Active matrix metalloproteases (MMPs) play a significant role in the pathogenesis of osteoarthritis (OA) via proteolytic degradation of cartilage. To investigate the activity of these metalloproteases in cartilage explant cultures, with and without stimulation by proinflammatory cytokines, e.g., interleukin-1a, we assessed a functional proteomic approach based on mass spectrometry and activity-based protein profiling-multidimensional identification technology (ABPP-MudPIT).  We first present the design and synthesis of chemical probes (Activity Based Probes (ABPs)) for efficient enrichment and elution of matrix metalloproteases. Using a quenched fluorescent substrate, we monitored the initial activities of the metalloproteases in the culture medium. The fluorescence was completely suppressed in the presence of ABPs. The multifunctional probes show an excellent labeling pattern of active MMPs in Western blots. The unique chemical properties of the probes enabled quantitative release of labeled active proteases from the avidin support, and active MMPs were identified in day 20 and day 30 cultured media samples, but not in control samples. We compared the active proteases with the proenzymes that were revealed by shotgun proteomics; this clearly indicates the predominance of proenzymes in the cultured medium.  The activation of endogenous MMPs by the activator 4-aminophenylmercuric acetate (APMA), and then profiling the activity of MMPs by ABPs, indicates that there are detectable levels of active MMPs in control as well as in the early days of IL-1a treated cultures. Antibodies further confirmed the identities of several active proteases after release from avidin supports.

Functional proteomics of matrix metalloproteases in a model of osteoarthritis

248th National Meeting (2014)

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