Detection of proteolytic activity by covalent tethering of fluorogenic substrates in zymogram gels

Abstract

Current zymographic techniques detect only a subset of known proteases due to the limited number of native proteins that have been optimized for incorporation into polyacrylamide gels. To address this limitation, we have developed a technique to covalently incorporate fluorescently labeled, protease-sensitive peptides using an azido-PEG3-maleimide crosslinker. Peptides incorporated into gels enabled measurement of MMP-2, -9, -14, and bacterial collagenase. Sensitivity analysis demonstrated that use of peptide functionalized gels could surpass detection limits of current techniques. Finally, electrophoresis of conditioned media from cultured cells resulted in the appearance of several proteolytic bands, some of which were undetectable by gelatin zymography. Taken together, these results demonstrate that covalent incorporation of fluorescent substrates can greatly expand the library of detectable proteases using zymographic techniques.

MDM2 Derived from Dedifferentiated Liposarcoma Extracellular Vesicles Induces MMP2 Production from Preadipocytes

“…Protein content of the isolated conditioned medium (CM) samples was quantified by mBCA according to the manufacturer's protocol (Thermo Fisher Scientific). Gelatin zymography was performed as described by Deshmukh and Toth (24,25). Briefly, 30 mg of protein collected from the CM was loaded onto precast gelatin zymography gels (10% polyacrylamide, 0.1% gelatin; Thermo Fisher Scientific).…”

Click chemistry functionalization of self‐assembling peptide hydrogels

“…The fluorogenic MMP biosensor Dabcyl‐GGPQG↓IWGQK‐Fluorescein‐AeeaC (where ↓ indicates the protease cleavage site) was synthesized using SPPS and functionalized with a quencher (dabcyl) and fluorophore (fluorescein) as previously described 50,62 . SAP hydrogels with the MMP biosensor clicked‐in were generated at a volume of 45 μl.…”

“…The fluorogenic MMP biosensor Dabcyl‐GGPQG↓IWGQK‐Fluorescein‐AeeaC (where ↓ indicates the protease cleavage site) was synthesized using SPPS and functionalized with a quencher (dabcyl) and fluorophore (fluorescein) as previously described. 50 , 62 SAP hydrogels with the MMP biosensor clicked‐in were generated at a volume of 45 μl. For experiments testing exposure to collagenase, gels were incubated in various concentrations of collagenase dissolved in PBS (ThermoFisher cat#17100–017 at 100, 50, 20, 10, 5, 2.5, 1, 0.1, 0.01 μg/ml) and incubated for 24 h at 37°C and 5% CO 2 .…”