High-throughput monitoring of in vitro enzymatic reactions using probe electrospray ionization-mass spectrometry

Probe electrospray ionization-mass spectrometry (PESI-MS) is an ambient ionization technique that enables rapid and simple mass spectrometry with minimal sample preparation. In this study, we evaluated the applicability of PESI-MS for the quantitative analysis of metabolites in in vitro enzyme reaction mixtures, which often contain high concentrations of proteins, salts, and buffers that are not suitable for conventional MS. Using glycolytic metabolites as a model, we optimized the type of organic solvents for mixing, data processing method, and investigated the effects of additives and reaction components on ionization efficiency and signal stability. Ethanol and 2-propanol were identified as suitable solvents for the detection of sugar phosphates, organic acids, and nucleotides. The quantitative reproducibility was improved by employing a total peak-height-based data processing method. The presence of proteins had a minimal influence on ionization. Although high concentrations of MgCl₂ and buffers suppressed the signal intensity, metabolite signals were still observable at 10 and 50 mM, respectively. The linearity of the calibration curves and the reproducibility of the analysis of glycolytic metabolites in the mock enzyme reaction solution were confirmed. Finally, PESI-MS was successfully used to monitor the time course of intermediates in an in vitro synthetic non-oxidative glycolysis system. The data trend obtained by PESI-MS was roughly consistent with the results of conventional LC-MS analysis. These findings demonstrate that PESI-MS is a simple, rapid, and robust method for analyzing in vitro enzymatic reactions.