Microfluidic mass spectrometry based fast chemical characterization of exosomes

基于微流控质谱的外泌体快速化学表征

• 批准号: 9231456
• 负责人: YIMING LIU
• 金额: $ 18.88万
• 依托单位: JACKSON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9231456
• 关键词: Annexin A1; Antibodies; Antigen-Antibody Complex; Biological Assay; Bone Marrow; Breast Cancer Cell; CD81 gene; Caliber; Cell Culture Techniques; Cell Line; Cells; Cellular Stress; Centrifugation; Chemicals; Cysteine; Cystine; DNA; Data; Detection; Development; Extracellular Space; Genomics; Glutamates; Glutamic Acid; Glutathione; Goals; Health Status; Incubated; Injectable; Injection of therapeutic agent; Knowledge; Lipids; Liquid substance; MCF7 cell; MDA MB 231; Magnetism; Malignant neoplasm of prostate; Mass Spectrum Analysis; Membrane; Membrane Proteins; Mesenchymal Stem Cells; Metabolism; Methods; Microchip Electrophoresis; Microfluidic Microchips; Microfluidics; Modeling; Molecular Analysis; Nature; PC3 cell line; Pathologic; Physiological; Principal Investigator; Process; Proteins; Proteomics; Protocols documentation; RNA; Research; Sampling; Sensitivity and Specificity; Serum; Streptavidin; Stress; System; Testing; Time; Validation; analytical method; base; cost effective; design; diagnosis evaluation; disease diagnosis; exosome; experimental study; improved; indexing; magnetic beads; nanovesicle; novel; organic acid; programs; public health relevance; signature molecule; small molecule; therapeutic evaluation

 DESCRIPTION (provided by applicant): The long-term goal of the research is to establish reliable and cost effective protocols based on profiling intra-exosomal metabolites for disease diagnosis and therapeutic evaluation. Exosomes are nanovesicles (30~100 nm in diameter) secreted from cells both in normal and in pathological conditions. Aberrant secretion and/or chemical composition of exosomes are associated with pathological conditions. Therefore, molecular analysis of exosomes in physiological fluids is potentially an invaluable approach for non-invasive disease diagnosis and therapeutic evaluation. While genomic, proteomic, and lipidomic studies on exosomes have generated mounting information on exosomal protein, DNA /RNA, and lipid compositions, there is a knowledge gap about the small molecule metabolites present in exosomes. The central hypothesis is that the profile of intra-exosomal metabolites is correlated with the health status of the cells of origin. The specific objective of this proposal i to develop microchip electrophoresis- mass spectrometric (MCE-MS) methods to quantify intra-exosomal metabolites. The rationale for the proposed research is that MCE-MS analytical methods that integrate exosomal sample manipulation (enrichment, injection, exosome lysing, etc.), electrophoretic separation, and mass spectrometric detection into a single platform are the methods of choice for profiling intra-exosomal metabolites. Two novel analytical strategies are proposed: 1) analysis of intact exosomes on a MCE-MS analytical platform, and 2) analysis of magnetic bead-bound exosomes obtained from immuno-purification on a magnetically active MCE-MS platform with significantly improved assay sensitivity. A metabolite panel that includes cellular energy molecules (i.e. ATP, ADP, and AMP), organic acids (i.e. glutamate, cysteine, and lactate), and sulphydryl compounds (i.e. glutathione and cystine) will be selected for the study. All these small molecules are involved in cellular metabolism. Their levels are direct results of various enzymatic processes within the cells. Therefore, collectively, it may serve well as an effective index of cell health status. Exosomes purified from various cell lines will be analyzed to determine the intra-exosomal metabolite profiles and to identify potential small molecule signatures for the originating cells. To gain the capability of analyzing exosome sub-populations with different membrane markers, a magnetically active MCE-MS analytical platform will be developed. Immuno-purification with biotinylated anti-bodies and streptavidin-coated magnetic beads will be performed to produce exosome sub-populations containing the respective membrane proteins. Magnetic bead-bound exosomes will then be injected into the magnetically active MCE-MS analytical platform, lysed, and analyzed. Metabolite profiling of exosomes isolated from mesenchymal stem cells (MSCs) and serum deprived MSCs (SD-MSCs) will add a translational component to the project to explore the correlation between the profile of intra-exosomal metabolites and the health status of cells by comparing MSCs and stressed serum deprived MSCs.

 描述（由申请人提供）：本研究的长期目标是建立基于分析外泌体内代谢物的可靠且具有成本效益的方案，用于疾病诊断和治疗评估。外泌体（Exosomes）是一种在正常和病理状态下由细胞分泌的纳米囊泡（直径30~100 nm）。外泌体的异常分泌和/或化学组成与病理状况相关。因此，对生理液体中的外泌体进行分子分析对于非侵入性疾病诊断和治疗评估而言是潜在的无价方法。虽然对外来体的基因组学、蛋白质组学和脂质组学研究已经产生了关于外来体蛋白质、DNA /RNA和脂质组成的大量信息，但关于外来体中存在的小分子代谢物存在知识缺口。中心假设是外泌体内代谢物的谱与来源细胞的健康状态相关。本提案的具体目标是开发微芯片电泳-质谱（MCE-MS）方法来定量外泌体内代谢物。所提出的研究的基本原理是，MCE-MS分析方法整合了外泌体样品操作（富集、注射、外泌体裂解等），电泳分离和质谱检测到一个平台是用于分析外来体内代谢物的方法。提出了两种新的分析策略：1）在MCE-MS分析平台上分析完整的外泌体，以及2）在具有显著提高的测定灵敏度的磁活性MCE-MS平台上分析从免疫纯化获得的磁珠结合的外泌体。本研究将选择包括细胞能量分子（即ATP、ADP和AMP）、有机酸（即谷氨酸、半胱氨酸和乳酸）和巯基化合物（即谷胱甘肽和胱氨酸）的代谢物组。所有这些小分子都参与细胞代谢。它们的水平是细胞内各种酶促过程的直接结果。因此，总的来说， 作为细胞健康状况的有效指标。将分析从各种细胞系纯化的外泌体以确定外泌体内代谢物谱并鉴定原始细胞的潜在小分子特征。为了获得分析具有不同膜标志物的外泌体亚群的能力，将开发磁活性MCE-MS分析平台。将使用生物素化抗体和链霉亲和素包被的磁珠进行免疫纯化，以产生含有相应膜蛋白的外泌体亚群。然后将磁珠结合的外泌体注射到磁活性MCE-MS分析平台中，裂解并分析。从间充质干细胞（MSC）和血清剥夺MSC（SD-MSC）分离的外泌体的代谢物谱将为该项目增加翻译组件，以通过比较MSC和应激血清剥夺MSC来探索外泌体内代谢物谱与细胞健康状态之间的相关性。