Mass spectrometric off-line reaction monitoring is a reliable and versatile technique for the determination of enzymatic reactions parameters. The examples presented were limited to its application to investigate the enzymatic cleavage of minimal substrates by RNase T1. However, mass spectrometric detection of reactants provides an almost unlimited choice of possible substrates and reaction products. Therefore, this technique can find applications in nearly every field of enzymatic investigations. Using an autosampler device and automated data processing - as presented here - this method can be used as a screening method to determine the characteristic of a large amount of enzyme variants or the characteristics of substrates or reaction modulators automatically.
The deviation of the parameters determined using the technique described from the data presented in literature can be explained with the different reaction environments and the different acquisition methods. A very low buffer concentration of only 10 mM has been chosen to minimise the influence of the buffer system to the ionisation. However, the main field of application of this method is the automated screening of enzyme variants, substrates or reaction modulators, thus applying an identical environment and testing method to all reactions under investigation, which allows a comparison of the data obtained. Furthermore, the automated sample preparation will enhance the reliability of the results by increasing the reproducibility of the method.