The application of mass spectrometry in the study of protein-lipid interactions

Résumé 0

Interactions between membrane proteins and their surrounding lipids play an integral role in coordinating membrane function. However, capturing and differentiating key lipid binding to membrane proteins within the complex and the dynamic membrane environment remains challenging for existing methods. Recent developments in native mass spectrometry have enabled non-covalent interactions of protein complexes to be maintained in the gas phase allowing critical membrane protein-lipid interactions in biological systems to be probed. In this thesis, native mass spectrometry is used throughout, sometimes coupled with liquid chromatography-mass spectrometry based lipidomics, to interrogate membrane proteins and their natural lipid environments. A protocol to control the degree of delipidation from membrane proteins was developed (Chapter 2). The resulting mass spectra revealed the target membrane proteins, MsbA and MdtK, in different lipid bound states, enabling the relationship between membrane proteins and neighbouring lipids to be observed. Next MS was used to characterise a novel detergent-free particle known as a lipodisq (Chapter 3). Lipodisqs encapsulate membrane proteins in their original lipid environment. By optimising lipodisq preparation and MS methodology, well-resolved MS spectra of rhodopsin and MdtK with bound lipids from their lipodisq preparations were obtained. Finally, MS was used in conjunction with cryo-EM to investigate a respiratory protein supercomplex (Chapter 4). MS not only provided mass information which assisted in the identification of the protein subunits and binding lipids, but also detected the non-covalent interactions between protein and other ligand molecules in the supercomplex system. Overall, the research reported in this thesis has contributed to broadening the applications of mass spectrometry in the emerging field of protein-lipid interactions.