A Quantitative Native Mass Spectrometry Platform for Deconstructing Hierarchical Organization of Membrane Proteins and Lipids

HYPOTHESIS: Hierarchical organization of protein and lipids at the cell membrane is essential for regulating a myriad of cellular signaling. The lateral heterogeneity of the biological membrane, both in terms of its lipid composition, as well as biophysical properties such as lateral pressure, curvature, fluidity has been shown to play regulatory role towards maintaining these assemblies of membrane proteins. This in turn imposes a challenging experimental prerequisite towards studying macromolecular assemblies of proteins and lipids at the membrane. It demands experimental approaches that (1) can unambiguously identify the associated proteins and lipids, along with their oligomeric states and binding stoichiometry, and (2) can be directly applied to a lipid-bilayer environments that mimics the lipid composition and critical biophysical properties of a target physiological membrane. METHODS: Addressing this need, we have developed lipid vesicle native mass spectrometry (nMS). This comprehensive tunable in vitro platform enables us to construct lipid bilayer, mimicking a target physiological membrane in terms of its composition and biophysical properties and directly subject it to nMS and MS/MS analysis to determine the oligomeric organization of the proteins present, identity of the bound lipids, and their binding stoichiometry; all directly from the membrane. PRELIMINARY DATA: First, to establish the broad applicability of our platform we chose a diverse set of membrane proteins with their oligomeric states ranging from monomer to pentamer, masses 12kDa-226kDa, and number transmembrane-helices 1-30. We further reconstituted them in a diverse set of lipid vesicles that are mimics of the eukaryotic ER, PM, Mitochondria, Golgi, and bacterial plasma membranes, as well as in vesicles of specific membrane curvature. In each of these cases, by directly subjecting these native-like vesicles to nMS we can directly determine the physiological oligomeric state of the proteins in the bilayer. Further, using the top-down MS/MS capability of our platform, we can unambiguously determine which of the membrane lipid associate with target membrane protein. Having established the broad and general applicability of our platform to study membrane proteins directly from a bilayer that can be customized to represent a target physiological membrane, we focused on applying this to two specific problems. First, we applied our approach to synaptic vesicle proteins and we show that through specific lipid binding VAMP2 induces clustering of specific lipid around its transmembrane and regulates the temporal scale of fusions. Next, applying our platform to a sugar transporter protein we quantitatively show that increase in cardiolipin % in the membrane leads to direct increase in functional oligomeric population. CONCLUSION: Our work presents a broad and generally applicable nMS platform that enables quantitative determination of molecular organization of protein and lipids directly from lipid membranes of desired composition and characteristics, identify of the specifically bound lipids, and determine how these specific bindings in turn regulate protein oligomerization. © FASEB.