AbstractObjectivesExtracellular vesicles (EVs) from rheumatoid arthritis (RA) synovial fluid (SF) have been reported to stimulate the release of pro‐inflammatory mediators from recipient cells. We recently developed a size exclusion chromatography (SEC)‐based method for EV isolation capable of high‐quality enrichments from human SF. Here, we employed this method to accurately characterise the SF EV proteome and investigate potential contributions to inflammatory pathways in RA.MethodsUsing our SEC‐based approach, SF EVs were purified from the joints of RA patients classified as having high‐level (n = 7) or low‐level inflammation (n = 5), and from osteoarthritis (OA) patients (n = 5). Protein profiles were characterised by mass spectrometry. Potential contributions of EV proteins to pathological pathways and differences in protein expression between disease groups were investigated.ResultsSynovial fluid EVs were present at higher concentrations in RA joints with high‐level inflammation (P‐value = 0.004) but were smaller in diameter (P‐value = 0.03) than in low‐level inflammation. In total, 1058 SF EV proteins were identified by mass spectrometry analysis. Neutrophil and fibroblast markers were overrepresented in all disease groups. Numerous proteins with potential to modulate inflammatory and immunological processes were detected, including nine citrullinated peptides. Forty‐five and 135 EV‐associated proteins were significantly elevated in RA joints with high‐level inflammation than in RA joints with low‐level inflammation and OA joints, respectively. Gene ontology analysis revealed significant enrichment for proteins associated with ‘neutrophil degranulation’ within SF EVs from RA joints with high‐level inflammation.ConclusionOur results provide new information about SF EVs and insight into how EVs might contribute to the perpetuation of RA.