Proteomics Analysis Of Serum-Derived Exosome In Patients With Ankylosing Spondylitis

Background:Ankylosing spondylitis(AS)is a chronic inflammatory disease characterized by specific invasion of the spine and sacroiliac joint.At present,the diagnosis of AS depends on clinical manifestations and imaging examinations,and there is still a lack of ideal blood diagnostic markers,so that the diagnosis of a large number of patients is delayed.Therefore,it is important to look for AS early diagnostic markers.Exsomes are a type of extracellular vesicles released by cells that can be found in body fluids such as blood,urine and saliva.Exosome can carry miRNA,proteins and other components,participate in immune response,inflammatory response,blood coagulation,angiogenesis and other functions,which can be applied to the diagnosis and treatment of diseases.Studies have shown that aquaporin-2 from urine-derived exosomes is significantly associated with autosomal dominant and autosomal recessive diabetes insipidus and can assist in the diagnosis of this disease.In the field of rheumatology,studies have found that specific proteins in synovial fluid and serum-derived exosomes of rheumatoid arthritis patients can be used to identify osteoarthritis or reactive arthritis.However,as for the proteome analysis of serum-derived exosomes in AS patients,there is no relevant research yet.Objective:Proteomic methods were used to analyze different protein in serum-derived exosomes from AS patients and normal subjects,and to further explore the biological significance of differential proteins.Methods:ExoQuick kits were used to extract exosomes from AS patients and normal human serum,and identified by transmission electron microscopy,Nanosight,and westen-blot.The protein in the exosomes was subjected to two-dimensional gel electrophoresis(2D)to analyze the gel map.Qualitative analysis of differential protein gel points by combined Matrix-Assisted Laser Desorption/Ionization Tandem Time of Flight Mass Spectrometry(MALDI-TOF-TOF-MS).At the same time,label free liquid chromatography mass spectrometry(LC-MS/MS)was used to quantitatively analyze the protein composition in exosomes.Volcano Plot.Protein-protein Interaction Network(PPI)analysis,GO analysis and KEGG signaling pathway analysis of differential proteins.The expression of differential proteins was further confirmed by enzyme-linked immunosorbent assay(ELISA).Results:In terms of exosome separation and identification,Nanosight showed that the exosomes were mainly 90-220 nm in diameter.The transmission electron microscopy showed that the exosomes were round-like vesicles,and the western-blot showed that the exosomes were positive for TSG101 and CD81.In the qualitative analysis of exosome different protein by 2D-MALDI-TOF-TOF-MS,the results of 2D gel electrophoresis showed that there were 184 protein spots in the AS group and 109 protein spots in the control group,with 90 matching protein spots.In the AS group,26 protein spots were highly expressed,and in the control group,1 protein protein was highly expressed(gray value)2 times).MALDI-TOF-TOF-MS analysis of the most different gel spots showed that Calcium-Binding Protein 39 was the most likely differential protein.In the label-free LC-MS/MS quantitative analysis of exosome different protein,the protein profiles of the two groups were significantly different.The Wayne diagram analysis showed that 65 proteins were only expressed in AS patients,and 67 proteins were only expressed in normal people.Among them,545 proteins were two groups of shared proteins;Volcano Plot showed that 31 proteins were up-regulated and 42 proteins were down-regulated in the AS group,up-regulated proteins including serum amyloid A-1(SAA1)and complement C4-B and other proteins,down-regulated proteins include tubulin ?-chain and periostin;PPI analysis showed that different protein can form a highly clustered protein network,with prothrombin and C-reactive protein as the two core proteins;GO analysis showed that the biological processes of differentially expressed proteins are mainly concentrated in the"protein activation cascade","complement activation"and"endocytosis",the molecular function of different prote:in is mainly concentrated in the"enzymatic activity of serine endopeptides"And the"serine-type peptidase activity",the cellular composition of different protein is mainly exosome,vesicle and microparticle-associated proteins;KEGG pathway analysis showed that different protein are mainly significant enrichment in the"Complement and coagulation cascade","ECM-receptor interaction","Staphylococcus aureus infection","Systemic lupus erythematosus"and"PI3K-Akt signaling pathway".ELISA results showed that the differential protein SAA1 was highly expressed:in exosomes of AS patients,consistent with the results of label-free LC-MS/MS analysis.Conclusion:The protein profiles of AS patients and normal human serum-derived exosomes are significant different.Bioinformatics analysis suggests that different protein are mainly involved:in the regulation of inflaimmatory response,and the differential protein SAA1 is highly expressed in exosomes of AS patients,in order to further explore the pathogenesis of AS.And the mining of AS diagnostic markers provides important clues.