Method Development And Application For Metabolomic Study Of Exosomes By High Resolution Mass Spectrometry

Exosomes are small vesicles enveloped by phospholipid bilayers that are abundant in various bodily fluids and contain significant biological information.They serve as active molecular carriers that enable intercellular communication and facilitate the exchange of materials between cells.Due to the heterogeneous nature of exosomes,the expression of their contents can vary between physiological and pathological conditions,making them a potential source of disease biomarkers.The metabolites contained within exosomes are highly effective in reflecting changes in biological phenotype and status as the end result of multiple substance reactions.Metabolomics is a crucial technique for studying metabolites,offering unique advantages over other omics research.The metabolome demonstrates greater timeliness and sensitivity to environmental perturbations,with changes in gene or protein expression being more prominently reflected within the metabolome.Therefore,the study of exosomal metabolomics is crucial for understanding the biogenesis of various diseases and for discriminating between different disease stages.However,a rapid,effective,and high-throughput method for exosomal metabolomics research is still lacking.In light of this context,this dissertation employs molecularly imprinted polymers(MIPs)for the enrichment of exosomes in bodily fluids,followed by their characterization using metabolomics techniques.We have developed a method for the metabolomic analysis of exosomes based on high-resolution mass spectrometry(HRMS)and conducted a practical application in the plasma exosomes of hepatocellular carcinoma.The following work was mainly carried out and achieved certain results:1.Method development,evaluation and optimization of exosome metabolomics based on high-resolution mass spectrometry.We employed MIPs to enrich exosomes from plasma and disrupted them via repeated freeze-thawing to release metabolites,which were subsequently extracted.The resulting metabolites were characterized using HRMS-based metabolomics techniques,and the obtained data were analyzed and evaluated from multiple dimensions before the method was further optimized.2.Non-targeted metabolomics of five body fluids and their corresponding exosomes were performed using the developed methods.Plasma,urine,saliva,amniotic fluid,and cerebrospinal fluid samples were analyzed,and statistical analysis from multiple perspectives revealed differences in the species and expression of metabolites between body fluids and their corresponding exosomes,indicating that they are not interchangeable.These findings highlight the high value and significance of metabolomic research on both body fluids and their exosomes.3.Metabolomic and lipidomic analyses were performed on plasma exosomes from patients with hepatocellular carcinoma(HCC)and healthy controls using the developed method.Eight HCC and eight healthy plasma samples were subjected to metabolomic analysis,while seven HCC and seven healthy samples were analyzed using lipidomics.Statistical analysis was employed to identify differential metabolites and lipids,and their corresponding metabolic pathways were explored.Ultimately,seven potential metabolic markers and seven lipid markers were identified,including sphingosine(d18:1),taurochenodeoxycholic acid(TCDCA),pipecolic acid(PA),indole piperine,4-hydroxynonenal(4-HNE),caffeine,TG(18:0/18:1/20:4),LPC(18:1/0:0),DG(22:1/0:0/18:2),TG(14:1/20:4/20:5),PC(15:0/20:5),PE(16:0/22:6),and SM(d18:0/22:3).