Mass spectroscopy - Research projects Fucini

Dissecting ribosome composition and stability using Mass spectrometry

Ribosome Stability
In collaboration with the group of Prof. Carol Robinson and Prof Christopher Dobson, we have projected intact Escherichia coli ribosomes into the gas phase of a mass spectrometer by means of nanoflow electrospray techniques.
Species with mass/charge ratios in excess of 20,000 were detected at the level of individual ions by using time-of-flight analysis. Once in the gas phase the stability of intact ribosomes was investigated and found to increase as a result of cross-linking ribosomal proteins to the rRNA. By lowering the Mg(2+) concentration in solutions containing ribosomes the particles were found to dissociate into 30S and 50S subunits. The resolution of the charge states in the spectrum of the 30S subunit enabled its mass to be determined as 852,187 +/- 3,918 Da, a value within 0.6% of that calculated from the individual proteins and the 16S RNA. Further dissociation into smaller macromolecular complexes and then individual proteins could be induced by subjecting the particles to increasingly energetic gas phase collisions. The ease with which proteins dissociated from the intact species was found to be related to their known interactions in the ribosome particle. The results show that emerging mass spectrometric techniques can be used to characterize a fully functional biological assembly as well as its isolated components.

Plant Ribosome composition
In collaboration with the group of Dr. Johan Gobom (Lehrach Department of MPI-MG) and Joachim Klose (Human Genetics Department at the Charité, Berlin), we have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to analyse the cytoplasmic 80S ribosomes from the model flowering plant Arabidopsis thaliana. In addition to the identification of the large majority of the protein components of the cytoplasmic 80S ribosome, we also observed a large heterogeneity within the ribosome, both in terms of the isoforms present as well as putative post-translational modifications.
Furthermore, we were able to identify a number of non-ribosomal proteins that pellet or co-migrate with the 80S ribosome. Among them, the nascent polypeptide associated complex (NAC) and RACK1, a protein found to promote efficient translation in yeast. All the data is accessible directly from the Gabi website.