Analyse der Sekundärmodifikationen von Komponenten der Phytochrom-Signaltransduktion in Arabidopsis thaliana
Final Report Abstract
Mapping of complex signal transduction networks has so far mainly relied on the analysis of mutants, studies of interactions in vitro and in vivo and on the biochemical characterisation of single components. Modern mass spectrometry allows the identification of single proteins and the characterisation of secondary modifications of peptides in complex mixtures. This allows a timeresolved analysis of the status of known signalling components. The signalling pathway of phytochrome A (phyA) can specifically be triggered by far-red light, thus uniquely allowing the observation of a single signalling chain. As this pathway is rather well known, it is possible to detect spatial and temporal changes of quantity and secondary modifications of key components in complex proteome samples and hence to elucidate their interplay with the help of single/selected reaction monitoring and high resolution mass spectrometry. In the period funded we were able to synthesize and purify unlabelled and 15N-labelled peptides of 4 target components and the photoreceptor itself, to obtain first peptide mass reads and to initiate calibration of the detection range with the help of the labelled peptides. Targeted analysis of complex Arabidopsis samples and the quantitative identification of the components in induced and non-induced seedling extracts as well as the mapping of specific secondary modifications were not accomplished. Since the approach is of considerable scientific interest, providing as it might a thorough interrogation of a complete signalling pathway, it could yield insights relevant well beyond the horizon of plant research. The project will thus be followed continued as soon as alternative mass spectrometry resources have been found and proven to deliver the necessary information reliably.
