Extraction of the cartilage with guanidine-HCl, representing the ECM-enriched proteome, was performed as previously described (54). Briefly, for mass spectrometric analyses, frozen cartilage sections were extracted using 4 M guanidine-HCl extraction buffer with 0.2% RapiGest (Waters Corporation, Milford, MA) for 24 hours on an orbital shaker at 4°C. Protein extracts were reduced with 4 mM dithiothreitol, alkylated with 16 mM iodoacetamide, and then precipitated with ethanol (9:1) overnight at 4°C. Samples were digested with 2 μg of trypsin, filtered through a 30-kDa filter (Pall Life Sciences, Port Washington, NY) and then a reversed-phase C18 column (The Nest group, Southborough, MA) to remove residual polysaccharides and salts.

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