Protease profiling using a fluorescent domino peptide cocktail

Five hexapeptides were prepared containing, in a domino-type arrangement, all 25 possible dipeptides between (1) aromatic, (2) hydrophobic, (3) positively charged, (4) negatively charged, and (5) small and polar amino acids. The peptides were fluorescence labeled at the N-terminus with a (7-coumaryl)oxyacetyl group, allowing the selective detection of N-terminal cleavage products. The five peptides were used as a cocktail reagent in an HPLC analysis. The cocktail produced specific cleavage patterns, or fingerprints, for a variety of proteases. This domino peptide cocktail can be used as a general reagent for protease identification and functional profiling.