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不良健康影响的环境化学品的关键目标。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。本申请的目标是开发和完善当前最先进的技术，用于全球检测环境化学物质诱导的低丰度蛋白质翻译后修饰（PTM）。我们假设拓扑学、化学和物理特征结合联合收割机来确定哪些蛋白质是化学加合的目标，并且这种加合导致蛋白质的结构/功能的变化，随后导致对化学暴露的毒理学反应。这些PTM以低丰度存在，并且不能通过常规方案检测。我们实验室目前正在开发专门分析低丰度PTM的MS方法。一系列分离和富集步骤结合MS和光谱处理算法的选择性检测将用于PTM的全局识别。将通过免疫沉淀分离加合蛋白，通过2D凝胶电泳分离，并通过MALDI和多维I-IPLC-ESI-MS/MS进行分析。SALSA（光谱分析的ALtax评分）将用于表征蛋白质修饰位点。有了明确的内收位点，我们将确定蛋白质内收的结果。本申请的目标是（i）开发MS方法以鉴定环境化学品的蛋白质靶标，（ii）确定使某些蛋白质或蛋白质内的基序（“亲电体结合基序”）易于化学加合的那些特征，以及（iii）确定加合到合理选择的“亲电蛋白”的潜在生物学/毒理学后果。对于后一个目标，我们将利用一个良好的化学诱导的毒性模型，以确定化学诱导的PTM的具体影响（a）核肌动蛋白的功能和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-细胞色素c对细胞凋亡的启动和进展的影响。

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.