Study On Proteome And Glycoprotein Identification Of Exosomes From Human Urine

Exosomes are currently the most popular vesicles.Exosomes exist in a variety of body fluids,such as blood,urine,saliva,amniotic fluid,lotion and so on.Exosomes are relatively easy to obtain,can selectively enrich their marker protein and RNA,making the exocrine become a very attractive non-invasive disease diagnosis method.At present,the study of exosomes has found a variety of diseases biomarkers,the exosomes is expected to bring new hope to early guidance of the disease and individualized treatment.In the past,the study of urine biomarkers focused on soluble proteins in urine,but the discovery and isolation of urinary exosomes has pushed the research of urine biomarkers into a whole new stage.But so far,there are no uniform isolation methods of urinary exosomes.In this paper,the method of isolating the exosomes from the urine based on the ultrafiltration is compared with the traditional methods of isolating the exosomes from the urine,which fully reflects the superiority based on the ultrafiltration to isolate urinary exosomes.Based on this isolation method,a stable and highly efficient human urine exosomes protein glycosylation was established.and the differential site information of N-glycosylation sites of urinary exosomes in healthy men and women was analyzed.The contents of this paper are as follows:In the first chapter,exosomes,the isolation technology of exosomes,proteomics and N-glycosylated modified proteins were reviewed.In the second chapter,the method of isolating the exosomes from the urine based on the 100 kDa ultrafiltration tube and the method of isolating the urinary exosomes,including the kit isolation method,the PEG6000 precipitation isolation method and the PEG10000 precipitation isolation method were evaluated.The expression of urinary exosomes marker proteins(TSG101,AQP2)were detected by the Western blot;The size and shape of urinary exosomes were analyzed by transmission electron microscopy;The range of particle size distribution of urinary exosomes was measured and calculated by nanoparticle tracking analysis;The expression profile of urinary exosomes protein was analyzed by mass spectrometry;The cellular compoent of urinary exosomes was analyzed by GO analysis.The research indicates that the isolation of urinary exosomes based on the ultrafiltration is a simple,economically effective method for the treatment of urinary exosomes in large-scale urine samples.Based on this method,1534 proteins were identified by mass spectrometry,and 97%of the exosomes in the ExoCarta database of 100 kinds of exosomes marker protein were identified.The third chapter is based on the ultrafiltration tube iaolation of urinary exosomes,the use of HILIC for urinary exosomes N-glycosylated peptide enrichment,compared to healthy men and women of the exosomes of the urinary exosomes N-glycosylation sites of the differential site information.A stable and highly efficient human urine exosomes protein glycosylation was established.The research strategy is:Based on the new method to isolation of healthy human urine exosomes from three men and three women,FASP digestion was performed on the isolated urinary exosomes.And then use HILIC for enrichment the urinary exosomes N-glycosylated peptides.Under this strategy,982 glycosylated proteins were identified from healthy men and 1189 glycosylated proteins were identified from healthy women.The data of N-glycosylated protein of urinary exosomes were analyzed by the volcanic map analysis and the heat map analysis,and the difference information of urinary exocrine N-glycosylation sites from healthy men and women was obtained.