Synovial fluid aspiration was performed by a board certified rheumatologist by selleck bio fine needle arthrotomy, and the syno vial fluid samples obtained were free from obvious con tamination with blood or debris. OA serum and synovial fluid samples were obtained from patients diagnosed with knee OA according to the 1985 criteria of Inhibitors,Modulators,Libraries the American Rheuma tism Association. For mass spectrometric analysis, OA synovial fluid samples were from five Caucasian men aged 50 to 75 years who met the 1985 OA criteria, exclusion criteria included radiographic evidence of chondrocalcinosis or evidence of crystals under polariz ing microscopy. Demographics and clinical characteris tics of these five individuals are shown in Table Inhibitors,Modulators,Libraries 1.

Synovial fluids from the other OA patients and from the RA patients were provided as de identified Inhibitors,Modulators,Libraries remnant clinical samples, and patient demographics were there fore unavailable for these samples. All RA patients met the 1987 American Rheumatism Association criteria for RA and had RA of less than 6 months duration, exclusion criteria included concurrent infectious or crys tal arthritis. Samples of normal serum were obtained from healthy individuals who had no joint pain and no radiographic evidence of knee arthritis. OA and normal sera were matched by Inhibitors,Modulators,Libraries age, sex, and BMI. Serum and synovial fluid samples were not matched but were derived from patients with the characteristics described earlier. All samples were aliquoted and stored at 80 C. Mass spectrometric analysis Synovial fluid proteins were separated by 1D or 2D polyacrylamide gel electrophoresis, trypsinized, and identified by liquid chromatography tandem mass spectrometry, as follows.

Fifty microliters of fro zen synovial fluid was diluted to a final volume of 1 ml in phosphate buffered saline containing Halt pro tease and phosphatase inhibitor, and then depleted of the Inhibitors,Modulators,Libraries highly abundant proteins albumin and immunoglobulin G by using selleck chemical Sunitinib the Pro teoPrep Immunoaffinity Albumin IgG Depletion Kit according to the manufacturers instructions. In brief, synovial fluids were twice passed over spin columns prepacked with a mixture of two beaded mediums containing recombinantly expressed, small, single chain antibody ligands. The flow through fractions containing synovial fluid depleted of albumin and IgG were diluted 1,1 with Laemmli Sample Buffer and then subjected to 1D PAGE or 2D PAGE analysis. Because a small number of proteins other than albumin and IgG may bind to the medium in the spin columns, the bound proteins were eluted with Laemmli sample buffer and also subjected to PAGE analysis. For 1D PAGE analysis, proteins were boiled for 10 minutes and separated on Precast Criterion XCT gels.