Fourier Analysis Method for Analyzing Highly Congested Mass Spectra of Ion Populations with Repeated Subunits.

Highly heterogeneous samples that are difficult to resolve chromatographically arise in many contexts, including hetero-oligomeric protein assemblies, chaperone-target and protein-lipid assemblies, and long-chain polymers. Native mass spectrometry has emerged as a powerful tool to probe the stoichiometry and structure of biomolecular ion complexes, including megadalton-sized assemblies and assemblies with dozens of subunits. However, mass spectra of these ions are often highly congested, obfuscating determination of charge state, total mass, or subunit mass with conventional analysis methods. Here, we present a fast Fourier transform-based algorithm that can be used to deconvolve highly congested mass spectra for heterogeneous ion populations with repeated subunits. The method is parameter-free and requires no initial guesses of charge states, total mass, or subunit mass. To demonstrate a range of applications, the method is applied to ubiquitin with multiple adductions of sodium and potassium, single and mixed polymers, and self-assembled native protein-lipid complexes (Nanodiscs). The algorithm facilitates identification of the charge states, subunit mass, and charge-state specific total mass distribution present in the ion population. Results from application of the algorithm to these analytes include the first reported mass spectra and lipid stoichiometries of intact Nanodiscs containing lipid-raft associated sphingomyelin. Advantages to using this method with ion assemblies that have undergone minimal gas-phase collisional "clean-up" to retain native-like stoichiometries are discussed.