Feasibility of Acetylcholinesterase Reaction Assay Monitoring in DIUTHAME-MS

DIUTHAME (desorption ionization using through hole alumina membrane) is a novel matrix-free laser desorption/ionization method that enables highly reproducible acquisition of mass spectra. This study aims to evaluate the applicability of DIUTHAME to the acetylcholinesterase reaction assay (AChE assay) commonly used in high-throughput screening (HTS) in the drug discovery process. The commercially available 9-ch DIUTHAME chip was applied to a series of AChE assays prepared with different reaction times. Numerous positive-mode TOF mass spectra were acquired from the raster-scanned sample spots of the AChE assays to analyze the progress of the enzyme reaction and to perform mass spectrometry imaging of the sample spots. The reaction kinetics plots obtained by DIUTHAME were found to reflect the time course of reaction progression as much as those obtained by MALDI and were found to have less error variation than MALDI. DIUTHAME allowed the already irradiated sample spots to be reused to reproduce the reaction kinetics plots by the second measurement conducted a week after, whereas MALDI was not able to properly reproduce the kinetics plots by remeasuring the irradiated sample spots. In DIUTHAME, which does not require a matrix, the experimental procedure for measuring AChE assay becomes extremely simple. DIUTHAME is potentially able to provide more precise AChE reaction kinetics plots than what have been accomplished by MALDI.