Highly Adaptable and Sensitive Protease Assay Based on Fluorescence Resonance Energy Transfer

Abstract

Proteases are widely used in analytical sciences and play a central role in several widespread diseases. Thus, there is an immense need for highly adaptable and sensitive assays for the detection and monitoring of various proteolytic enzymes. We established a simple protease fluorescence resonance energy transfer (pro-FRET) assay for the determination of protease activities, which could in principle be adapted for the detection of all proteases. As proof of principle, we demonstrated the potential of our method using trypsin and enteropeptidase in complex biological mixtures. Briefly, the assay is based on the cleavage of a FRET peptide substrate, which results in a dramatic increase of the donor fluorescence. The assay was highly sensitive and fast for both proteases. The detection limits for trypsin and enteropeptidase in Escherichia coli lysate were 100 and 10 amol, respectively. The improved sensitivity for enteropeptidase was due to the application of an enzyme cascade, which leads to signal amplification. The pro-FRET assay is highly specific as even high concentrations of other proteases did not result in significant background signals. In conclusion, this sensitive and simple assay can be performed in complex biological mixtures and can be easily adapted to act as a versatile tool for the sensitive detection of proteases.

Determining bacteriophage endopeptidase activity using either fluorophore-quencher labeled peptides combined with liquid chromatography-mass spectrometry (LC-MS) or Förster resonance energy transfer (FRET) assays

“…by proteolytic activity) the quencher is no longer able to inhibit the fluorophore, resulting in a fluorescent signal. This has been commonly used to study several proteases [12–15], and recently for a bacterial autolysin with amidase activity [9], but has yet to be evaluated for the study of phage endopeptidases. While this method is useful to determine if a cleavage has occurred within a FRET-modified peptide, it does not reveal precisely where the cleavage has occurred.…”

Enhanced Colorimetric Immunoassay Accompanying with Enzyme Cascade Amplification Strategy for Ultrasensitive Detection of Low-Abundance Protein

“…We reasoned that if such systems could be extended to detect species other than the intracellular reaction, it might be generally useful for other types of highly sensitive and selective assays. Zuchner et al developed a simple protease fluorescence resonance energy transfer for highly sensitive and rapid detection of enteropeptidase based on dual-enzyme cascade amplification32. To the best of our knowledge, there is no report focusing on enzyme cascade amplification strategy for the development of colorimetric immunoassay until now.…”

Research Techniques Made Simple: Methodology and Applications of Förster Resonance Energy Transfer (FRET) Microscopy

“…FRET biosensors can also be generated to examine the enzymatic activity of a protein of interest (Zauner et al, 2011). For example, a biosensor can be designed to include a peptide containing the cleavage sequence of a protease of interest.…”