OFF-LINE REACTION monitoring using mass spectrometry is a method to determine kinetic parameters of an enzymatic reaction. In this method, the reaction is conducted in a reaction vessel and samples are taken at definite points in time. These samples are analysed to obtain concentration information for all compounds involved in the reaction. The advantage of mass spectrometric off-line reaction monitoring is the capability of the mass spectrometer to obtain the desired information about the samples without any preceding separation like in HPLC thus increasing the possible time resolution. Furthermore, the mass spectrometer allows the simultaneous investigation of several reactants with almost no limitation regarding the compounds used and monitored. Using an autosampler device, this method allows a widely automated acquisition of data for several reactions simultaneously, only limited by the desired time resolution, as the acquisition of one sample takes approximately two minutes (including the equilibrium run).
To show the capabilities of this method, kinetic parameters for the hydrolysis of minimal substrates by RNase T1 were determined. In addition to the standard assay, the transesterification of 5',3'-GpC to 2',3'-cGMP and cytidine, the transesterification 5',3'-ApC to 2',3'-cAMP and cytidine by a variant of RNase T1 (RNase T1 RV) and the hydrolysis of 2',3'-cGMP by RNase T1 were investigated.