Development of an Integrated Intermediary Metabolomics and Metabolic Flux Core

项目总结

Project Summary This proposal seeks funding for a Liquid Chromatography (LC), Differential Mobility Spectroscopy (DMS), Quadrupole Time of Flight (LC-DMS-QToF) SCIEX 6600 mass spectrometer (AB Sciex LLC, Framingham, MA) to build an integrated system describing the metabolic traffic in cells, plasma and tissues. The essential endogenous biochemicals of life are confined to enzymatically delineated metabolic pathways shared across most species. The collective flow of nutrient substrates into synthetic or catabolic pathways that best describes key functions, transformations, differentiations, adaptations, or disease processes of a cell, tissue, or organism. The challenge then is interpreting the flow form concentration data, since the concentration of a metabolite may have little to do with its flow through a metabolic pathway. The lack of robust high-throughput analytical techniques to assess the flow of metabolic traffic through these pathways is a major limitation to improving our understanding of cellular and organismal physiology. Yale lacks a dedicated metabolomics core. The acquisition of the LC-DMS-QToF integrated system will provide a core with: 1) a dedicated intermediary metabolomics platform on the KEGG metabolic map (CoMBI) that through integrated omics can be reinforced by orthogonal transcriptomics, proteomics, phosphoproteomics or genomics data sets, 2) an expansion of Mass Isotopomeric MultiOrdinate Flux Analysis (MIMOSA) developed by my laboratory to quantitatively measure intracellular fluxes through sequential position-specific label transfer of stable isotopes, and 3) an in vivo inter-tissue metabolic flux analysis designed for genetic screens of metabolism. The combination of DMS with the high resolution and speed of ToF and mass fragmentation minimize isobaric interference which otherwise plague stable isotope studies. We have had the opportunity of having the requested instrument consigned to our laboratory at Yale through an academic/industry technology alliance with SCIEX as a way to generate preliminary proof-of-principle data and establish our pipelines. A bioinformatic alliance with Elucidata has developed a customized, open-sourced, cloud-based platform with an intuitive user interface to support our unique computational demands. The instrument operates in our existing Islet, Oxygen consumption, Mass Isotopomer flux Core analytical core (IOMIC) through chargebacks and with institutional support at cost under an established, university-approved Internal Service Provider (ISP) account. This unique and transformative platform will integrate the tens of 1000s of mass spectroscopic features, correct for natural abundance, and track positional movement of mass labels through cells to generate a high resolution “Google maps” of metabolism where the rate and direction of metabolic flow are quantified with demonstrated application to microbiology, plants, fruit flies, cell culture, cancer and human tissue. These capabilities are absolutely critical in order to sustain, and increase, the current pace of understanding metabolism in various tissues.