MASS SPECTROMETRY is an approved tool for the analysis of biopolymers such as proteins, nucleic acids, lipids and polysaccharides. Furthermore, this technique is also used to characterise natural products. Nevertheless, there are only few applications for investigating enzymatic reactions using the special capabilities of mass spectrometry.
The aim of this work was to develop methods for the investigation of enzymatic reactions using mass spectrometric techniques, electrospray ionisation MS and MALDI MS, in particular. The main focus of this work is to evolve methods which are useful for the further investigation of RNase T1, as the standard assays (see 1.2.4) are insufficient for investigations with higher substrate concentration or longer oligonucleotides as substrate. Furthermore, methods are required that allow a rapid investigation of the enzymatic properties of RNase T1 variants with respect to altered specificity or reaction mechanism. For the investigation of variants obtained by semi-rational or evolutionary methods, fast automated methods for the determination are needed.
To show the capabilities and limitations of the methods developed, these methods were applied to RNase T1.