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.
Molecular analysis of circulating exosomes in physiological fluids is potentially an invaluable approach for non-invasive disease diagnosis and therapeutic evaluation. However, little study has been done on small molecule metabolites present in exosomes due to the lack of effective analytical methods. We propose to develop microchip electrophoresis-mass spectrometric methods for metabolite assay of exosomes. The methods will provide a reliable and cost-effective means for profiling intra-exosomal metabolites, which will open up a new avenue for studies on circulating exosome-based non-invasive disease diagnosis and therapeutic evaluation.
Showing the most recent 10 out of 37 publications
