School of Chemistry and Molecular Engineering , East China University of Science and Technology , Shanghai , 200237 , P. R. China.
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering , Nanjing University , Nanjing 210023 , P. R. China.
Anal Chem. 2019 Aug 6;91(15):9910-9915. doi: 10.1021/acs.analchem.9b01570. Epub 2019 Jul 10.
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/μL 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).
蛋白激酶在调节几乎所有细胞过程中起着关键作用。在这里，我们开发了一种基于野生型 Aerolysin 纳米孔的新型一步法，它能够在不标记/修饰、固定、协同酶和复杂步骤的情况下检测激酶活性。由于 Aerolysin 纳米孔的正电荷限制，激酶诱导的磷酸肽被专门捕获，而正电荷的底物肽可能会被电场从孔中移走。结合内标法，磷酸肽的事件频率与激酶呈剂量依赖性响应。以蛋白激酶 A 为模型靶标，检测限达到 0.005 U/μL。该方法还允许激酶抑制剂筛选、细胞裂解液中激酶活性检测以及在单分子水平上实时监测激酶催化的磷酸化，这将进一步有益于基础生化研究、临床诊断和激酶靶向药物发现。此外，这种纳米孔传感器对改变底物电荷的其他酶（如磺化、羧化或酰胺化）具有很强的检测能力。
