Elucidating the role of detergents, lipids and post-translational modifications in membrane protein stability by mass spectrometry

Mass spectrometry in combination with ion mobility are utilised to characterise the stability of membrane proteins in the gas phase and to elucidate protein-lipid interactions. Detergents are most commonly used to transport membrane proteins from solution into the gas phase. Using a set of membrane proteins solubilised in several detergents, an ionisation mechanism of membrane protein-detergent complexes is proposed. This suggests that detergents serve as charge carriers in both positive and negative polarity. A better understanding of the underlying mechanism allows the identification of detergents that are removed at low energies and reduce Coulombic repulsion. The activation energy and charge density are found to be important parameters that dictate the stability of membrane proteins in the gas phase, allowing conditions to be found that preserve native-like conformations. The stability of membrane proteins in the gas phase is also influenced by bound lipids. Methods to identify endogenous lipids directly from membrane protein-lipid complexes were developed. The stability provided by lipid binding is quantified allowing the identification of lipids that enhance the stability of particular membrane proteins. Ionic protein-lipid interactions are investigated in the context of variability in pH. Protonation of acidic residues plays an important role in selectivity of lipid binding and enhances the stability at low pH of active conformations present in physiological conditions. The methods developed are used to determine the oligomeric state of cytochrome c oxidase, pushing the boundaries of what is possible with non-denaturing mass spectrometry to the study of highly complex mammalian membrane proteins. By preserving non-covalent interactions in the gas phase evidence of equilibrium between monomeric and dimeric species of cytochrome c oxidase is found. Mass deviations can be attributed to a large lipid entity suggesting the role of lipids in stabilising the native oligomeric state. Using conventional mass spectrometry methods new lipids and phosphorylation sites are identified. Acetylation sites that have not been reported previously were located in close proximity to the dimeric interface, suggesting a potential role in stabilising this large dimeric interface.