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Native ion mobility-mass spectrometry (IM-MS) typically introduces protein ions into the gas phase through nano-elecrospray ionization (nESI). Many nESI setups have mobile stages for tuning the ion signal and extent of co-solute and salt adduction. However, tuning the position of the emitter capillary in nESI can have unintended downstream consequences for collision-induced unfolding or collision-induced dissociation (CIU/D) experiments. Here we show that relatively small variations in the nESI emitter position can shift the midpoint potential of CID breakdown curves and CIU transitions; by as much as 8 V on commercial instruments. 

Viroporins are small viral ion channels that play important roles in the viral infection cycle and are proven antiviral drug targets. Matrix protein 2 from influenza A (AM2) is the best-characterized viroporin, and the current paradigm is that AM2 forms monodisperse tetramers. Here, we used native mass spectrometry and other techniques to characterize the oligomeric state of both the full-length and transmembrane (TM) domain of AM2 in a variety of different pH and detergent conditions. Unexpectedly, we discovered that AM2 formed a range of different oligomeric complexes that were strongly influenced by the local chemical environment. Native mass spectrometry of AM2 in nanodiscs with different lipids showed that lipids also affected the oligomeric states of AM2. Finally, nanodiscs uniquely enabled the measurement of amantadine binding stoichiometries to AM2 in the intact lipid bilayer. These unexpected results reveal that AM2 can form a wider range of oligomeric states than previously thought possible, which may provide new potential mechanisms of influenza pathology and pharmacology. 

Native electrospray mass spectrometry is a powerful method for determining the native stoichiometry of many polydisperse multi-subunit biological complexes, including multi-subunit protein complexes and lipid-bound transmembrane proteins. However, when polydispersity results from incorporation of multiple copies of two or more different subunits, it can be difficult to analyze subunit stoichiometry using conventional mass spectrometry analysis methods, especially when m / z distributions for different charge states overlap in the mass spectrum. It was recently demonstrated by Marty and co-workers (K. K. Hoi, et al. , Anal. Chem. , 2016, 88 , 6199–6204) that Fourier Transform (FT)-based methods can determine the bulk average lipid composition of protein-lipid Nanodiscs assembled with two different lipids, but a detailed statistical description of the composition of more general polydisperse two-subunit populations is still difficult to achieve. This results from the vast number of ways in which the two types of subunit can be distributed within the analyte ensemble. Here, we present a theoretical description of three common classes of heterogeneity for mixed-subunit analytes and demonstrate how to differentiate and analyze them using mass spectrometry and FT methods. First, we first describe FT-based analysis of mass spectra corresponding to simple superpositions, convolutions, and multinomial distributions for two or more different subunit types using model data sets. We then apply these principles with real samples, including mixtures of single-lipid Nanodiscs in the same solution (superposition), mixed-lipid Nanodiscs and copolymers (convolutions), and isotope distribution for ubiquitin (multinomial distribution). This classification scheme and the FT method used to study these analyte classes should be broadly useful in mass spectrometry as well as other techniques where overlapping, periodic signals arising from analyte mixtures are common.