High Throughput Lipidomics Analysis by MALDI/Ion Mobility Mass Spectrometry

描述（由申请人提供）：代谢组学的研究领域包括广泛的生物分子研究，这些生物分子是细胞内酶促反应的产物和底物。细胞代谢物的一个主要亚类是脂类，脂类本身是多种分子，包括脂肪酸、类固醇、甘油脂、甘油磷脂、鞘脂和戊烯醇。在过去的十年中，由于质谱法和各种辅助技术的进步，脂质测量有了显著的改善。脂质在代谢组学领域中是一类重要的化合物，因为这些分子不仅通过形成脂质双层来分离细胞内的隔室，而且还发挥着特定的作用，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，甚至是驱动正常细胞生物学关键过程的蛋白质识别的实体。在每一类脂质中，都有密切相关的分子，称为分子种，这增加了脂质混合物的复杂性和分析的挑战性。基于我们在使用MALDI成像质谱方面取得的最新进展，我们建议可以显著提高脂质分析的吞吐量，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学信息。此外，我们建议该平台可以调整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，以解决涉及脂质合成和代谢的酶促途径的特定问题。在某种程度上，这种方法将利用霰弹枪脂质组学领域取得的进步，再加上MALDI质谱的高通量能力，从而获得定性和定量信息。初步数据表明，即使是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，而无需除稀释外的任何进一步样品处理。该数据可以在不到一分钟的时间内获得，其形式可以很容易地集成到工具中，以自动识别脂质以及定量存在的脂质丰度。为了开展这些研究，将使用先进的质谱技术，包括离子迁移技术，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。待开发的先进质谱技术可以在许多实验室中实施，即使没有离子迁移率；然而，随着离子迁移率变得越来越广泛，它提供了目前其他质谱系统所不具备的独特能力。

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.