Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer

Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative understanding of these processes requires both detections of oligomeric organization of IMPs and lipids directly from intact membranes and determination of key membrane components and properties that regulate them. Addressing this, we have developed a platform that enables native mass spectrometry (nMS) analysis of IMP–lipid complexes directly from intact and customizable lipid membranes. Both the lipid composition and membrane properties (such as curvature, tension, and fluidity) of these bilayers can be precisely customized to a target membrane. Subsequent direct nMS analysis of these intact proteolipid vesicles can yield the oligomeric states of the embedded IMPs, identify bound lipids, and determine the membrane properties that can regulate the observed IMP–lipid organization. Applying this method, we show how lipid binding regulates neurotransmitter release and how membrane composition regulates the functional oligomeric state of a transporter. Editor summary: A native-mass-spectrometry-based approach analyzes integral membrane protein–lipid complexes directly from near-physiological membrane conditions, providing information about protein oligomeric states, lipid identities, and membrane properties.