Development of an Integrated Intermediary Metabolomics and Metabolic Flux Core

项目摘要 该提案旨在为液相色谱（LC），差分迁移率光谱（DMS）， 四极杆飞行时间（LC-DMS-QToF）SCIEX 6600质谱仪（AB Sciex LLC，Fraudal，MA） 建立一个描述细胞、血浆和组织中代谢运输的综合系统。的基本 生命的内源性生化物质仅限于酶促描绘的代谢途径， 大多数物种营养基质集体流入合成或分解代谢途径， 细胞、组织或生物体的关键功能、转化、分化、适应或疾病过程。 然后，挑战是解释流形式浓度数据，因为代谢物的浓度可能 与其通过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研究的主要限制。 了解细胞和有机体生理学。 耶鲁大学缺乏专门的代谢组学核心。LC-DMS-QToF集成系统的收购将 提供核心：1）KEGG代谢图谱（CoMBI）上的专用中间代谢组学平台 通过整合组学可以通过正交转录组学，蛋白质组学，磷酸化蛋白质组学 或基因组学数据集，2）开发了质量同位素多纵标通量分析（MIMOSA）的扩展 我的实验室通过顺序位置特异性标记转移定量测量细胞内通量 和3）设计用于遗传筛选的体内组织间代谢通量分析， 新陈代谢. DMS与ToF和质量碎裂的高分辨率和速度的组合 最大限度地减少同量异位素干扰，否则困扰稳定同位素研究。我们有机会 通过学术/工业技术将所需的仪器委托给我们在耶鲁的实验室 我们与SCIEX结盟，以生成初步的原理证明数据并建立我们的管道。一 生物信息学联盟与Elucidata开发了一个定制的，开源的，基于云的平台， 直观的用户界面，以支持我们独特的计算需求。该仪器在我们现有的 胰岛、耗氧量、质量同位素通量核心分析核心（IOMIC）通过退款和 在一个既定的，大学批准的内部服务提供商（ISP）帐户下的成本机构支持。 这个独特的变革性平台将整合数十个1000个质谱特征，对吗？ 并跟踪质量标记在细胞中的位置移动，以生成高分辨率 新陈代谢的“谷歌地图”，其中代谢流的速率和方向被量化， 应用于微生物学、植物、果蝇、细胞培养、癌症和人体组织。这些能力 绝对至关重要，以维持，并增加，目前的速度了解代谢在各种 组织中

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.