Identification of Proteases by Combined Fluorescent Gel Zymography and Proteomics

Identifying proteases and the substrate-peptides they degrade can provide valuable information to harness the enzymatic responses of specific cell types. Previous approaches to protease identification are reliant on experimentation with protease inhibitors or are limited to classification by molecular weight. Our method, which combines fluorescent gel zymography with proteomic analysis provides a platform approach to identify unknown proteases and confirm protease-substrate pairs. This method involves incorporating peptides, synthesized with a fluorophore on the N-terminus and a quencher on the C- terminus, into polyacrylamide gels. Samples such as conditioned media or cells can be run on these peptide-containing gels by electrophoresis. Cleavage events are visualized by an increase in fluorescence caused by separation of the quencher from the fluorophore. Following, fluorescent bands are excised, and peptides are digested by a tryptic digestion. Finally, using Liquid Chromatography – Mass Spectroscopy (LC-MS) and proteomic analysis samples of digested peptides are analyzed to determine protease identity. Using this approach, we have been able to demonstrate the effective use of fluorescent gel zymography to visualize cleavage of two peptide substrates by THP-1 conditioned media, THP-1 cells, and Collagenase. Furthermore, by coupling zymography with several cell secreted proteases and membrane bound proteases have been identified by combining zymography with proteomics.