Enzyme reaction rates determined by fast atom bombardment mass spectrometry

Abstract

Fast atom bombardment (FAB) mass spectrometry has been used to measure the reaction rate of trypsin with the synthetic substrate p-toluenesulfonyl-L-arginine methyl ester (TAME). Rates were measured by FAB mass spectrometry both in real time by monitoring the reaction as it proceeded on the probe inside the mass spectrometer, and also by individual analysis of a series of aliquots removed with time from a batch reaction. The results were then compared to the rates measured using the usual spectrophotometric (UV) assay run with the same reaction mixture. The results showed that the initial rates of trypsin cleavage were the same for these three methods at approximately 0.15 μmol product produced min−1 μg−1 enzyme. After 5 min, the rates determined by FAB mass spectrometry using timed aliquots and the UV assay remained about the same, whereas the real-time measurement decreased substantially to approximately 0.002 μmol product min−1 μg−1 enzyme. The results are discussed with respect to the particular advantages and disadvantages of these methods for following enzyme reactions.