Identification and Significance of Protein Adducts

蛋白质加合物的鉴定和意义

• 批准号: 6875939
• 负责人: Serrine S Lau
• 金额: $ 27.38万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6875939
• 关键词: SDS polyacrylamide gel electrophoresis; actins; adduct; alkylation; apoptosis; chemical models; chemical reaction; cytochrome c; high performance liquid chromatography; immunoprecipitation; laboratory rat; matrix assisted laser desorption ionization; method development; posttranslational modifications; protein structure function; tissue /cell culture; two dimensional gel electrophoresis; western blottings

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.

描述（由申请人提供）：长期以来，蛋白质一直被认为是对健康产生不利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得快速、高通量的蛋白质分析成为可能。该应用的目标是开发和完善当前最先进的技术，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起来确定哪些蛋白质是化学加合的目标，并且这种加合会导致蛋白质结构/功能的变化，从而导致对化学暴露的毒理学反应。这些 PTM 丰度较低，无法通过常规方法检测到。我们的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与 MS 选择性检测和光谱处理算法相结合，将用于 PTM 的全局识别。加合的蛋白质将通过免疫沉淀分离，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（光谱分析评分算法）将用于表征蛋白质修饰位点。通过明确的加合位点，我们将确定蛋白质加合的后果。该应用的目标是 (i) 开发 MS 方法来识别环境化学物质的蛋白质靶标，(ii) 确定使某些蛋白质或蛋白质内的基序（“亲电子结合基序”）易于化学加合的特征，以及 (iii ）以确定与合理选择的“亲电蛋白”内合的潜在生物/毒理学后果。对于后一个目标，我们将利用一个充分表征的化学诱导毒性模型来确定化学诱导的 PTM 对 (a) 核肌动蛋白功能和 PTM-肌动蛋白对染色质重塑的影响的具体影响，以及 (b) ) 细胞色素 c 的功能，以及 PTM-细胞色素 c 对细胞凋亡启动和进展的影响。