快速高分离度液相色谱/质谱方法筛选组织蛋白酶B的抑制剂

Inhibitor Screening of Cathepsin B by Rapid Resolution Liquid Chromatography/Mass Spectrometry

1引言组织蛋白酶B（CB）是溶酶体内半胱氨酸蛋白水解酶,属于木瓜蛋白酶家族,其特异性底物为Z-Arg-Arg-7-氨基-4-甲基香豆素[1],CB催化底物肽键断裂生成7-氨基-4-甲基香豆素。近年来的研究表明,CB在肿瘤的发展中起作用的一类关键酶[2],CB通过多种机制促进血管形成,从而促进肿瘤生长,同时降解基底膜主要成分以及激活胶原酶、尿激酶等其它蛋白酶发生协同作用,使肿瘤易侵袭、转移和扩散。

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 min× 1.8 μm） 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.