A method based on rapid resolution liquid chromatography/mass spectrometry was developed for the inhibitor screening of Cathepsin B (CB) from natural products by detecting 7-amino-4-methylcoumarin, the product catalysed by CB. First, 8 nmol/L CB reacted with 20 μmol/L Z-Arg-Arg-7-amino-4-methylcoumarin at 37 ℃for 20 min, then 1% trifluoroacetic acid was added to quench the reaction and final, 1 μmol/L 7-Amino-4-(methoxymethyl) coumarin was added as internal standard. Quantitative analysis was carried out for the analyte by MS after the chromatographic separation on Agilent ZORBAX Extend-C18 (2.1 mm× 50 mm× 1. 8 μrn) column with methanol-water gradient elution. The limit of quantitation of 7-amino-4- methylcoumarin was 100 nmol/L and linear range was 100 nmol/L-20 μmol/L. As a method of inhibitor screening based on quantitative analysis of enzyme catalyzed product, it is rapid, accurate, sensitive, and adapt to high throughput screening inhibitors of CB from natural products or compounds library of combinatorial chemistry.
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