Integrative Mass Spectrometry Approaches to Monitor Protein Structures, Modifications, and Interactions

This thesis illustrates the current standing of mass spectrometry (MS) in molecular and structural biology. The primary aim of the herein described research is to facilitate protein characterization by combining mass spectrometric methods among each other and with complementary analytical strategies. In Chapter 1, an overview of mass spectrometric methods that are most useful for studying the organization of protein assemblies is provided. Based on recent examples from literature, it illustrated how these MS methods can be integrated with other molecular and structural biology techniques. In Chapter 2, factors confining the obtainable mass resolution in native MS are identified. It is shown that mass resolution is mainly limited by inefficient analyte desolvation, i.e., the incomplete disruption of unwanted non-covalent analyte-small molecule adducts. It is furthermore discussed that a higher mass resolution is primarily required for native MS analysis of species with very small mass differences, e.g., protein complexes carrying different kinds and numbers of post-translational modifications. In Chapter 3, the benefits of high-resolution native MS are demonstrated by studying phosphorylation reactions in binary protein-ligand and protein-protein systems up to 155 kDa. Complementation of high-resolution native MS with bottom-up and top-down proteomics is shown to enable the characterization of enzymatic protein phosphorylation with respect to concurring non-covalent interactions, overall reaction kinetics and the sequence and completeness of residue-specific modification events. In Chapter 4, the scope of this approach is further extended by inclusion of cross-linking MS and ion mobility spectrometry MS to analyze the interdependence of multisite phosphorylation, interaction dynamics and conformational changes during the interplay of Polo-like kinase 1, Aurora kinase A and Bora. Uniting the data from all applied MS methods yields a mechanistic model of this three-protein reaction, illustrating that integrative MS-based approaches can provide a dynamic view on protein structures and interactions. This analytical angle is complementary to X-ray crystallography and cryo-electron microscopy data, suggesting a unique niche of MS in structural biology. In Chapter 5, potential functions of MS in hybrid structural biology approaches are further investigated. The analyses of the LRP1-RAP and KaiCBA complexes illustrate the value of complementing traditional biochemical and biophysical techniques with native MS to assess assembly states as well as condition-dependent association and dissociation processes. Both studies also show that cross-linking MS renders valuable insights into protein conformations and binding interfaces, especially when high-resolution structural characterization is not feasible. In all, it is demonstrated that MS is able to illuminate various facets of protein structures, modifications and interactions. Therefore, MS will continue to play a vital role in exploring the structural and chemical principles of biological processes