Triple-FRET multi-purpose fluorescent probe for three-protease detection

A new, robust and reliable methodology for three-protease screening in a single-enzyme mode has been developed and verified, employing a multi-purpose peptide probe with three selectively cleavable sites furnished with four fluorophores. A triple-FRET-based single-excitation quadruple-emission concept for unambiguous sensing of trypsin, chymotrypsin and caspase-8 in the lowest detectable concentrations of 0.5 ng mL(-1), 0.2 mu g mL(-1), and 2 U mL(-1), respectively, has been applied and graphically depicted. Then the developed 4-dye probe has been also studied from the perspective of simultaneous two-protease screening, which was found only partially feasible, primarily due to unselective chymotrypsin cleavage.

Automated summary

A new, robust and reliable method for screening three proteases in a single-enzyme mode has been developed using a multi-purpose peptide probe and multiple fluorophores. The method allows for the detection of trypsin, chymotrypsin and caspase-8 at very low concentrations. Additionally, the simultaneous screening of two proteases using the developed probe was found to be only partially feasible, mainly due to unselective chymotrypsin cleavage.