Quantitative analysis capability of DIUTHAME mass spectrometry verified by acetylcholinesterase enzyme-catalyzed reaction assays

Abstract

Matrix-assisted laser desorption/ionization (MALDI) is advantageous for mass spectrometry applications where throughput is important. However, quantitative analysis is essentially problematic for MALDI-MS whose results depend on the intrinsically stochastic microcrystalline state of the matrix. High-throughput screening (HTS) of drug candidates is a typical example that requires high throughput. The application of MALDI-MS to HTS, which is quantitative analysis, imposes restrictions on designing an experimental system. Surface-assisted laser desorption/ionization (SALDI) methods, which do not depend on matrix crystal formation, are expected to be applied to quantitative analyses such as HTS. A recently developed one type of SALDI methods, desorption ionization using through hole alumina membrane (DIUTHAME), possesses a distinct feature that the surface microstructure effective for ionization is formed by through holes. In this study, the quantitative analysis capability of DIUTHAME was verified by applying DIUTHAME to enzyme-catalyzed reaction measurements, which are also used for HTS. Quantitative DIUTHAME-MS was conducted on various conditions of acetylcholinesterase-catalyzed reaction solutions containing cow milk as a substitute of biological media. Even for the enzyme-catalyzed reaction solutions containing complex additives that make the quantitative analysis extremely difficult, DIUTHAME based on the through hole structure enables quantitative measurements of the analytes by applying the reaction solutions to the back side of the laser exposed surface. In comparison with those obtained by MALDI-MS, the results obtained by DIUTHAME-MS showed less variability of data and delivered a better linearity of the Lineweaver-Burk plots and a more reasonable value of the Michaelis constant. Accordingly, it was demonstrated that DIUTHAME-MS possesses the quantitative analysis capability much better than that of MALDI-MS.

Keywords: DIUTHAME; MALDI; SALDI; enzyme-catalyzed reaction; quantitative analysis.