Hierarchical organization of membrane proteins and lipids on organellar membranes is fundamental to membrane-associated signaling. These associations are often templated by weak lipid-protein interactions that form heterogeneous, polydisperse assembly states of protein-lipid signalosomes. Capturing these assemblies demands experimental platforms with dynamic molecular detection range to detect these polydisperse assembly states with precise molecular resolution, directly from the membranes. Addressing this, we present a single-ion native mass spectrometry platform that can capture both the membrane-associated macromolecular assembly states of integral and peripheral membrane proteins, as well as the weak interactions between proteins and lipids that template these states, directly from customizable membranes mimicking organellar surfaces. Further, the single-ion detection provides key molecular resolution to unambiguously identify the molecular constituents and their stoichiometry in the observed polydisperse and heterogeneous assembly states. Taking several examples of integral and peripheral membrane proteins, we demonstrate how this platform can capture membrane-associated macromolecular complexes held through weak protein-lipid interactions. The application of this platform to synaptic vesicle proteins reveals how specific lipid binding templates key functional assembly states of SNAREs and SNARE-associated protein assemblies on the membrane. Through functional assays, we demonstrate how these lipid bindings regulate protein assemblies at synaptic membranes. In an exciting application of this platform, we discovered how specific rare disease mutations, which cause neurocognitive disorders, impair the functional assembly states of SNAREs and associated protein assemblies. Separately, applying the platform to a peripheral membrane kinase, we demonstrate how weak-affinity interactions with plasma membrane lipids recruit the kinase and amplify the kinase output by increasing the local membrane-bound concentration. Together, we present a technological platform for determining heterogeneous, macroscopic organization states of membrane proteins and lipids directly from a customizable lipid membrane.
Shepherd et al. (Sun,) studied this question.