Real‐Time and Label‐Free Measurement of Deubiquitinase Activity with a MspA Nanopore

Covalently attaching ubiquitin (Ub) to cellular proteins as a post‐translational modification can result in altered function of modified proteins. Enzymes regulating Ub as a post‐translational modification, such as ligases and deubiquitinases, are challenging to characterize in part due to the low throughput of in‐vitro assays. Single‐molecule nanopore based assays have the advantage of detecting proteins with high specificity and resolution, and in a label‐free, real‐time fashion. Here we demonstrate the use of a MspA nanopore for discriminating and quantifying Ub proteins. We further applied the MspA pore to measure the Ub‐chain disassembly activity of UCH37, a proteasome associated deubiquitinase. The implementation of this MspA system into nanopore arrays could enable high throughput characterizations of unknown deubiquitinases as well as drug screening against disease related enzymes. A MspA nanopore is demonstrated as a sensor for discriminating and quantifying various ubiquitin proteins. Real‐time measurement of polymeric ubiquitin chain disassembly by a deubiquitinating enzyme was performed and enzyme kinetics were determined.