方法来识别环境化学品的蛋白质靶标，(Ii)确定那些使某些蛋白质或蛋白质中的基序易于被化学加成的特征，以及(Iii)确定加合到合理选择的“亲电素”上的潜在生物/毒理学后果。为达到后一目的，我们将利用化学诱导的毒性模型来确定化学诱导的PTMS对(A)核肌动蛋白的功能和PTM-肌动蛋白对染色质重塑的影响，以及(B)细胞色素c的功能，以及PTM-细胞色素c对细胞凋亡的启动和发展的影响。

DESCRIPTION (provided by applicant): 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.