Enrichment and Proteomic Profiling of Stromal‐Associated Extracellular Vesicle Subpopulations From Arthritic Synovial Fluid by Size‐Exclusion Chromatography Coupled to CD90‐Directed Immunocapture

ABSTRACT Extracellular vesicles (EVs) are emerging as key mediators of disease‐associated intercellular communication and as promising biomarker sources across many disease conditions, including joint disorders such as rheumatoid arthritis (RA) and osteoarthritis (OA). Molecular profiling of synovial tissue has uncovered disease‐driving cellular states, but tissue biopsies are invasive and not routinely available. EVs in synovial fluid may offer a minimally invasive and complementary window into joint pathobiology. However, the molecular complexity of synovial fluid and the heterogeneity of EV populations have hindered the analysis of defined disease‐relevant EV subsets. To address this challenge, we developed a refined size‐exclusion chromatography‐ultrafiltration (SEC‐UF) workflow coupled to magnetic bead‐based immunocapture for targeted enrichment of cell type‐associated EV subpopulations from arthritic synovial fluid. As proof‐of‐concept, we targeted a stromal‐associated EV population using CD90/THY1, a surface marker expressed by discrete synovial fibroblasts subsets implicated in arthritis pathobiology. In vitro validation using synovial fibroblast‐derived EVs confirmed surface‐accessible CD90 and demonstrated selective immunocapture of defined EV subpopulations. Application to patient‐derived synovial fluid showed that SEC‐UF pre‐enrichment improves the robustness of CD90 + EV recovery from this complex biofluid. Liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) profiling established broad EV‐associated proteome coverage in synovial fluid (SF)‐EV preparations and identified multiple proteins reflecting the inflamed arthritic synovial environment. Fraction‐resolved proteomics enabled comparison of the CD90‐immunocaptured SF‐EV subset with the non‐captured CD90 − /CD90 low SF‐EV pool. Differential proteomics and surfaceome‐informed cell‐of‐origin analysis supported enrichment of stromal‐associated surface markers in the CD90 + fraction, whereas the non‐captured SF‐EV pool retained broader immune‐associated and residual stromal EV inputs. Together, this fit‐for‐purpose workflow provides a strategy to resolve EV heterogeneity patient synovial fluid and supports future biomarker‐oriented studies of defined subpopulations in arthritic diseases.