Study On Finding Serum Exosome Protein Markers Of Asthma Based On Mass Spectrometry

Background:Asthma is a heterogeneous disease characterized by chronic airway inflammation,of which the pathogenesis involves a variety of cells and cell components.Recent researches have found that exosome,which is a kind of extracellular vesicle,plays a vital role in the pathogenesis of asthma.Exosome,range from 30-150 nm in diameter,is a kind of membranous nano-sized vesicle.It is secreted from different cell types into the extracellular environment after the fusion of multivesicular bodies and cell membrane.Exosomes and their internal "cargos" proteins,miRNA,lipids and other biomolecular participate in a variety of physiological,pathological and other biological processes.It has been confirmed that exosomes exist in BALF of asthmatics,and their related proteins,lipids and miRNA are involved in the pathophysiological processes of asthma,such as inflammation and airway remodeling.After being stimulated by allergen,circulating dendritic cells,CD4+T cells,eosinophils etc.are involved in the airway inflammation and immune response of the body.And some studies showed that dendritic cells in peripheral blood from asthmatic patients could promote Th2 differentiation by releasing exosomes.Considering that exosomes can be released to extracellular regions and involved in intercellular communication.And proteins act as an executor of physiological functions,so,we speculate that there are different proteins derived from exosomes in the serum of asthmatic patients and healthy people.What's more,the serous exosomes between severe and non-severe asthmatic patients may have different proteins expressions.So far,no study on peripheral blood exosomes proteomics in asthmatics has been reported.Objective:We aim to explore the differences in serous exosomes proteins expression between patient with asthma and healthy people,patients with severe asthma and non-severe asthma,then,to find out the differentially expressed proteins and to explore the biological functions in each comparison group by bioanalysis.Moreover,we expect to find some novel proteins,which can be deemed as a new ideal biomarker for asthma diagnosis,disease monitoring and treatment.Methods:First of all,we collected venous blood(5ml per sample)from healthy control subjects(n=10)and patients of asthma,including patients of severe asthma(n=5)and non-severe asthma(n=5).Then we separated serum by centrifugation.Exosomes were isolated from serums by secant volume exclusion chromatography.The exosomes were identified by transmission electron microscope and nanoparticle tracking analysis(NTA).Then we analyzed the proteome of each exosome sample by 4D LFQ technology of liquid chromatography-mass spectrometry(LC-MS/MS).The data were retrieved and processed by Maxquant software(v1.6.6.0)to screen the differentially expressed proteins with statistical significance.Finally,we predicted the function and the critical paths of differentially expressed proteins according to GO classification and KEGG pathway analysis.Results:1.Serum exosomes in the asthma group and the healthy control group showed that round or unrepresentative round structure with double-layer membrane under the electron microscope.The NTA test result of selected samples suggested that the exosomes in the asthma group have a diameter of about 107 nm and the concentration is 9.7 ×109/L.In the healthy control group,its serum exosomes' diameter is about 153.5 nm and the concentration is 8.9×109/L.2.Using label-free quantitative proteomics technology,we identified 700 proteins in serum exosomes,of which 538 quantifiable proteins.3.When defining that the differential expression was>1.5 as the threshold of up-regulation and<1/1.5 as the threshold of down-regulation.,compared with the healthy control group,44 proteins were up-regulated and 39 proteins were down-regulated in exosomes of asthma group.Besides,5 proteins were up-regulated and 10 proteins were down-regulated in serous exosomes of severe asthma group when compared with non-severe asthma groups.4.In GO enrichment analysis,the differential proteins of serum exosomes between asthma group and healthy control group were related to the stimulation of immune response,cell migration,regulation of inflammatory response and formation of membrane attack complex,in contrast,the differential proteins between severe asthma group and non-severe asthma group were mainly involved in the regulation of epithelial cell formation and differentiation,as well as cell response to lipids.5.KEGG pathway enrichment analysis showed that the differential proteins between asthma group and healthy control group were mainly involved in "asthma","PI3K-Akt signal pathway","NF-?B signal pathway","Fc-? RI signal pathway" and"asthma related mechanism' What's noteworthy is that the up-regulated proteins IGHV3-7 and IGHV6-1 were involved in the regulation all of pathways above.And the differential proteins between severe asthma and non-severe asthma,such as CES1,were related to the drug metabolic pathway.Conclusion:There were differences in the expression of serum exosome protein between patients with asthma and healthy controllers.Bioinformatics analysis suggested that the differential proteins of this compare group mainly involved in inflammatory immune response.Proteins such as IGHV3-7 and IGHV6-1 in serum exosomes of asthma group were up-regulated and participated in the regulation of "asthma","PI3K-AKT signal pathway","NF-?B signal pathway" and "Fc-? RI signal pathway".There are differences in serum exosome protein expression between severe asthma group and non-severe asthma group.Bioinformatics analysis showed upregulated protein CES1 of serum exosomes in severe asthma,compared with non-severe asthma was involved in regulating the pathway of drug metabolism.