Advancing Protein Characterisation: Integrating Native, Top-Down, and Ion Mobility Mass Spectrometry for Biological Insight

Complex biological processes cannot take place without the help of proteins. Proteins perform essential tasks in our cells, primarily through interactions with other proteins, other biomolecules, or small molecules. The functionality of this sophisticated system is mainly based on the right protein being in the right place at the right time with the correct folding. Errors in this system are in many cases triggers of disease either directly or through downstream processes. Therefore, expanding the understanding of all involved processes, especially the initiating ones, and potential errors is essential in research. Two levels play an important role in proteins. The first level involves the amino acid sequence with all potential modifications and is called the primary structure of proteins. The second deals with folding, conformation, and interaction partners of proteins. The terms secondary, tertiary, and quaternary are often used here, or, in summary, higher-order structure. Many different techniques have been and continue to be used to analyse proteins, and all have their characteristic strengths and weaknesses. The technique that plays the main role in this thesis is mass spectrometry. In addition to determining the mass of the analyte, mass spectrometry can provide further interesting insights into protein characteristics. If proteins are analysed intact without prior digestion, and then fragmented into smaller pieces in the mass spectrometer, this is referred to as the top-down approach. This approach allows the elucidation of the primary structure as well as the localization of post-translational modifications and is therefore one of the most effective techniques for the characterization of proteins and proteoforms. However, not only proteoform identification plays an important role in the understanding of biological processes, but also their higher-order structure is essential. Since this higher-level structure is often determined by labile, non-covalent interactions, the measurement conditions must be adapted to obtain it. These efforts have given rise to the field of native mass spectrometry. Native mass spectrometry allows the transfer of proteins and complexes in their native conformation into the mass spectrometer. In combination with the top-down approach, also known as native top-down analysis, information on proteoform identity can then be linked to 3D structural information. Another technique used in combination with mass spectrometry for protein structure elucidation is ion mobility spectrometry. This provides an additional separation dimension and is particularly useful for investigating differences in the conformation or folding of proteins. In the following work, the techniques described were used either alone or in combination to contribute to the development of methods in the relevant fields. One aim is to make methods accessible to a wider range of users and to establish standards and controls. This requires collaborative initiatives, and this thesis includes the result of such an endeavour. In addition, work was done to improve existing methodologies, and a review was published that explains the basics for beginners in the field. The challenge of obtaining sufficient fragmentation in top-down protein analysis was also targeted in one subsection. The last part of the thesis presents an application of the methodologies for basic research in the field of Alzheimer's dementia. Here, various proteoforms of the amyloid β peptide play an essential role in the disease and its progression. Native mass spectrometry in combination with ion mobility was used to investigate the differences between these proteoforms and to better understand their interactions. In summary, this thesis aims to show the possibilities and opportunities of native mass spectrometry, top-down protein analysis, and ion mobility, and above all, their application in protein characterisation.