Table 1_Proteomic analysis of infiltrating neutrophils from rheumatoid arthritis synovial fluid and their contribution to protein carbamylation.xlsx

IntroductionCarbamylated proteins and dysregulated neutrophils are implicated in rheumatoid arthritis (RA) pathogenesis. Herein, we characterized the neutrophils present in RA synovial fluid (SF) using proteomic techniques and evaluated their contribution to protein carbamylation.
MethodsRA-SF neutrophil proteomic profile and SF proteome signature were investigated using high-resolution mass spectrometry. Carbamylated proteins and the degree of protein carbamylation were evaluated by mass spectrometric analysis. ELISA and chemiluminescence kits were used to examine myeloperoxidase (MPO) activity, and hydrogen peroxide (H2O2) generation.
ResultsSF neutrophils exhibited a shift in proteomic cargo with up-regulated proteins involved in defense responses, neutrophil degranulation, and reactive oxygen species metabolic processes, while proteins down-regulated were associated with megakaryocyte differentiation, leukocyte migration, and integrin-mediated signaling pathway. Elevated levels of neutrophil-derived proteins were detected in RA-SF. In addition, we specifically identified many carbamylated proteins and observed an increased frequency of protein carbamylation in RA-SF samples. Functionally, neutrophils from RA-SF showed a significantly increased level of MPO release and HH2O2 generation. Moreover, MPO activity was higher in RA-SF than in autologous blood samples, which correlated well with the degree of protein carbamylation in RA-SF.
DiscussionSynovial neutrophils were found to be activated and increased releasing protein cargo, including MPO and ROS, into the synovial fluid. Presence of many carbamylated proteins in RA-SF and an increased MPO activity showed a strong correlation to the degree of protein carbamylation, suggesting neutrophil-derived MPO in promoting generation of aberrantly carbamylated proteins.

**引言**：氨甲酰化蛋白质与功能失调的中性粒细胞均与类风湿关节炎（RA）的发病机制密切相关。本研究采用蛋白质组学技术，对类风湿关节炎滑液（RA-SF）中的中性粒细胞进行了系统表征，并评估了其在蛋白质氨甲酰化过程中的作用。

**方法**：本研究通过高分辨质谱法，分析了类风湿关节炎滑液中性粒细胞的蛋白质组谱及滑液蛋白质组特征。采用质谱分析对氨甲酰化蛋白质及其氨甲酰化程度进行评估。使用酶联免疫吸附试验（ELISA）与化学发光试剂盒，检测了髓过氧化物酶（MPO）活性及过氧化氢（H₂O₂）的生成水平。

**结果**：类风湿关节炎滑液中的中性粒细胞其蛋白质组内容物发生显著重塑：上调表达的蛋白质主要参与防御反应、中性粒细胞脱颗粒及活性氧代谢过程；而下调表达的蛋白质则与巨核细胞分化、白细胞迁移及整合素介导的信号通路密切相关。在类风湿关节炎滑液中可检测到高水平的中性粒细胞源性蛋白质。此外，本研究特异性鉴定出多种氨甲酰化蛋白质，并观察到类风湿关节炎滑液样本中蛋白质氨甲酰化的发生频率显著升高。功能实验显示，类风湿关节炎滑液来源的中性粒细胞其MPO释放及过氧化氢生成水平均显著升高。进一步研究发现，类风湿关节炎滑液中的MPO活性高于自体血液样本，且该活性与滑液中蛋白质的氨甲酰化程度呈显著正相关。

**讨论**：本研究发现，类风湿关节炎滑液中的中性粒细胞处于激活状态，其向滑液中释放包括髓过氧化物酶（MPO）与活性氧（ROS）在内的蛋白质内容物的水平显著升高。类风湿关节炎滑液中存在多种氨甲酰化蛋白质，且MPO活性升高与蛋白质氨甲酰化程度呈显著正相关，这提示中性粒细胞来源的MPO可促进异常氨甲酰化蛋白质的生成。