Proteomic Analysis of Infiltrating Neutrophils from Rheumatoid Arthritis Synovial Fluid and Their Contribution to Protein Carbamylation

123 Cxt ¹ Rong Du ² Pan Wang ¹ Wenlin Qiu ² Lu Chen ¹ Jian Wan ¹ Hiu Qiu ³ Li Xiong ¹ Kutty Selva Nandakumar ⁴ Rikard Holmdahl ⁵ Hui Geng ^1*

• ¹ School of Life Science, Central China Normal University, Wuhan, China
• ² Huazhong University of Science and Technology, Wuhan, Hubei Province, China
• ³ Wuhan University, Wuhan, Hubei Province, China
• ⁴ Halmstad University, Halmstad, Halland, Sweden
• ⁵ Karolinska Institutet (KI), Solna, Stockholm, Sweden

Carbamylated proteins and dysregulated neutrophils are implicated in rheumatoid arthritis (RA) pathogenesis. Herein, we characterize neutrophils in RA synovial fluid (SF) using proteomics and evaluated their contribution to protein carbamylation. Mass spectrometric analysis revealed that SF neutrophils exhibited a shift in proteomic cargo with up-regulated proteins involved in defense responses, neutrophil degranulation, and reactive oxygen species induced 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. We specifically identified many carbamylated proteins and observed an increased frequency of protein carbamylation in RA-SF samples. Functionally, neutrophils from RA-SF showed an increased level of MPO release and H2O2 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, suggesting neutrophil-derived MPO in promoting generation of aberrantly carbamylated proteins.