Pioneering Mass Spectrometry-Based Footprinting to Reveal Human Membrane Protein Structures in Native Membranes and Tissues - Project Summary/Abstract Our research focuses on deciphering the higher-order structure (HOS) and function of human integral membrane proteins (IMPs) in their native cellular and tissue environments. IMPs are central to many biological processes, such as transport, catalysis, and signal transduction. They are the primary targets for 60% of FDA-approved drugs. However, their HOS and functional structural properties (e.g., conformational changes, ligand interactions, solvent accessibility, and dynamics) under physiological conditions remain poorly understood, particularly for the transmembrane (TM) regions. Traditional structural approaches (e.g., cryo-EM and crystallography) often require the extraction of IMPs from membranes using detergents, which disrupt their native conformations and limit the ability to study these proteins in their native state within living systems. Our hypothesis is that the membrane environment is crucial for maintaining the native structure of human IMPs, which is essential for regulating their physiological functions. Capturing the structural information of IMPs in their native settings provides a more accurate reflection of their functional states and more effectively guides drug development. To address this critical knowledge gap, our lab is pioneering innovative mass spectrometry (MS)-based footprinting techniques to capture the native states of IMPs in cells and tissues. MS-based footprinting is emerging as a powerful means to answer biological questions about IMPs by affording sufficient structural information on their dynamics, conformations, and interactions. However, major challenges—such as the unreactive and protected TM domains and the low footprinting efficiency in membrane environments—have hindered its biomedical application. We propose to design and improve novel footprinting methods that provide high coverage of IMPs in native settings. We will then apply these methods to various human IMPs (e.g., enzymes, transporters, and transducers) to investigate their structural properties and reveal drug interactions. Additionally, we propose to integrate these approaches with MS imaging (MSI), creating a novel paradigm—spatial structural proteomics—which will provide insights into IMPs' distribution, content, and HOS within their biological contexts, thereby revealing their roles in disease progression. Over the next five years, we will develop a suite of robust MS-based tools for investigating the HOS, spatial localization, and functional interactions of IMPs in their native environments. By combining structural biology, spatial structural proteomics, and data science, our research will pave the way for transformative biomedical applications, including the discovery of new therapeutics and a deeper understanding of IMP-associated diseases.
