Development of a novel 2D proteomics approach for the identification of proteins secreted by primary chondrocytes after stimulation by IL-1 and oncostatin M.
OBJECTIVE
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.
Musculoskeletal Research Group, School of Clinical Medical Sciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.
J B
JB
Catterall
A D
AD
Rowan
S
S
Sarsfield
J
J
Saklatvala
R
R
Wait
T E
TE
Cawston
D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000032:analysis; D002478:Cells, Cultured; D019902:Chondrocytes / Q000276:immunology / Q000378:metabolism*; D002852:Chromatography, Ion Exchange / Q000379:methods; D021984:Cyclophilin A / Q000032:analysis; D016207:Cytokines / Q000494:pharmacology*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005260:Female; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000032:analysis; D019278:Matrix Metalloproteinase 3 / Q000032:analysis; D008970:Molecular Weight; D053683:Oncostatin M; D010449:Peptide Mapping / Q000379:methods; D010544:Peroxidases / Q000032:analysis; D054464:Peroxiredoxins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013268:Stimulation, Chemical
D015415:Biomarkers; D016207:Cytokines; D007375:Interleukin-1; C508351:OSM protein, human; D053683:Oncostatin M; D010544:Peroxidases; D054464:Peroxiredoxins; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1; D021984:Cyclophilin A