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）：研究领域称为代谢组学的研究包括对细胞内酶促反应的产物和底物的广泛生物分子的研究。细胞代谢物的主要亚类之一是脂质，它们本身是一类分子，范围从脂肪酸，类固醇，甘油脂质，甘油磷脂，鞘脂，鞘脂脂和前醇。在过去的十年中，脂质的测量显着改善，这在很大程度上是由于质谱和各种辅助技术的进步。由于这些分子起着的核心作用，不仅在通过形成脂质双层的脂质中分离细胞内的隔室，而且通过特定的作用，这些分子可以作为信号分子的前体来确定驱动式化学，并且是通过特定的驱动力来确定驱动力，并且可以通过特定的作用，因此，脂质构成了代谢性领域内的重要一类化合物，不仅可以通过特定的作用来分离细胞内部的隔室，而且可以通过特定的作用来作为指定性分子的前体来依赖于驱动力的驱动力，依赖于驱动式化学，并在驱动器中的驱动力，均可驱动式化学，甚至是dnna的驱动器。在正常细胞生物学中。在每类脂质中，AE ae密切相关的分子称为分子物种，这些分子会增加脂质混合物的复杂性和具有挑战性的分析。根据我们在使用MALDI成像质谱法方面取得的最新进展，我们建议可以显着增加脂质分析的吞吐量，以便在相当常规的基础上每天从几百个样本中获取重要的代谢性信息。此外，我们建议可以调整该平台以提高对特定类脂质类别的灵敏度，并用于高吞吐量脂质组分析，以解决有关涉及脂质合成和代谢涉及的酶促途径的特定问题。在某种程度上，这种方法将利用在shot弹枪脂质组学领域取得的进步，并伴随着MALDI质谱的高吞吐量能力，以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这种液体中存在的脂质复杂混合物的信息，而没有任何进一步的样品处理，除非稀释。可以在不到一分钟的时间内以可以轻松集成到工具中以自动识别脂质并定量存在的丰度O脂质的形式，以在不到一分钟的时间内获得此数据。为了进行这些研究，将使用先进的质谱技术，包括离子迁移率技术将脂质离子与MALDI离子化过程中产生的其他化合物和杂质分开。即使没有离子移动性，也可以在许多实验室中实施要开发的高级质谱技术。但是，由于离子迁移率变得越来越广泛，因此提供了其他质谱系统目前不存在的独特能力水平。 公共卫生相关性：开发高吞吐量方法，以测量生物样品中存在的脂质的复杂混合物，包括血清，血浆，尿液以及细胞和组织，从脂质成分的复杂程度的角度来看，这具有挑战性，并且还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离子化质谱平台的新方法来生成有关脂质的定量信息，并使用数据库搜索来识别脂质和内标定量，以自动将质谱信号转换为脂质单个分子物种的定量测量。分析吞吐量的改善可能与降低这些特定代谢产物的成本降低可能​​有很大的关系，因此可以分析大量样本，包括在特定人类疾病研究中收集的1000种样本，以揭示脂质生物化学参与疾病过程。