Development of an Integrated Intermediary Metabolomics and Metabolic Flux Core

项目摘要 这项提案寻求为液相色谱(LC)、差分迁移率光谱(DMS)、 飞行时间四极杆(LC-DMS-QToF)SCIEX 6600质谱仪(AB Science ex LLC，马萨诸塞州弗雷明翰) 建立一个描述细胞、血浆和组织中代谢运输的综合系统。最重要的 生命的内源性生物化学物质被限制在酶描述的代谢途径上，这些代谢途径 大多数物种。营养底物进入合成或分解代谢途径的集体流动最好地描述 细胞、组织或生物体的关键功能、转变、分化、适应或疾病过程。 然后，挑战是解释流动形式的浓度数据，因为代谢物的浓度可能 与其通过新陈代谢途径的流动几乎没有关系。缺乏强大的高通量分析 评估通过这些途径的代谢流量的技术是改善我们的 了解细胞和生物体生理学。 耶鲁大学缺乏专门的代谢组学核心。收购LC-DMS-QToF集成系统将 提供核心：1)KEGG代谢图(COMII)上的专用中间代谢组学平台 通过集成组学可以通过正交转录组学、蛋白质组学、磷蛋白质组学来加强 或基因组数据集，2)发展了质量等聚体多重通量分析(MIMOSA)的扩展 由我的实验室通过顺序特定位置标记转移来定量测量细胞内的通量 3)体内组织间代谢通量分析，设计用于遗传筛查 新陈代谢。DMS与ToF的高分辨率、高速度和质量粉碎的结合 尽量减少同量异压干扰，否则会困扰稳定同位素研究。我们有机会 通过学术/行业技术将所需的仪器寄送到我们在耶鲁的实验室 与SCIEX结盟，作为生成初步原则证明数据和建立我们的管道的一种方式。一个 与Elucidata的生物信息联盟开发了一个定制的、开源的、基于云的平台， 直观的用户界面，支持我们独特的计算需求。该仪器在我们现有的 胰岛、氧气消耗、质量同种异构体熔剂堆芯分析核心(IOMIC) 在大学批准的已建立的内部服务提供商(ISP)帐户下按成本提供机构支持。 这个独特的、变革性的平台将整合数万个质谱学特征，对吗？ 用于自然丰度，并跟踪质量标签在单元格中的位置移动以生成高分辨率 新陈代谢的“谷歌地图”，其中新陈代谢流动的速度和方向是用演示的 应用于微生物学、植物、果蝇、细胞培养、癌症和人体组织。这些功能包括 对于维持和提高目前对各种新陈代谢的理解速度来说是绝对关键的 纸巾。

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.