PROTEIN TARGETS OF ENVIRONMENTAL CHEMICALS

环境化学品的蛋白质目标

• 批准号: 6525216
• 负责人: Daniel C Liebler
• 金额: $ 21.35万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-15 至 2003-12-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6525216
• 关键词: SDS polyacrylamide gel electrophoresis; adduct; biotransformation; chemical carcinogen; chemical conjugate; electrospray ionization mass spectrometry; environment related neoplasm /cancer; environmental toxicology; epoxides; haloalkane; hepatotoxin; high performance liquid chromatography; laboratory rat; liver disorder; method development; monocrotaline; peptide library; peptides; protein sequence; protein structure function; proteolysis; proteomics; quinones; technology /technique development; toxicant interaction

Proteins long have been appreciated as critical targets of environmental chemicals that produce toxicity and cancer. Recent developments in mass spectrometry ionization methods and instrumentation have driven the growth of proteomics, the study of the protein complement of the genome. The overall goal of this project is to use mass spectrometry methods for proteomics to identify the protein targets of environmental chemicals. We propose to selectively identify adducted peptides derived from proteolysis of adducted proteins and to characterize adduction at the level of amino acid sequence. We will use these approaches to test the hypothesis that specific cellular patterns of toxicity are linked to covalent modification of specific protein targets. In specific aim 1, we will develop methods for detecting and identifying protein targets of environmental chemicals. Work in this aim is sub-divided into four major emphasis areas. la) Peptide adduct libraries will be prepared from synthetic peptides site-specifically adducted by the reactive intermediates 1,4-benzoquinone, dehydromonocrotaline, 4-vinylcyclohexene diepoxide, and reactive glutathione conjugates derived from 1,1-dichloroethylene and used to establish characteristic MS-MS fragmentation of adducted peptides. lb) Instrument control algorithms will be developed for rapid, selective detection and sequencing of adducted peptides in HPLC-MS-MS analyses. 1c) Proteomics analysis methods based on electrophoresis- or HPLC-based separations, combined with proteolytic digestion and MS-MS analysis will be adapted to the identification of adducts. 1d) The applications of "shotgun sequencing" methods to the identification of protein adducts in complex mixtures by electrospray HPLC-MS-MS will be explored. In specific aim 2, we will characterize the protein targets of 1,1-dichloroethylene (DCE) in rat liver. We will identify DCE-derived adducts with hepatocyte canalicular membrane to test the hypothesis that specific adduction leads to selective canalicular injury. The methods developed in this project will open important, yet largely unexplored aspects of chemical biology to mechanistic investigation.

长期以来，蛋白质一直被认为是产生毒性和癌症的环境化学物质的关键目标。 质谱电离方法和仪器的最新发展推动了蛋白质组学（基因组蛋白质补体的研究）的发展。 该项目的总体目标是利用蛋白质组学的质谱方法来识别环境化学品的蛋白质靶标。 我们建议选择性地鉴定源自加合蛋白的蛋白水解的加合肽，并在氨基酸序列水平上表征加合。 我们将使用这些方法来检验特定细胞毒性模式与特定蛋白质靶标的共价修饰有关的假设。 在具体目标1中，我们将开发检测和识别环境化学品蛋白质靶标的方法。 实现这一目标的工作分为四个主要重点领域。 la) 肽加合物文库将由合成肽制备，该合成肽由反应中间体 1,4-苯醌、脱氢野百合碱、4-乙烯基环己烯二环氧化物和源自 1,1-二氯乙烯的反应性谷胱甘肽缀合物进行位点特异性加合，并用于建立特征 MS-加合肽的 MS 断裂。 lb) 将开发仪器控制算法，用于在 HPLC-MS-MS 分析中快速、选择性地检测和测序加合肽。 1c) 基于电泳或 HPLC 分离的蛋白质组学分析方法，结合蛋白水解消化和 MS-MS 分析，将适用于加合物的鉴定。 1d) 将探索“鸟枪测序”方法在电喷雾 HPLC-MS-MS 鉴定复杂混合物中蛋白质加合物中的应用。 在具体目标 2 中，我们将表征大鼠肝脏中 1,1-二氯乙烯 (DCE) 的蛋白质靶标。 我们将鉴定 DCE 衍生的与肝细胞小管膜的加合物，以检验特异性内收导致选择性小管损伤的假设。 该项目开发的方法将为化学生物学的重要但很大程度上尚未探索的方面提供机制研究。