Probing the Composition, Assembly and Activity of Protein Molecular Machines using Native Mass Spectrometry

Native mass spectrometry and mass spectrometry in general, are powerful analytical tools for studying proteins and protein complexes. Native mass spectrometry may provide accurate mass measurements of large macromolecular assemblies enabling the investigation of their composition and stoichiometry. This composition can be unraveled further by the use of bottom-up and top down MS and several other techniques that use mass spectrometry as a readout, for instance to map interactions in protein complexes (crosslinking MS), detect conformational changes (ion-mobility MS) and discover ligand binding sites (HDX-MS). Several (simple) mass measurements can provide a wealth of information making mass spectrometry one of the, if not the, most versatile analytical chemical techniques. This thesis describes both the development and application of the mass spectrometry toolbox for studying biomolecular assemblies. Although there is an emphasis on native mass spectrometry and in particular its use to study very large protein complexes with high resolution, the majority of the findings described here can benefit studies of other proteins and protein complexes as well