Samantha Shepherd
Yale University
Detection Dynamic Assemblies of Membrane Proteins and Lipids on Cellular Membranes
Ultrastructural organization of proteins and lipids in cellular membranes is fundamental to all membrane-associated signaling. Detecting these dynamic, heteromeric, and transient assemblies necessitates experimental approaches with dynamic detection range to detect these assembly states and unbiased molecular resolution to determine their composition. Further, compositions and properties of the membranes often play key role in their biogenesis, demanding analysis directly from the lipid membrane. Addressing this, we have combined native mass spectrometric (nMS) with single ion measurements and top-down proteomics to deliver an experimental platform that can detect macromolecular assemblies of membrane proteins and lipids directly from lipid membrane. Our platform relies on three key technological advancements; first, we discovered a gas phase chemistry that enables flying intact lipid vesicle into MS and gently ablate embedded/associated protein-lipid complexes for their downstream MS detection. Next, we combine this with charge detection-based single ion detection approaches to directly detect heterogeneous and polydisperse assembly states of multiprotein-lipid signaling complexes formed on membranes. Finally, integrating these with top-down proteomics enables unambiguous identification of molecular architecture and composition of the detected complexes. Taking examples of ion channels, neuronal SNAREs, peripheral membrane kinases, we demonstrate how this platform offers a broadly applicable experimental avenue to capture dynamic and heterogeneous macromolecular assembly states membrane proteins and lipids formed on the membrane.
Ultrastructural organization of proteins and lipids in cellular membranes is fundamental to all membrane-associated signaling. Detecting these dynamic, heteromeric, and transient assemblies necessitates experimental approaches with dynamic detection range to detect these assembly states and unbiased molecular resolution to determine their composition. Further, compositions and properties of the membranes often play key role in their biogenesis, demanding analysis directly from the lipid membrane. Addressing this, we have combined native mass spectrometric (nMS) with single ion measurements and top-down proteomics to deliver an experimental platform that can detect macromolecular assemblies of membrane proteins and lipids directly from lipid membrane. Our platform relies on three key technological advancements; first, we discovered a gas phase chemistry that enables flying intact lipid vesicle into MS and gently ablate embedded/associated protein-lipid complexes for their downstream MS detection. Next, we combine this with charge detection-based single ion detection approaches to directly detect heterogeneous and polydisperse assembly states of multiprotein-lipid signaling complexes formed on membranes. Finally, integrating these with top-down proteomics enables unambiguous identification of molecular architecture and composition of the detected complexes. Taking examples of ion channels, neuronal SNAREs, peripheral membrane kinases, we demonstrate how this platform offers a broadly applicable experimental avenue to capture dynamic and heterogeneous macromolecular assembly states membrane proteins and lipids formed on the membrane.
