Project Summary The overall objective of this project is to develop new microfluidic devices capable of producing lipid nanodiscs (NDs) having detailed and tunable compositions, and utilize these NDs as vehicles for improved structural mass spectrometry (SMS) and proteomics assays of membrane proteins (MPs). Despite positions of prominence in both biochemistry and the pharmaceutical sciences, our understanding of MPs lags significantly behind our knowledge of soluble proteins and their functional complexes. This has led to a critical imbalance, where MPs, which account for greater than 60% of drug targets, account for ~3% of the structural entries in the protein data bank (PDB). As a result, MP-targeted drug discovery is slowed, and treatment strategies go undiscovered. To bridge this gap, we propose the development of monolithic microfluidic tools capable of producing NDs over a wide range of lipid compositions, having narrow size distributions. We will then immediately deploy these tailored NDs to study the structure and biophysics of cytochrome P450 (CYP), a monotopic membrane protein found in all kingdoms of life, and responsible for the metabolism of most small molecule drugs in humans. The means by which CYPs are able to metabolize such a wide range of xenobiotics, and the role that cellular membranes play in the apparent structural plasticity of CYPs, remains unknown. We will develop ion mobility-mass spectrometry (IM-MS) and collision induced unfolding (CIU) methods that, in our preliminary data, have been able to detect the first evidence for structural shifts in CYP as a function of its local lipid environment. Our efforts will further extend to build NDs into robust extraction devices for improved coverage of the membrane proteome. To complement our IM-MS workflows, we will also deploy and optimize chemical cross-linking (CXL) approaches for use with MPs housed within NDs. The tools discussed above will then be applied to the study of the MP complexes within the mitochondrial membrane. Specifically, we will target the protein assemblies associated with the electron transport chain (ETC), as well as the vast array of protein-protein interactions (PPIs) that have either been observed or predicted for CYP. Our work will seek to provide new structural information on complexes that have been studied extensively in the past (e.g. ATP Synthases), as well as seek to discover new mitochondrial MP complexes, with potentially broad implications for cellular function.