A Wild-Type Nanopore Sensor for Protein Kinase Activity.

ABSTRACT

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).