Membrane proteins play critical biochemical roles but remain challenging to study. Recently, native or nondenaturing mass spectrometry (MS) has made great strides in characterizing membrane protein interactions. However, conventional native MS relies on detergent micelles, which may disrupt natural interactions. Lipoprotein nanodiscs provide a platform to present membrane proteins for native MS within a lipid bilayer environment, but prior native MS of membrane proteins in nanodiscs has been limited by the intermediate stability of nanodiscs. It is difficult to eject membrane proteins from nanodiscs for native MS but also difficult to retain intact nanodisc complexes with membrane proteins inside. Here, we employed chemical reagents that modulate the charge acquired during electrospray ionization (ESI). By modulating ESI conditions, we could either eject the membrane protein complex with few bound lipids or capture the intact membrane protein nanodisc complex-allowing measurement of membrane protein oligomeric state within an intact lipid bilayer environment. The dramatic differences in the stability of nanodiscs under different ESI conditions opens new applications for native MS of nanodiscs.
Lipoprotein nanodiscs are ideally suited for native mass spectrometry because they provide a relatively monodisperse nanoscale lipid bilayer environment for delivering membrane proteins into the gas phase. However, native mass spectrometry of nanodiscs produces complex spectra that can be challenging to assign unambiguously. To simplify interpretation of nanodisc spectra, we engineered a series of mutant membrane scaffold proteins (MSP) that do not affect nanodisc formation but shift the masses of nanodiscs in a controllable way, eliminating isobaric interference from the lipids. Moreover, by mixing two different belts before assembly, the stoichiometry of MSP is encoded in the peak shape, which allows the stoichiometry to be assigned unambiguously from a single spectrum. Finally, we demonstrate the use of mixed belt nanodiscs with embedded membrane proteins to confirm the dissociation of MSP prior to desolvation.
Noncovalent
interactions between biomolecules are critical to their
activity. Native mass spectrometry (MS) has enabled characterization
of these interactions by preserving noncovalent assemblies for mass
analysis, including protein–ligand and protein–protein
complexes for a wide range of soluble and membrane proteins. Recent
advances in native MS of lipoprotein nanodiscs have also allowed characterization
of antimicrobial peptides and membrane proteins embedded in intact
lipid bilayers. However, conventional native electrospray ionization
(ESI) can disrupt labile interactions. To stabilize macromolecular
complexes for native MS, charge reducing reagents can be added to
the solution prior to ESI, such as triethylamine, trimethylamine oxide,
and imidazole. Lowering the charge acquired during ESI reduces Coulombic
repulsion that leads to dissociation, and charge reduction reagents
may also lower the internal energy of the ions through evaporative
cooling. Here, we tested a range of imidazole derivatives to discover
improved charge reducing reagents and to determine how their chemical
properties influence charge reduction efficacy. We measured their
effects on a soluble protein complex, a membrane protein complex in
detergent, and lipoprotein nanodiscs with and without embedded peptides,
and used computational chemistry to understand the observed charge-reduction
behavior. Together, our data revealed that hydrophobic substituents
at the 2 position on imidazole can significantly improve both charge
reduction and gas-phase stability over existing reagents. These new
imidazole derivatives will be immediately beneficial for a range of
native MS applications and provide chemical principles to guide development
of novel charge reducing reagents.
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