The use of matrix‐assisted laser desorption/ionization mass spectrometry in enzyme activity assays and its position in the context of other available methods

Abstract

Activity assays are indispensable for studying biochemical properties of enzymes. The purposes of measuring activity are wide ranging from a simple detection of the presence of an enzyme to kinetic experiments evaluating the substrate specificity, reaction mechanisms, and susceptibility to inhibitors. Common activity assay methods include spectroscopy, electrochemical sensors, or liquid chromatography coupled with various detection techniques. This review focuses on the use of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) as a growing and modern alternative, which offers high speed of analysis, sensitivity, versatility, possibility of automation, and cost‐effectiveness. It may reveal reaction intermediates, side products or measure more enzymes at once. The addition of an internal standard or calculating the ratios of the substrate and product peak intensities and areas overcome the inherent inhomogeneous distribution of analyte and matrix in the sample spot, which otherwise results in a poor reproducibility. Examples of the application of MALDI‐TOF MS for assaying hydrolases (including peptidases and β‐lactamases for antibiotic resistance tests) and other enzymes are provided. Concluding remarks summarize advantages and challenges coming from the present experience, and draw future perspectives such as a screening of large libraries of chemical compounds for their substrate or inhibitory properties towards enzymes.

Maximizing analytical precision: exploring the advantages of ratiometric strategy in fluorescence, Raman, electrochemical, and mass spectrometry detection

“…5) Versatility: Ratiometric methods, incorporating various detection techniques such as fluorescence, absorbance, electrochemistry, Raman scattering, and mass spectrometry, emerge as a powerful tool for achieving accurate and sensitive measurements in diverse research areas. With its broad applicability, a ratiometric sensing proves valuable in a variety of analytical chemistry applications, including environmental monitoring, biomedical analysis, and materials science (Bigdeli et al, 2019;Jin et al, 2021;Wei et al, 2022;Šebela, 2023). allowing ratiometric sensing and imaging.…”