Studying small molecule-induced protein dynamics using high-resolution mass spectrometry

Abstract

Mass spectrometry (MS)-based proteomics has come a long way since mass spectrometry was first applied to investigate proteins. New technical solutions have emerged that allow for faster and more accurate mass spectra acquisition alongside artificial intelligence (AI)-assisted software for data evaluation and processing. With these advancements, it is now possible to explore an increasingly comprehensive part of the cellular proteome. Many cellular processes rely on complex dynamics of protein-protein interactions, relocations, regulation, and conformational changes of proteins. Some of these protein dynamics can be modulated by interactions with small molecules that are able to specifically target certain proteins, or often more specifically, enzymes. There are many MS-based proteomics approaches that already allow investigation of protein dynamics but only few methods have been established to investigate small molecule-protein interactions. The integration of different methods can be a key to gain relevant information on dynamic processes and small molecule-protein interactions. In this cumulative thesis, new, integrated MS-based methods were developed and applied to investigate a diverse range of research questions spanning Parkinson’s disease (PD) over Angelman syndrome (AS) to eukaryotic ribosome biogenesis. Across all three parts of this thesis, I integrated and optimized existing MS-based proteomics methods and successfully applied the developed approaches to elucidate dynamic processes. In this context, I investigated various scientific problems from complex environments such as the inhibition of the LRR kinases 1 and 2 and its impact on PPIs on proteome level, over dynamics of macromolecular machinery in the biogenesis of the 60S ribosome to the interaction of ubiquitin ligase E6AP with small molecule activators on a molecular level. The novel integrated methods are especially beneficial to investigate the interaction between small molecule activators or inhibitors and their target enzymes. The elucidation of these dynamics on a molecular level is important for our understanding of how enzyme dysfunction can lead to pathogenicity, and how it can be addressed by small molecules activators or inhibitors. This information is especially valuable regarding therapeutic applications of these small molecules. A good example is the vitamin-B12 derivative cyanocobalamin that we identified as activator of E6AP ubiquitin ligase activity in our study and that could prove beneficial in the therapy of patients with Angelman syndrome.