Beyond pre-analytical and analytical concerns in the study of synovial fluid proteome: description of an optimized Gel-based protocol

Purpose: This study aims to define, for the first time, a standard sample processing protocol for synovial fluid proteome analysis for a gel based-approach by two-dimensional gel electrophoresis (2DE). Methods: 29 synovial fluid knee samples collected from 29 patients (mean age: 68.19 years old; female: 55.17%; male: 44.83%) undergoing total knee arthroplasty (TKA) were analysed All the patients underwent a clinical, radiological and laboratory evaluation according to the current clinical practice. Five-hundred μl aliquoted SF was sample treated with two different hyaluronidase concentrations (1μg/ml vs 1mg/ml). The 1μg/ml hyaluronidase treated samples underwent two different high-abundance protein depletion techniques: affinity columns (ProteoPrep®, Sigma-Aldrich) or protein-enrichment kit (ProteoMinerTM, Bio-Rad Laboratories). Standardization of SF two-dimensional gel electrophoresis (2DE) map wa performed. Results: SF treated with 1μg/mL hyaluronidase showed a significantly higher protein spots number compared to 1mg/mL hyaluronidase treated samples (p=0.001). A significantly higher protein spots number was obtained in SF treated with ProteoMinerTM, compared to SF albumin and IgG-depleted (p=0.001). Moreover, ProteoMinerTM-enrichement provided 334 unique mean protein spots in SF proteome, which could not be detected nor in SF samples without depletion, nor in samples treated with affinity depletion.