High Throughput Lipidomics Analysis by MALDI/Ion Mobility Mass Spectrometry

通过 MALDI/离子淌度质谱法进行高通量脂质组学分析

• 批准号: 8415669
• 负责人: ROBERT Carl MURPHY
• 金额: $ 38.71万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-15 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8415669
• 关键词: Acetyl Coenzyme A; Address; Area; Biochemical Reaction; Biological; Carbon; Categories; Cells; Cellular biology; Chemistry; Cholesterol; Cholesterol Esters; Clinical; Clinical Trials; Coenzyme A; Complex; Complex Mixtures; Coupled; DNA; Data; Databases; Development; Diphosphates; Disease; Dolichol; Esters; Fatty Acids; Fatty-acid synthase; Glycerol; Glycerophospholipids; Goals; Grant; Human; Image; Individual; Institutes; Ions; Isoprene; Laboratories; Lipid Bilayers; Lipid Biochemistry; Lipids; Liquid substance; Maps; Mass Spectrum Analysis; Measurement; Measures; Metabolism; Methods; Minor; Molecular; Normal Cell; Pathway interactions; Plasma; Play; Process; Proteins; Research; Resolution; Robotics; Role; Sampling; Shotguns; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sphingolipids; Spottings; Steroids; System; Techniques; Technology; Tissue Extracts; Tissues; Ubiquinone; Urine; base; cost; design and construction; high throughput analysis; improved; insight; interest; ion mobility; ionization; lipid biosynthesis; metabolomics; microbial alkaline proteinase inhibitor; novel; peroxidation; prenol; tool

DESCRIPTION (provided by applicant): The area of research termed metabolomics encompasses the study of a wide range of biomolecules that are products and substrates of enzymatic reactions within cells. One of the major subclasses of cellular metabolites are lipids, which they themselves are a diverse class of molecules that range from fatty acids, steroids, glycerolipids, glycerophospholipids, sphingolipids, and prenols. The measurement of lipids has significantly improved over the past decade, largely due to advances in mass spectrometry and various ancillary techniques. Lipids constitute an important class of compounds within the metabolomic sphere of interest because of the central role these molecules play, not only in separating compartments within cells through formation of lipid bilayers, but also through specific roles these molecules can play as precursor of signally molecules, targets for peroxidation chemistry, regulators of DNA expression, and even entities that are recognized by proteins that drive critical processes in normal cell biology. Within each class of lipids, there ae closely related molecules termed molecular species which increase the complexity of the lipid mixture and challenging analysis. Based upon recent advances we have made in using MALDI imaging mass spectrometry we propose that it will be possible to significantly increase the throughput of lipid analysis so that it will be possible to obtain important metabolomic information from several hundreds of samples per day on a fairly routine basis. Furthermore, we propose that this platform can be tuned to increase sensitivity for specific classes of lipids and used for high throughput lipidomic analysis to address specific questions concerning enzymatic pathways involved in lipid synthesis and metabolism. In part this approach will take advantage of the advances that have been made in the area of shotgun lipidomics coupled to the high throughput capability of MALDI mass spectrometry to yield both qualitative and quantitative information. Preliminary data shows that even untreated serum and plasma can yield information about the complex mixture of lipids present in this fluid without any further sample treatment except dilution. This data can be obtained within less than a minute in a form that can readily integrate into tools to automatically identify lipids as well as quantitate the abundance o lipids present. In order to carry out these studies, advanced mass spectrometric techniques will be used, including ion mobility technology to separate lipid ions from other compounds and impurities generated during the MALDI ionization process. The advanced mass spectrometric techniques to be developed can be implemented in many laboratories even without the ion mobility; however, ion mobility, as it becomes more widely available, provides a unique level of capability not currently present with other mass spectrometric system. PUBLIC HEALTH RELEVANCE: Development of high throughput methods to measure the complex mixture of lipids present in biological samples, including serum, plasma, urine, as well as cells and tissues is challenging from the stand point of the degree of complexity of lipid components, but also the need to understand the quantitative levels of lipids present in these samples. A novel method to generate quantitative information concerning lipids is proposed based upon a MALDI ionization mass spectrometry platform and the use of database searching to identify lipids and internal standard quantitation to automatically convert mass spectrometric signals into quantitative measures of individual molecular species of lipids. The improvement of analytical throughput could have a significant bearing upon the reduction of costs in measuring these specific metabolites so that a large number samples including those in the 1,000s that have been collected in studies of specific human disorders can be analyzed to reveal the involvement of lipid biochemistry in disease processes.

描述（由申请人提供）：代谢组学的研究领域涵盖了对细胞内酶促反应的产物和底物的各种生物分子的研究。细胞代谢物的主要亚类之一是脂质，它们本身是多种分子，范围包括脂肪酸、类固醇、甘油脂、甘油磷脂、鞘脂和异戊烯醇。在过去的十年中，脂质的测量有了显着的改进，这主要归功于质谱法和各种辅助技术的进步。脂质是代谢组学领域内一类重要的化合物，因为这些分子发挥着核心作用，不仅通过形成脂质双层来分隔细胞内的区室，而且通过特定的作用，这些分子可以作为信号分子的前体、过氧化化学的靶标、DNA表达的调节剂，甚至是被驱动关键过程的蛋白质识别的实体。 在正常细胞生物学中。在每一类脂质中，都有一些密切相关的分子，称为分子种类，这增加了脂质混合物的复杂性和挑战性的分析。基于我们在使用 MALDI 成像质谱法方面取得的最新进展，我们认为将有可能显着提高脂质分析的通量，从而可以在相当常规的基础上每天从数百个样品中获得重要的代谢组信息。此外，我们建议可以调整该平台以提高对特定类别脂质的敏感性，并用于高通量脂质组学分析，以解决有关脂质合成和代谢中涉及的酶途径的特定问题。在某种程度上，这种方法将利用鸟枪脂质组学领域取得的进步以及 MALDI 质谱的高通量能力来产生定性和定量信息。初步数据表明，即使未经处理的血清和血浆也可以产生有关该液体中存在的复杂脂质混合物的信息，无需任何进一步的样品处理（除了稀释）。这些数据可以在不到一分钟的时间内获得，并且可以轻松集成到工具中，以自动识别脂质并定量存在的脂质丰度。为了进行这些研究，将使用先进的质谱技术，包括离子淌度技术，将脂质离子与 MALDI 电离过程中产生的其他化合物和杂质分离。即使没有离子淌度，所开发的先进质谱技术也可以在许多实验室中实施；然而，随着离子淌度的应用越来越广泛，它提供了目前其他质谱系统所不具备的独特功能水平。 公共卫生相关性：开发高通量方法来测量生物样品（包括血清、血浆、尿液以及细胞和组织）中存在的脂质复杂混合物具有挑战性，从脂质成分的复杂程度来看，而且需要了解这些样品中存在的脂质的定量水平。提出了一种基于 MALDI 电离质谱平台并使用数据库搜索来识别脂质和内标定量来生成有关脂质定量信息的新方法，以自动将质谱信号转换为脂质的各个分子种类的定量测量。分析通量的提高可能对降低测量这些特定代谢物的成本产生重大影响，从而可以分析大量样本，包括在特定人类疾病研究中收集的数千个样本，以揭示脂质生物化学在疾病过程中的参与。