Search

Summary Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane-mimetic systems at the single-particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterizing the particle size, sample purity, and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane-protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers—in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation., Graphical Abstract, Highlights • We introduce a label-free, single molecule approach for membrane-protein characterization • Mass photometry quantifies membrane proteins in different membrane-mimetic systems • MP reveals carrier and protein heterogeneity • It helps distinguish different functional states of membrane proteins, The Bigger Picture Membrane proteins are some of the most important biological molecules, carrying out vital functions and being frequent drug targets. Yet, preferring lipid environments and so requiring solubilization, they are challenging to study. Here, we show that mass photometry can characterize the heterogeneity of membrane proteins and the carriers in which they are solubilized. It can also distinguish different functional states of membrane proteins. Our approach thus opens the door to more comprehensive studies of function, structure, and interaction of these critical proteins in their native membrane environment at the single-molecule level., Membrane proteins are important in cell signaling and disease. They are also difficult to study as they require solubilization from lipids by membrane-mimetic systems. We show that mass photometry can facilitate the study of membrane proteins in various mimetic systems. With this method, we can distinguish different oligomeric and functional states of membrane proteins, opening the door for in vitro functional studies, structural characterization, and protein-protein interaction analysis of membrane proteins.