General protease assay method coupling solid phase substrate extraction and capillary electrophoresis
General protease assay method coupling solid phase substrate extraction and capillary electrophoresis
复制标题
DOI：
10.1021/ac9801061
复制
发表时间：
1998-09-15
影响因子：
7.4
通讯作者：
Dovichi, NJ
中科院分区：
文献类型：
--
作者：
Craig, DB;Wong, JCY;Dovichi, NJ
毛细管电泳与激光诱导荧光检测用于开发一种通用的，高度特异性的蛋白酶测定。在这种方法中，在n端生物素化的肽，在c端附近的赖氨酸残基上用荧光素标记。通过固相萃取固定链霉亲和素的底物，然后进行大量洗涤，从荧光标记混合物中去除杂质。纯化的荧光底物与链霉亲和素分离，并与蛋白酶孵育。生物素和荧光标记之间的肽序列包含感兴趣的蛋白酶的裂解序列。裂解后，荧光产物不含生物素基团。第二次固相萃取用于去除未反应的底物，以显著降低背景信号。该产物通过毛细管电泳检测，对非特异性蛋白酶产生的产物具有很强的鉴别能力。以胰凝乳蛋白酶为试验蛋白酶，在10 μ L样品体积下，用10 pg/mL（4.6 × 10(-13) M）胰凝乳蛋白酶或5 amol酶检测产物。
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-mu L sample volume.