Proteominer small capacity beads

Synovial fluid sample collection was carried out as previously described [16 (link)]. The samples without detectable hemolysis were centrifuged at 400× g at 4 °C for 5 min to remove cells and debris, then aliquoted and stored at −80 °C until use.
A five hundred μL aliquoted sample was thawed and treated with 1 μg/mL hyaluronidase (Sigma-Aldrich, Gillingham, UK) at 37 °C for 1 h [16 (link)], then centrifuged at 10,000× g, at 5 °C for 10 min. The Bradford method measured the total SF protein concentration by eluding a mean of 13 μg/μL (±1.4).
The SF samples treated with 1 μg/mL hyaluronidase were concentrated by 3-kDa cut-off Amicon filter (Millipore, Billerica, MA, USA) to reach the concentration of about 25 μg/μL and treated with ProteoMiner™ Small Capacity beads (Bio-Rad Laboratories, Hercules, CA, USA). Five mg of SF’s proteome were appropriately adapted to its subcolumn and processed according to the manufacturer’s instructions. Finally, the enriched SF protein concentration was determined.

SF (1 ml) was treated with 1 μg/mL of hyaluronidase⁵ (link). The supernatant was centrifuged through a 0.22 μm cellulose acetate membrane (Costar Spin-X, Corning, Tokyo, Japan) tube filter (5000× g/15 min) to remove insoluble material. Protein concentrations of SF were determined by Bradford assay (Thermo Scientific, Rockford, IL, USA).
For peptide-based depletion, 5 mg SF was bound to its own spin column containing ProteoMiner™ Small Capacity beads (Bio-Rad Laboratories, Hemel Hempstead, UK) with on-bead trypsin digestion.