New analytical methods for global and targeted metabolomics

现有的在一次分析中综合研究数百或数千种代谢物的分析方法目前不发达，更适合于中到高丰度的稳定代谢物，并且缺乏充分的验证和质量控制程序，这反过来可能导致错误的生物学结论。拟议研究计划的长期目标是改进全球代谢组学和靶向代谢组学的分析方法，主要侧重于改进使用高分辨率质谱仪检测和识别生物液和组织中低丰度和不稳定的代谢物。为了实现这一目标，我们将利用液-质联用技术(LC-MS)对离子液体进行系统的研究，以确定在代谢组学中最有用的新提取材料。特别是，我们将调查我们最近令人兴奋的发现，即PF6离子脂适合于脱脂，并可能代表一类新的脂类澄清剂。我们还将进一步改进和微型化我们的顺序提取方案，将复杂的代谢组分为脂类、阴离子、两性离子、阳离子和中性组分。其次，由于目前还没有足够灵敏和准确的MS方法，目前许多激素肽的检测依赖于多克隆抗体的低质量免疫分析。我们将通过结合固相微萃取(SPME)、纳米LC和最先进的衍生化策略，为这些具有挑战性的分析物开发新型的高灵敏度分析方法，以获得适合于在生物体液中检测它们的检测限。最后，我们将通过设计能够保存这些重要代谢物类别的生物库设备，并通过识别新发现的不稳定代谢物，来扩大我们对不稳定代谢物的研究计划，如激素多肽、氧磷脂和硫醇。该项目开发的创新分析技术、设备和方法将在改善靶向和非靶向代谢组学方面发挥关键作用，而我们的超灵敏LC-MS平台将为激素多肽分析提供突破性技术。这一研究计划将回答一些重要问题，包括如何(I)定制新材料以提高对传统方法的代谢物覆盖率，(Ii)使我们新的顺序提取方案小型化，使其适用于即使在样本有限的情况下也能深入挖掘代谢组，以及(Iii)设计新的分析方法和设备，以保存和分析不稳定的代谢物，特别是与氧化应激和炎症有关的激素肽和代谢物。总而言之，这些方法将有助于代谢组学方法的合理设计和选择，以及发现以前未确定的代谢物，从而为分析代谢组学领域做出重要而迫切需要的贡献。

The existing analytical approaches for comprehensive study of hundreds or thousands of metabolites in a single analysis are currently underdeveloped, better-suited for medium to high-abundance stable metabolites and lack adequate validation and quality control procedures, which can in turn lead to erroneous biological conclusions. The long-term objective of the proposed research program is to improve analytical methods in both global and targeted metabolomics with the primary focus on improving the detection and identification of low abundance and unstable metabolites in biological fluids and tissues using high-resolution mass spectrometry. To achieve this objective, we will examine systematically ionic liquids to identify the most useful new extraction materials in metabolomics by liquid chromatography - mass spectrometry (LC-MS). In particular, we will investigate our recent exciting finding that PF6 ionic lipids are suitable for lipid removal and may represent a new class of lipid clarification agents. We will also further refine and miniaturize our sequential extraction protocols to subdivide a complex metabolome into lipid, anion, zwitterion, cation and neutral fractions. Secondly, many hormone peptides are currently detected by poor quality immunoassays relying on polyclonal antibodies because sufficiently sensitive and accurate MS methods do not yet exist. We will develop novel highly sensitive assays for these challenging analytes by combining solid-phase microextraction (SPME), nano LC and state-of-the-art derivatization strategies to achieve limits of detection suitable for their detection in biofluids. Finally, we will expand our research program for unstable metabolites, such as hormone peptides, oxylipins, and thiols, by designing biobanking devices that can preserve these important classes of metabolites and by identifying newly discovered unstable metabolites. The innovative analytical techniques, devices and methods developed in this program will play a critical role to improve both targeted and untargeted metabolomics, while our ultra-sensitive LC-MS platfrom will provide breakthrough technology for hormone peptide analysis. This research program will answer important questions including how (i) to customize emerging materials to improve metabolite coverage over classical approaches, (ii) to miniaturize our new sequential extraction protocols to render them suitable for deep mining of the metabolome even in sample-limited situations, and (iii) to design new analytical methods and devices for preservation and analysis of unstable metabolites, especially hormone peptides and metabolites involved in oxidative stress and inflammation. Collectively, these approaches will aid in rational design and selection of metabolomics methods and discovery of previously-uncharacterized metabolites, thus presenting an important and critically-needed contribution to the field of analytical metabolomics.