MASS SPECTROMETRIC on-line reaction monitoring is a versatile method to investigate the mechanism of enzymatic reaction. This technique allows a continious observation of an enzymatic reaction with a time resolution of a few milliseconds, thus providing almost uninterrupted reaction data. The special features of mass spectrometers allow the simulataneous monitoring of several compounds, applying tandem mass spectrometry it is even possible to distinguish between isomers.
As enzymatic reactions are normally conducted in buffered aqueous solutions, mass spectrometric on-line monitoring requires an ion source which allows the ionisation of buffered aqueous solutions. NANOES has these properties and was therefore used for on-line reaction monitoring. Because of the low flowrate of these sources, even investigations of several hours can be undertaken with a small sample amount. However, long time monitoring for more than an hour increases the risk of protein denaturation and thus a loss in activity.
As described in 3.1.3.1, compound quantification is difficult without an internal standard. As the ionisation process and the sample composition differs between FIA (where a volatile acidic solvent is present) which was used for offline-reaction monitoring and NANOES, the calibration data obtained in 3.1.3.1 cannot be used without reservation. However, the almost linear correlation of relative intensity and relative concentration still exists, which allows a estimation of concentrations, which is normally sufficient for the elucidation of reaction mechanisms.
To demonstrate the capabilities of this technique, the enzyme catalysed turnover of minimal substrates for RNase T1 wild type (wt) and the variant RV was investigated.