Monitoring Stepwise Proteolytic Degradation of Peptides by Supramolecular Domino Tandem Assays and Mass Spectrometry for Trypsin and Leucine Aminopeptidase

Abstract

A label-free optical detection method has been designed that allows direct monitoring of enzymatic peptide digestion in vitro. The method is based on the addition of a reporter pair, composed of the macrocyclic host cucurbit[7]uril (CB7) and the fluorescent dye acridine orange (AO), to detect the proteolytic degradation of peptides. The enzymatic activity of trypsin and leucine aminopeptidase (LAP) was investigated using H-LSRFSWGA-OH as a substrate. The substrate as well as the intermediary and final products (i.e., H-FSWGA-OH and phenylalanine) formed during its enzymatic hydrolysis differ in their binding affinity to the receptor CB7, which results in varying degrees of dye displacement and, therefore, different fluorescence intensities. CB7 showed a relatively weak binding constant of K ≈ 10⁴ M^–1 with the substrate, a relatively strong binding constant of K ≥ 10⁶ M^–1 with H-FSWGA-OH (which is a final product formed by trypsin digestion and the intermediary product formed during the enzymatic activity of LAP), and a moderate binding constant of K ≤ 10⁵ M^–1 with phenylalanine. Owing to this differential binding affinity of CB7 with the substrate and the corresponding products, the digestion of a peptide by trypsin was followed as a decrease in fluorescence signal, while the complete degradation of the peptide by LAP was monitored as a decrease and a subsequent increase in fluorescence signal. The k_cat/ K _M value for trypsin (2.0 × 10⁷ min^–1M^–1) was derived from the change in fluorescence signal with time. Additionally, the complete degradation of the peptide by LAP was also followed by mass spectrometry. The use of a supramolecular sensing ensemble (macrocyclic host and dye) as a fluorescent reporter pair gives this method the flexibility to adapt for monitoring the stepwise degradation of different biologically relevant peptides by other proteases.