Mass spectrometry (MS) has emerged as a powerful tool to study membrane protein complexes and protein-lipid interactions. Because they provide a precisely-defined lipid bilayer environment, lipoprotein Nanodiscs offer a promising cassette for membrane protein MS analysis. However, heterogeneous lipids create several potential challenges for native MS: additional spectral complexity, ambiguous assignments, and differing gas-phase behaviors. Here, we present strategies to address these challenges and streamline analysis of heterogeneous-lipid Nanodiscs. We show that using two lipids of similar mass limits the complexity of the spectra in heterogeneous Nanodiscs and that the lipid composition can be determined by using a dual Fourier transform approach to obtain the average lipid mass. Further, the relationship between gas-phase behavior, lipid composition, and instrumental polarity was investigated to determine the effects of lipid head group chemistry on Nanodisc dissociation mechanisms. These results provide unique mechanistic and methodological insights into characterization of complex and heterogeneous systems by mass spectrometry.
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