Investigation of Protein-Ligand Complexes by Native Mass Spectrometry and Ion Mobility-Mass Spectrometry

by Melanie Gth

Institution: Freie Universitt Berlin
Year: 2017
Posted: 02/01/2018
Record ID: 2154276
Full text PDF: http://edocs.fu-berlin.de/diss/receive/FUDISS_thesis_000000106075


Intermolecular interactions of proteins with each other or with other molecules play a key role in all processes in living organisms. Therefore, proteins represent important therapeutic targets and their structural elucidation is essential for the development of new drugs. Native mass spectrometry (MS) is an attractive tool for the investigation of proteins and their complexes. Molecules are ionized gently and transferred from solution into the gas phase with the aim to maintain inter- and intramolecular non-covalent interactions and the three-dimensional structure. By measuring the mass-to-charge ratio of the ionic species, information on mass, charge, and stoichiometry can be obtained. Native MS is readily compatible with other gas-phase techniques, such as ion mobility spectrometry (IMS) and the combination of both methods, so-called ion mobility-mass spectrometry (IM-MS), provides further structural information on the overall size and shape of a molecule. In this thesis, protein-ligand complexes were investigated using native MS and IM-MS with the aim of evaluating their potential for application in high-throughput analysis for drug discovery. First, native MS was used to elucidate the influence of the standard solvent dimethyl sulfoxide (DMSO) on the protein gas-phase structure and protein-ligand affinity. It was shown that the protein charge-state distribution and likely the gas-phase structure is altered depending on the DMSO concentration. In addition, the protein-ligand affinity decreased with increasing DMSO levels. In a second study, the potential of native MS for fragment-based drug discovery was evaluated. This approach is based on the screening of small molecular fragments against a target and promises higher hit rates and smaller library sizes compared to standard high-throughput screening. Data on four protein systems showed that native MS currently presents a medium- to low-throughput method but can provide valuable insights into protein-ligand interactions that are inaccessible by other techniques. In combination with IMS, the influence of a proteins microenvironment on its gas-phase structure was investigated. To do so, crown-ether molecules were attached non-covalently to microsolvate positively charged protein side chains, preventing them from collapsing onto the protein backbone. Using tandem MS and IM-MS, the crown-ether binding sites were identified and it was shown that specific side chains stabilize the gas-phase structure even without crown-ether binding. In the last study, IM-MS was successfully tested as a tool for conformational screening of protein-peptide complexes. Only subtle structural differences between the complexes were observed, and a further investigation by collision-induced unfolding and collision-induced dissociation displayed differences in complex stability and unfolding behavior. In summary, native MS and IM-MS are valuable tools for the characterization of protein-ligand interactions. Currently, the methods are limited to a small number of samples, but ongoing