Microfluidic mass spectrometry based fast chemical characterization of exosomes

 描述(由申请人提供)：这项研究的长期目标是建立可靠和成本有效的方案，基于对疾病诊断和治疗评估的外体内代谢物进行分析。Exosome是由正常和病理条件下的细胞分泌的直径为30~100 nm的纳米囊泡。外切体的异常分泌和/或化学成分与病理条件有关。因此，生理体液中外切体的分子分析对于非侵入性疾病的诊断和疗效评估具有潜在的价值。虽然对外切体的基因组、蛋白质组和脂体学研究已经产生了越来越多的关于外切体蛋白质、DNA/RNA和脂组成的信息，但对于外切体中存在的小分子代谢物还存在着知识差距。中心假设是，外体内代谢物的分布与来源细胞的健康状况相关。这项建议的具体目标是开发微芯片电泳质谱(MCE-MS)方法来定量外体内代谢物。提出这项研究的理由是，MCE-MS分析方法将胞外样本处理(浓缩、注射、外切体裂解等)、电泳分离和质谱学检测整合到一个平台中，是分析外切体内代谢物的首选方法。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切体；2)在磁活性MCE-MS平台上分析免疫纯化获得的磁珠结合外切体，显著提高了分析灵敏度。将选择一个包括细胞能量分子(即ATP、ADP和AMP)、有机酸(即谷氨酸、半胱氨酸和乳酸)以及巯基化合物(即谷胱甘肽和半胱氨酸)的代谢物小组进行研究。所有这些小分子都参与了细胞新陈代谢。它们的水平是细胞内各种酶过程的直接结果。因此，总的来说，它可能会起到很好的作用 作为细胞健康状况的有效指标。从各种细胞系中提纯的外切体将被分析，以确定外切体内代谢物的特征，并识别原始细胞的潜在小分子特征。为了获得分析不同膜标记的外体亚群的能力，将开发一个磁活性MCE-MS分析平台。用生物素标记的抗体和链霉亲和素包裹的磁珠进行免疫纯化，以产生包含各自膜蛋白的外体亚群。然后，磁珠结合的外切体将被注入磁性激活的MCE-MS分析平台，进行裂解和分析。从间充质干细胞(MSCs)和去血清MSCs(SD-MSCs)分离的外切体的代谢物图谱将在该项目中增加一个翻译成分，通过比较MSCs和应激去血清MSCs来探索外切体内代谢物图谱与细胞健康状态之间的相关性。

 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.