Phosphoproteomic Analysis Of Peripheral Blood Mononuclear Cell In Ankylosing Spondylitis

Objective Ankylosing spondylitis(AS)is a common rheumatic autoimmune disease that mainly invades the central axis bones,with sacroiliac joints being the most severe,accompanied by a series of extra-articular manifestations such as uvitis,ulcerative colitis,aortic disease,and osteoporosis.The pathogenesis of AS is complex and has not been completely clarified.In recent years,many scholars have conducted in-depth studies on the pathogenesis of AS from the genetic level,molecular level,cellular level,cell-cell interactions,and microorganisms,and have made a series of progress.Protein phosphorylation modification is a very important post-translational modification of proteins,which has an important impact on the structure and function of proteins,and is a key pathway for intracellular signal transduction.In order to better understand the pathogenesis of AS,we extracted proteins from peripheral blood mononuclear cells(PBMCs)of patients with AS and and performed phosphorylation enrichment analysis.In order to study the role of differentially phosphorylated proteins in the pathogenesis of AS,and to elaborate the underlying pathogenesis of AS.Methods Peripheral blood of patients with untreated AS(20 cases),patients with AS(15 cases)and healthy people(20 cases)were collected,and mononuclear cells in peripheral blood were extracted by density gradient centrifugation.The protein in the mononuclear cells is then extracted and digested with trypsin.The peptide fragments after enzymatic hydrolysis were enriched with Ti O2 metal affinity column,followed by liquid chromatography tandem mass spectrometry.Use Max Quant(v1.6.3.4)software to analyze and quantify the mass spectrometry data,and use bioinformatics analysis methods to explore potential signal pathways related to the pathogenesis of AS.Then we screened out the phosphorylated proteins related to the disease of as,and analyzed the expression level of these proteins after treatment.Results Through quantitative phosphorylation proteomics,a total of 1561 significantly differential phosphorylated peptides were detected,of which 756 peptides from 472 proteins were up-regulated,and 805 from 363 proteins were down-regulated.Gene annotation enrichment analysis showed that the proteins with altered phosphorylation are mainly localized in the cell junctions,cytoskeleton,outer cell membrane,and pseudopods,which mainly affect the chemotactic,deformation and adhesion functions of mononuclear cells;the main biological processes involved are GTPase and small GTPase-mediated signal transduction,RNA splicing,neutrophil activation,actin activity,and platelet aggregation,High-coagulation state,etc.In addition,KEGG pathway enrichment analysis showed that that T cell receptor signaling pathway,B cell receptor signaling pathway,endocytosis signaling pathway,phagocytosis signaling pathway,Platelet activation and activation signaling pathways may be closely related to the disease.Conclusion We use quantitative phosphorylation proteomics to study and systematically characterize the changes in protein phosphorylation in peripheral blood mononuclear cells from patients with AS.The experimental results show that the pathogenesis of AS is closely related to the role of the immune system,and is also related to the platelet signaling pathway.These findings have important implications for understanding the pathogenesis of AS and disease progression.