Optimized Metabolite Extraction, Separation, and Identification for Metabolomics

描述(由申请人提供)：本提案的总体目标是帮助确定肝脏代谢组中的关键代谢变化，以帮助研究各种疾病，包括肝脏脂肪变性、炎症和药物毒性。随着肥胖症、II型糖尿病以及药物和酒精滥用的日益普遍，这些肝脏疾病给医疗保健系统带来了越来越大的负担。代谢组学，或对生物过程留下的化学指纹的研究，是一门快速发展的学科，它极有希望提高我们对这些病理疾病的理解。然而，在我们对提取方法和分离方法如何影响下游化学计量学分析中使用的数据的理解方面仍然存在重大差距。因此，我们设计了四个具体的目标，从根本上解决任何基于液相色谱结合质谱仪的代谢组学研究的基本方面：提取、分离和鉴定，并特别参考哺乳动物的肝脏。我们假设，可以开发出与疾病状态无关的可重复提取和分离的方法。到目前为止，还没有一个有组织的、协调一致的努力来优化和标准化进行代谢研究时的这些最基本的步骤。虽然这里我们主要关注肝组织，但这些方法将作为其他组织和生物液以及包括核磁共振光谱和气相色谱-质谱仪在内的平台的基础。基于三个在代谢组学领域拥有公认专业知识的独立实验室(剑桥大学和英国医学研究委员会的Griffin实验室、国家癌症研究所的Gonzalez实验室和宾夕法尼亚州立大学的Patterson实验室)的努力，我们计划对一系列代谢物类别、极性和代谢途径的每个步骤(代谢物提取、高效液相色谱分离和质谱分析)进行系统地处理和优化，从而确保向大量现有的代谢物数据分析平台提供高质量的数据。此外，除了在同行评议的期刊上发表外，我们还将利用社区主导的开源设施，如欧洲生物信息研究所的MetbraLight(实验代谢组学数据的中央储存库)和ISA-TAB倡议(用于描述基因组实验的元数据的统一工具)，以开放的格式向包括学术界和制药业在内的更广泛的科学界免费提供我们的协议和数据。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to help define key metabolic changes in the liver metabolome to aid research into a variety of diseases including hepatic steatosis, inflammation, and drug toxicity. These diseases of the liver are an increasing burden on the healthcare system with growing prevalence of obesity, type II diabetes, and drug and alcohol abuse. Metabolomics, or the study of chemical fingerprints that biological processes leave behind, is a rapidly advancing discipline that has great promise to improve our understanding of these pathological conditions. However, there remain significant gaps in our understanding of how extraction methodologies and separation approaches influence the data used in downstream chemometric analyses. Therefore, we have designed the four specific aims that address fundamentally essential aspects of any metabolomic study based on liquid chromatography coupled with mass spectrometry: extraction, separation, and identification with specific reference to the mammalian liver. We hypothesize that reproducible extraction and separation methodologies can be developed that are independent of disease state. To date, there has not been an organized, concerted effort to optimize and standardize these most essential steps when conducting a metabolomic study. While here we focus on liver tissue, these approaches will serve as a foundation for other tissues and biofluids as well as platforms including nuclear magnetic resonance spectroscopy and gas chromatography coupled with mass spectrometry. Based on the efforts of three independent laboratories with well-recognized expertise in metabolomics (Griffin Lab at the University of Cambridge and the UK Medical Research Council, Gonzalez Lab at the National Cancer Institute, and the Patterson Lab at Penn State University) we plan to systematically address and optimize each step (metabolite extraction, separation by liquid chromatography, and identification by mass spectrometry) across a range of metabolite classes, polarities and metabolic pathways, thus ensuring the delivery of high quality data to the vast array of already existing metabolomic data analysis platforms. Furthermore, in addition to publication in peer-reviewed journals, we will make our protocols and data freely available to the wider scientific community, including both academia and pharma, in an open format by making use of community-led open source facilities such as the European Bioinformatics Institute's MetaboLight (central repository for experimental metabolomics data) and the ISA-TAB initiative (a unified tool for meta data description of omic experiments).