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Rheumatology Advance Access published online on March 27, 2006
Rheumatology, doi:10.1093/rheumatology/kel060
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received July 12, 2005
Accepted January 31, 2006

Original Papers

Development of a novel 2D proteomics approach for the identification of proteins secreted by primary chondrocytes after stimulation by IL-1 and oncostatin M

J. B. Catterall 1, A. D. Rowan 1, S. Sarsfield 2, J. Saklatvala 2, R. Wait 2, and T. E. Cawston 1 *

1 Musculoskeletal Research Group, School of Clinical Medical Sciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK
2 Kennedy Institute, Imperial College, London, UK

* To whom correspondence should be addressed.
T. E. Cawston, E-mail: T.E.Cawston{at}ncl.ac.uk


  Abstract

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, {beta}2-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.

Keywords: Chondrocyte; IL-1; MMP; OSM; Proteomics..
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