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OBJECTIVES: To develop a proteomics approach to study changes in the secreted protein levels of primary human chondrocytes after stimulation by the pro-inflammatory cytokines interleukin-1 and oncostatin M. METHODS: Using both the primary human articular and bovine nasal chondrocyte-conditioned mediums, methods were investigated to enable the separation of proteins by two-dimensional (2D) gel electrophoresis. Differentially regulated proteins were identified using tandem electrospray mass spectrometery. RESULTS: We discovered that proteoglycans and glycosylaminoglycans (GAGs) secreted by chondrocytes significantly interfered with 2D gel focusing. Several different methods for GAG removal were attempted including enzymic digestion, cetyl pyridinium chloride precipitation and anion exchange in high salt. The anion exchange proved to be the most effective. Even from these initial gels, we were able to identify eight proteins produced by human chondrocytes: matrix metalloproteinase (MMP)-1, MMP-3, YKL40, cyclophilin A, beta2-microglobulin, transthyretin, S100A11, peroxidine 1 and cofilin. MMP-1, MMP-3, YKL40 and cyclophilin A were all identified as processed, smaller peptide fragments. CONCLUSIONS: We were able to develop a novel sample preparation protocol to allow the reproducible sample preparation of secreted proteins from human chondrocytes. From the initial data, we were able to show that at least some of the proteins produced were cleaved to smaller fragments as a result of proteolysis. Therefore, this technique provides valuable information about protein processing which gene-based arrays do not.

Original publication

DOI

10.1093/rheumatology/kel060

Type

Journal article

Journal

Rheumatology (Oxford, England)

Publication Date

09/2006

Volume

45

Pages

1101 - 1109

Addresses

Musculoskeletal Research Group, School of Clinical Medical Sciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.

Keywords

Cells, Cultured, Chondrocytes, Humans, Cyclophilin A, Peroxidases, Biological Markers, Interleukin-1, Cytokines, Chromatography, Ion Exchange, Peptide Mapping, Electrophoresis, Gel, Two-Dimensional, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Stimulation, Chemical, Molecular Weight, Aged, Aged, 80 and over, Female, Male, Matrix Metalloproteinase 3, Matrix Metalloproteinase 1, Oncostatin M, Peroxiredoxins