文献类型：期刊文献

英文题名：A Wild-Type Nanopore Sensor for Protein Kinase Activity

作者：Meng, Fu-Na[1,3];Ying, Yi-Lun[1,2];Yang, Jie[1];Long, Yi-Tao[1,2]

机构：[1]East China Univ Sci & Technol, Sch Chem & Mol Engn, Shanghai 200237, Peoples R China;[2]Nanjing Univ, Sch Chem & Chem Engn, State Key Lab Analyt Chem Life Sci, Nanjing 210023, Jiangsu, Peoples R China;[3]Heze Univ, Heze City 274015, Shandong, Peoples R China

年份：2019

卷号：91

期号：15

起止页码：9910

外文期刊名：ANALYTICAL CHEMISTRY

收录：;EI(收录号：20193507363012);WOS:【SCI-EXPANDED(收录号:WOS:000480499200079)】；

基金：This research was supported by the National Natural Science Foundation of China (21834001 and 61871183), and Excellent Research Program of Nanjing University (ZYJHO04). Dr. Yi-Lun Ying is sponsored by National Ten Thousand Talent Program for Young Top -Notch Talent, Shanghai Rising-Star Program (19QA1402300) and "ChenGuang" Project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation (17CG27).

语种：英文

外文关键词：Diagnosis - Electric fields - Enzyme immobilization - Carboxylation - Clinical research

摘要：Protein kinases play a critical role in regulating virtually all cellular processes. Here, we developed a novel one-step method based on a wild-type aerolysin nanopore, which enables kinase activity detection without labeling/modification, immobilization, cooperative enzymes and complicated procedures. By virtual of the positively charged confinement of the aerolysin nanopore, the kinase-induced phosphopeptides are specially captured while the positively charged substrate peptides might move away from the pore by the electric field. Combining with internal standard method, the event frequency of the phosphopeptides exhibited a dose-dependent response with kinases. The detection limit of 0.005 U/mu L has been achieved with protein kinase A as a model target. This method also allowed kinase inhibitor screening, kinase activity sensing in cell lysates and the real-time monitoring of kinase-catalyzed phosphorylation at singe molecule level, which could further benefit fundamental biochemical research, clinical diagnosis and kinase-targeted drug discovery. Moreover, this nanopore sensor shows strong capacity for the other enzymes that altered substrate charge (e.g., sulfonation, carboxylation, or amidation).