Integral Membrane Proteins and Lipids Ejected from the Membranes of Native Tissues

Our overall objective is to make it possible to study membrane proteins in their native context. We will implement the ejection of mammalian membrane protein assemblies intact from their native tissue environments (without first dissolving the bilayer) for interrogation using new mass spectrometry (MS) approaches. This will enable us to connect small-molecule regulation of membrane proteins to the true modified status of the protein, which may include oligomerisation, post-translational modifications (PTMs) and clustering of the protein with itself or other biomolecules. In so doing, we will transition MS of membrane protein assemblies from an approach that relies on using detergent to extract individual components of assemblies to one capable of analysing those components as an ensemble. It will involve directly ejecting intact assemblies from the membranes of human cells and tissues in healthy and diseased states. Why is this programme needed? Despite the development of numerous high-throughput and expansive genomic, transcriptomic, and proteomic methods to identify the underpinnings of disease, it is currently not possible to define whether, or how, PTMs and single-nucleotide polymorphisms (SNPs) impact membrane proteins' interactions with the array of lipids, cofactors and other potential interactors they encounter in cell membranes. Maintaining such interactions - and defining how membrane protein PTMs and SNPs affect interactions with other biomolecules in the membrane - is, however, critical for informing therapeutic choices. Why now? Because we have just discovered how to preserve a signalling pathway across a lipid bilayer and have new powerful MS technology ready to uncover new signalling pathways and propose interventions. State-of-the-art SoLVe methodology: Motivated by the desire to conduct research in ever-more-native environments, we demonstrated that we could eject proteins directly from bacterial and mitochondrial membranes without recourse to chemical (detergent) intervention. We achieved this by applying short Sonication pulses to Lipid Vesicles (SoLVe) and introducing vesicle fragments directly into a mass spectrometer. Using this approach, we uncovered differences in the subunit composition of bacterial complexes (c.f. over-expressed proteins in micelles), and unexpected lipid and cofactor binding. In our most recent application of SoLVe, we captured bovine rhodopsin signalling to its downstream effectors across native membrane fragments. Together with novel MS developments, in which we have begun to link small molecules with membrane proteins, we have amassed several critical technological advances and are now poised to implement further MS breakthroughs. At the time of our first SoLVe publication, in which we ejected complexes from bovine mitochondria, we were unable to sequence the ejected proteins and so used co-factor binding and subunit masses to inform our assignments. Questions raised about the reliability of this approach prompted us to continue collaborating with Thermo Fisher to develop further the Tribrid technology. We can now apply top-down sequencing to intact complexes such that we can confirm their identity and link small molecule binding to the PTM status of proteins in true native environments. Continued development with Thermo Fisher has involved close collaboration - first to troubleshoot the ejection of membrane protein complexes from detergent micelles and then to develop ejection from membranes. We have filed two joint patent applications and secured access to the prototype instrument that we helped design, to be installed in our laboratory in the summer of 2022. Equipped with an infra-red laser, allowing us to synchronise laser exposure time and power, the instrument enables us to fine-tune the ejection of assemblies from membrane environments.