Quantification of human alcohol metabolizing enzymes by mass spectrometry

通过质谱法定量人体酒精代谢酶

• 批准号: 7386190
• 负责人: WILLIAM F BOSRON
• 金额: $ 21.72万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-15 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7386190
• 关键词: Acetaldehyde; Address; Alcohol dehydrogenase; Alcohols; Aldehyde dehydrogenase (NAD+); Applications Grants; Cells; Cytochromes; Development; Drug Kinetics; Enzymes; Escherichia coli; Esophageal Tissue; Esophagus; Ethanol; Ethanol toxicity; Experimental Designs; Exploratory/Developmental Grant; Freezing; Generations; Genetic Polymorphism; Genotype; Grant; Guidelines; Human; Individual; Injury; Investigation; Ions; Isoenzymes; Label; Liver; Liver Extract; Mass Spectrum Analysis; Metabolic; Metabolism; Methods; Monitor; National Institute on Alcohol Abuse and Alcoholism; Peptides; Predisposition; Proteins; Proteomics; Reaction; Resistance; Sampling; Standards of Weights and Measures; Tissue Sample; Tissues; Variant; alcohol content; aldehyde dehydrogenases; human tissue; instrument; novel; protein expression; response; tumor

DESCRIPTION (provided by applicant): This is an R21 exploratory/developmental grant application to develop methods to quantify the content of alcohol metabolizing enzymes and variants (initially alcohol dehydrogenase) in human tissues (liver and esophagus). We believe that the application fits the R21 guidelines because we will use newly developed mass spectrometry proteomics methods to address the specific aims. The application is also in response to PA-05-074, Mechanisms of Alcohol-Induced Tissue Injury, because it provides important protein expression information to support the investigation of alcohol metabolizing enzyme genotypes that confer susceptibility or resistance to ethanol-induced cell/tissue damage. Our central hypothesis is that the expression/content of alcohol dehydrogenases (ADHs) and variants determine the pharmacokinetics and cellular toxicity of ethanol and acetaldehyde in tissues. In Aim #1, we will develop proteomics methods to quantify the expression of six ADHs in liver and esophagus tissue by triple quadruple linear ion trap LC/MS/MS and multiple reaction monitoring (MRM) with appropriate isotopic labeled ADH protein standards. We will develop a novel method of expressing isotopic labeled ADH protein standards in E. coli and using their peptide MRM transitions to quantify specific isoenzymes as well as groups of isoenzymes. In Aim #2, we have collected 22 frozen liver and 16 frozen esophagus samples (8 matched tumor and normal esophagus samples) from donors. The samples will be genotyped at the ADH loci. In Aim #3, we will perform a preliminary analysis to see if there is any correlation between ADH expression (Aim #1) and genotype (Aim #2) in tissues. We will estimate the individual contributions of six ADHs to alcohol metabolic capacity in liver and see if this capacity varies with genotype. We will assess the feasibility of extending the proteomics and genotyping analyses to other alcohol metabolizing enzymes like aldehyde dehydrogenase and cytochrome P450s involved in alcohol/acetaldehyde metabolism in humans.

描述（由申请人提供）：这是一项 R21 探索性/开发资助申请，旨在开发量化人体组织（肝脏和食道）中酒精代谢酶和变体（最初是乙醇脱氢酶）含量的方法。我们相信该应用程序符合 R21 指南，因为我们将使用新开发的质谱蛋白质组学方法来解决特定目标。该申请也是对 PA-05-074（酒精引起的组织损伤的机制）的回应，因为它提供了重要的蛋白质表达信息，以支持对酒精代谢酶基因型的研究，这些基因型赋予对乙醇引起的细胞/组织损伤的易感性或抵抗力。我们的中心假设是，乙醇脱氢酶 (ADH) 和变体的表达/含量决定组织中乙醇和乙醛的药代动力学和细胞毒性。在目标#1中，我们将开发蛋白质组学方法，通过三重四极杆线性离子阱 LC/MS/MS 和多重反应监测 (MRM) 以及适当的同位素标记的 ADH 蛋白标准品来量化肝脏和食道组织中六种 ADH 的表达。我们将开发一种在大肠杆菌中表达同位素标记的 ADH 蛋白标准品的新方法，并使用其肽 MRM 转换来量化特定同工酶以及同工酶组。在目标 2 中，我们从捐赠者那里收集了 22 个冷冻肝脏和 16 个冷冻食管样本（8 个匹配的肿瘤和正常食管样本）。样本将在 ADH 基因座上进行基因分型。在目标#3中，我们将进行初步分析，看看组织中的ADH表达（目标#1）和基因型（目标#2）之间是否存在相关性。我们将估计六种抗利尿激素对肝脏酒精代谢能力的个体贡献，并观察这种能力是否随基因型而变化。我们将评估将蛋白质组学和基因分型分析扩展到其他酒精代谢酶（如参与人类酒精/乙醛代谢的乙醛脱氢酶和细胞色素 P450）的可行性。