Table_1_TMT-Based Quantitative Proteomics Analysis of Synovial Fluid-Derived Exosomes in Inflammatory Arthritis.docx

ObjectivesTo compare the proteomics of synovial fluid (SF)-derived exosomes in rheumatoid arthritis (RA), axial spondyloarthritis (axSpA), gout, and osteoarthritis (OA) patients.MethodsExosomes were separated from SF by the Exoquick kit combined ultracentrifugation method. Tandem mass tags (TMT)-labeled liquid chromatography mass spectrometry (LC-MS/MS) technology was used to analyze the proteomics of SF-derived exosomes. Volcano plot, hierarchical cluster, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted.ResultsA total of 1,678 credible proteins were detected. Sixty-nine differentially expressed proteins were found in gout, compared with OA, axSpA, and RA simultaneously. Twenty-five proteins were found highly expressed in gout uniquely, lysozyme C and protein S100-A9 included, whose bioinformatic analysis was significantly involved in “neutrophil degranulation” and “prion diseases”. Eighty-four differentially expressed proteins were found in axSpA, compared with OA, gout, and RA simultaneously. Thirty-nine proteins were found highly expressed in axSpA uniquely, RNA-binding protein 8A and protein transport protein Sec24C included, whose bioinformatic analysis was significantly involved in “acute-phase response” and “citrate cycle”. One hundred and eighty-four differentially expressed proteins were found in RA, compared with OA, gout, and axSpA simultaneously. Twenty-eight proteins were found highly expressed in RA uniquely, pregnancy zone protein (PZP) and stromelysin-1 included, whose bioinformatic analysis was significantly involved in “serine-type endopeptidase inhibitor activity” and “complement and coagulation cascades”. Enzyme-linked immunosorbent assay (ELISA) result showed that the exosome-derived PZP level of SF in RA was higher than that in OA (p < 0.05).ConclusionOur study for the first time described the protein profiles of SF-derived exosomes in RA, axSpA, gout, and OA patients. Some potential biomarkers and hypothetical molecular mechanisms were proposed, which may provide helpful diagnostic and therapeutic insights for inflammatory arthritis (IA).

本研究旨在比较类风湿性关节炎（RA）、轴向强直性脊柱炎（axSpA）、痛风和骨关节炎（OA）患者关节滑液（SF）来源的细胞外囊泡的蛋白质组学。研究方法包括通过Exoquick试剂盒联合超速离心法从SF中分离细胞外囊泡，并利用串联质谱标签（TMT）标记的液相色谱-质谱联用（LC-MS/MS）技术分析SF来源细胞外囊泡的蛋白质组学。研究采用了火山图、层次聚类、基因本体（GO）和京都基因与基因组百科全书（KEGG）通路分析。研究结果显示，共检测到1,678种可信蛋白。与OA、axSpA和RA相比，痛风中发现69种差异表达蛋白。其中，25种蛋白在痛风中特异性高表达，包括溶菌酶C和S100-A9蛋白，其生物信息学分析显著涉及“中性粒细胞脱颗粒”和“朊病”。与OA、痛风和RA相比，axSpA中发现84种差异表达蛋白。其中，39种蛋白在axSpA中特异性高表达，包括RNA结合蛋白8A和蛋白转运蛋白Sec24C，其生物信息学分析显著涉及“急性期反应”和“柠檬酸循环”。与OA、痛风和axSpA相比，RA中发现184种差异表达蛋白。其中，28种蛋白在RA中特异性高表达，包括妊娠区带蛋白（PZP）和基质金属蛋白酶-1，其生物信息学分析显著涉及“丝氨酸型内肽酶抑制活性”和“补体和凝血级联反应”。酶联免疫吸附测定（ELISA）结果显示，RA患者SF中由细胞外囊泡衍生的PZP水平高于OA患者（p < 0.05）。结论：本研究首次描述了RA、axSpA、痛风和OA患者SF来源细胞外囊泡的蛋白质谱。提出了一些潜在的生物标志物和假设的分子机制，这些可能为炎症性关节炎（IA）的诊断和治疗提供有益的见解。