Design and Synthesis of Hetero-Bicephalic Detergents for Native Mass Spectrometry of Membrane Proteins

Native mass spectrometry is playing a central role in characterizing the noncovalent interactions of membrane proteins, enabling the discovery of lipids that regulate membrane protein function and modulate interactions with other molecules. The use of specialized charge-reducing detergents and/or molecular additives is critical to mitigate overactivation during native MS to facilitate proper analysis. Here, we present the design and synthesis of a new class of dual-headgroup detergents featuring two distinct hydrophilic moieties. One headgroup, the spermine moiety, is designed to promote lower charge states of membrane proteins, thereby enhancing the preservation of labile complexes in the mass spectrometer. The other hydrophilic headgroup─either a maltoside or a tetraethylene glycol moiety─is commonly found in traditional detergents used for the extraction and solubilization of membrane protein complexes. Using a diverse set of membrane proteins varying in size, topology, and oligomeric state, we demonstrate that these novel dual-headgroup detergents exhibit exceptional charge-reducing properties, particularly when introduced to membrane proteins solubilized in different detergents. Additionally, we highlight their effectiveness in preserving the complex of a G-protein-coupled receptor with a nanobody that selectively binds to its active state. Collectively, these high-performance detergents advance the rational design of detergents for native MS and expand the capabilities for studying membrane proteins in diverse biochemical environments.