Development of ESI-MS-based continuous enzymatic assay for real-time monitoring of enzymatic reactions of acetylcholinesterase

•Development of MS-based continuous assay for real-time monitoring of enzymatic reactions.•Kinetic parameters of enzyme and inhibitor were measured without quenching reactions.•This assay provides a simply and accurate way to measure enzyme activity.

The continuous enzymatic assay based on ESI-MS was developed to real-time monitoring of enzymatic reactions of acetylcholinesterase (AChE). The changes of product concentrations were continuously measured. Calibration curves were established for quantitative calculation. By this method, the Michaelis constant (Km) of acetylcholinesterase was determined to be 70.60 ± 0.93 μM and Huperzine A as an effective inhibitor of acetylcholinesterase displayed a mixed inhibition with competitive and noncompetitive inhibition behaviors. The half maximal inhibitory concentration (IC50) and inhibition constant (Ki) value of Huperzine A were also calculated as 48.51 ± 1.16 nM and 26.73 ± 0.27 nM, respectively. This method provides the rapid and accurate ways to monitor enzyme reactions.