General Protease Assay Method Coupling Solid-Phase Substrate Extraction and Capillary Electrophoresis

Abstract

Capillary electrophoresis with laser-induced fluorescence detection was used to develop a universal, highly specific protease assay. In this method, a peptide, biotinylated at the N-terminus, is labeled with fluorescein at a lysine residue near the C-terminus. Impurities are removed from the fluorescence labeling mixture by solid-phase extraction of the substrate on immobilized streptavidin, followed by extensive washing. The purified fluorescent substrate is dissociated from the streptavidin and incubated with the protease. The peptide sequence between the biotin and fluorescent label contains the cleavage sequence of the protease of interest. After cleavage, the fluorescent product does not contain a biotin group. A second solid-phase extraction is used to remove unreacted substrate to dramatically lower the background signal. The product is detected by capillary electrophoresis, which provides powerful discrimination against products generated by nonspecific proteases. With chymotrypsin as a test protease, product was detected with as little as 10 pg/mL (4.6 x 10(-13) M) chymotrypsin, or 5 amol of enzyme in the 10-microL sample volume.

Characterization of the Streptococcal C5a Peptidase Using a C5a-Green Fluorescent Protein Fusion Protein Substrate

“…Many enzymatic assays using immobilized substrates have been described previously, but they often require radiometric or complex procedures for detection of the reporter (11,14,16,19,30,31). The assay described here has the advantage of a simple protocol and quick measurement of the reporter.…”

Capillary isoelectric focusing with pH 9.7 cathode for the analysis of gastric biopsies

“…1 and 2). Fluorescent labeling using neutral or anionic reagents disrupts both the zone electrophoresis and the isoelectric focusing profile of standards [42–44]. We employed the cationic fluorogenic reagent Chromeo P540 in this experiment; this reagent converts cationic lysine residues to cationic products, generating clean electrophoretic profiles [5, 17].…”

A Method for Rapid Protease Substrate Evaluation and Optimization

“…Our assay is based on the depletion of biotin containing substrates from the solution by incubation with streptavidin immobilized on magnetic beads [21,22] (see Scheme 1). The peptide sequences were labeled on the carboxy terminus with a biotin moiety and upon protease cleavage, the biotin containing part of the molecule was detached from the rest of peptide substrate containing the fluorescent label.…”

“…All substrates are mapped to the space of 96 substrates. As can be clearly seen, even a small selection of substrates can distinguish between all tested proteases (each protease has a unique pattern of cleaved substrates), and identification of specific enzymes by their "fingerprint" [21,[28][29][30][31] is feasible. The assay system described here for interrogation of protease activities using synthetic substrates is very simple and provides a rapid and accurate assessment of substrate qualities of multiplicity of peptides.…”