Monitoring enzyme reaction and screening of inhibitors of acetylcholinesterase by quantitative matrix-assisted laser desorption/ionization fourier transform mass spectrometry

Abstract

A matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS)-based assay was developed for kinetic measurements and inhibitor screening of acetylcholinesterase. Here, FTMS coupled to MALDI was applied to quantitative analysis of choline using the ratio of choline/acetylcholine without the use of additional internal standard, which simplified the experiment. The Michaelis constant (K m ) of acetylcholinesterase (AChE) was determined to be 73.9 mol L Ϫ1 by this approach. For Huperzine A, the linear mixed inhibition of AChE reflected the presence of competitive and noncompetitive components. The half maximal inhibitory concentration (IC 50 ) value of galantamine obtained for AChE was 2.39 mol L Ϫ1 . Inhibitory potentials of Rhizoma Coptidis extracts were identified with the present method. In light of the results the referred extracts as a whole showed inhibitory action against AChE. The use of high-resolution FTMS largely eliminated the interference with the determination of ACh and Ch, produced by the low-mass compounds of chemical libraries for inhibitor screening. The excellent correlation with the reported kinetic parameters confirms that the MS-based assay is both accurate and precise for determining kinetic constants and for identifying enzyme inhibitors. The obvious advantages were demonstrated for quantitative analysis and also high-throughput characterization. This study offers a perspective into the utility of MALDI-FTMS as an alternate quantitative tool for inhibitor screening of

Screening and structural characterization of α-glucosidase inhibitors from hawthorn leaf flavonoids extract by ultrafiltration LC-DAD-MSn and SORI-CID FTICR MS

“…In recent years, mass spectrometry-based assays have attracted great attention for high-throughput screening. These methods, which include quantitative MALDI-FT-MS [23], enzyme affinity selection procedure followed by ESI-FT-MS [24], frontal affinity chromatography-MS [25], size-exclusion chromatography-MS [26], capillary electrophoresis-MS [27], and pulsed-ultrafiltration MS [28 -30], all have been successfully used to determine potentially active compounds. Ultrafiltration LC-MS/MS is a powerful tool to screen biologically active compounds from botanical extracts because of its high throughput on-line screening ability, and its high sensitivity and selectivity necessary for the characterization of compounds present at low concentrations in highly complex matrices without time-consuming purification procedures [28 -30].…”

High-resolution mass spectrometry for detecting Acetylcholine in Arabidopsis

“…High-resolution mass spectral analysis of ACh has been applied to either mammals or in vitro enzyme assays previously. 16,17 However, there have been no reports on the detection of ACh from plant tissues using mass spectrometry at high resolution where the level of ACh is generally much lower compared to animal tissues. In this study, we detected various peaks which have m/z values quite similar, but clearly distinct from that of ACh by ESI-orbitrap FT-MS (Fig.…”

Kinetic Modelling of Enzyme Catalyzed Biotransformation Involving Activations and Inhibitions

“…Greis et al studied the phosphorylation catalysed by kinases using this technique for accurate prediction of the kinetics of reaction [93]. This technique has been used in combination with various chromatographic techniques such as capillary isoelectric focusing, frontal affinity chromatography and size exclusion chromatography to analyse the inhibition of the enzyme in direct or indirect manner [93][94][95][96]. This method is free of laborious work of labelling or derivatization and can be done in short period of time.…”