New analytical methods for global and targeted metabolomics

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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.