Structural investigation of protein(-RNA) assemblies by mass spectrometry

The study of the three-dimensional arrangement of proteins and RNA is of utmost importance for understanding the molecular details of their function. In this thesis, structural proteomics approaches in combination with mass spectrometry were applied to the study of a set of protein(-RNA) complexes of increasing complexity and size. Hydroxyl radical based footprinting (HRF), UV-induced cross-linking and lysine directed protein-protein chemical cross-linking in combination with mass spectrometry were applied to study of the aggregation of the human prion protein, the activation of the DEAH box helicase Prp43 by its effector protein Ntr1 and the determination of protein interaction networks of human spliceosomal complexes. The results helped identify amino acid residues of the solvent protected core in the amyloid fibrils and propose a model for the binding and activation of Prp43 to Ntr1. The large scale protein cross-linking studies resulted in more than 500 identified proximal contacts among different spliceosomal proteins and in more than 25 identified RNA contacts in U snRNPs, which constitutes the largest number identified in a spliceosomal RNP using mass spectrometry to date.