Background/Purpose: Juvenile idiopathic arthritis (JIA) is a chronic disease marked by persistent synovial inflammation, often leading to structural joint damage. While numerous proteases produced by synovial cells, chondrocytes, macrophages, and polymorphonuclear cells were implicated in joint damage in rheumatoid arthritis, few studies explored similar mechanisms in JIA. Targeting proteolysis represents a promising disease-modifying strategy and may yield novel biomarkers for JIA, but this approach requires a comprehensive understanding of proteolytic activity. The study objective was to utilize N-terminomics to define the proteolytic landscape of JIA joints.

Methods: Synovial fluid samples (n=5) were collected from JIA patients under an approved IRB protocol. Proteins were labeled at their N-termini using reductive dimethylation. Samples underwent trypsin digestion, followed by enrichment of N-terminally labeled or modified peptides using HPG-ALD polymer. Peptides were further separated by high-pH off-line fractionation and analyzed on a Bruker TimsTOF Pro mass spectrometer. High-confidence peptides were identified using a stringent false discovery rate and mapped to full-length protein sequences to determine cleavage sites. Proteases present in the samples were also identified.

Results: This analysis of JIA synovial fluid identified 13,268 proteolytically cleaved peptides from 854 proteins. Of these, 5,805 peptides (44%) originated from secreted proteins, reflecting the prevalence of circulating proteins in synovial fluid. Notably, 2,732 peptides (19.2%) were detected in all five samples. Comparison with the previously characterized osteoarthritis (OA) synovial fluid degradome showed 6,978 cleavage sites (32.9%) from 325 proteins to be unique in JIA synovial fluid. Ingenuity Pathway Analysis of peptides unique to JIA identified neutrophil degranulation as the most significant pathway. In total, 95 proteases were detected in JIA synovial fluid, including neutrophil-derived proteases and others distinct from OA synovial fluid.

Conclusion: Our findings demonstrate a vast proteolytic landscape of JIA synovial fluid, providing an unprecedented expansion of proteolytic events and identifying potentially causative proteases. Implication of neutrophil activity in protein and joint breakdown highlights the relatively neglected role of innate immunity in JIA and identifies numerous potential biomarkers of their impact. Continued degradome analysis of larger JIA cohorts and stratification by clinical variables will be crucial for elucidating affected pathways and advancing both diagnostic and therapeutic strategies for JIA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mass-spectrometry-based-n-terminomics-uncovers-proteolytic-signatures-and-pathways-in-juvenile-idiopathic-arthritis-synovial-fluid/