Quantification of human alcohol metabolizing enzymes by mass spectrometry

描述（由申请人提供）：这是一项R21探索性/发展性拨款申请，旨在开发量化人体组织（肝脏和食道）中酒精代谢酶及其变体（最初是酒精脱氢酶）含量的方法。我们相信该应用符合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的目标。该应用也响应了PA-05-074，酒精诱导的组织损伤机制，因为它提供了重要的蛋白质表达信息，以支持酒精代谢酶基因型的研究，这些基因型赋予对酒精诱导的细胞/组织损伤的易感性或抗性。我们的中心假设是乙醇脱氢酶（ADHs）及其变体的表达/含量决定了乙醇和乙醛在组织中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，通过三重四重线性离子阱LC/MS/MS和多重反应监测（MRM），以适当的同位素标记ADH蛋白标准，量化肝脏和食管组织中6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蛋白标准物的新方法，并利用它们的肽MRM过渡来量化特定的同工酶和同工酶群。在目标2中，我们从供体中收集了22个冷冻肝脏和16个冷冻食管样本（8个匹配肿瘤和正常食管样本）。样本将在ADH位点进行基因分型。在目标3中，我们将进行初步分析，看看组织中ADH表达（目标1）和基因型（目标2）之间是否存在任何相关性。我们将估计六种ADHs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的可行性，如醛脱氢酶和参与人类酒精/乙醛代谢的细胞色素p450。

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.