Mechanistic information about how gaseous ions are formed from charged droplets has been difficult to establish because direct observation of nanodrops in a size range relevant to gaseous macromolecular ion formation by optical or traditional mass spectrometry methods is challenging owing to their small size and heterogeneity. Here, the mass and charge of individual aqueous nanodrops between 1-10 MDa (15-32 nm diameter) with ∼50-300 charges are dynamically monitored for 1 s using charge detection mass spectrometry. Discrete losses of minimally solvated singly charged ions occur, marking the first direct observation of ion emission from aqueous nanodrops in late stages of droplet evaporation relevant to macromolecular ion formation in native mass spectrometry. Nanodrop charge depends on the identity of constituent ions, with pure water nanodrops charged slightly above the Rayleigh limit and aqueous solutions containing alkali metal ions charged progressively below the Rayleigh limit with increasing cation size. MS2 capsid ions (∼3.5 MDa; ∼27 nm diameter) are more highly charged from aqueous ammonium acetate than from its biochemically preferred, 100 mM NaCl/10 mM Na phosphate solution, consistent with ion emission reducing the nanodrop and resulting capsid charge. The extent of charging indicates that the capsid partially collapses inside the nanodrops prior to the charging and formation of the dehydrated gaseous ions. These results demonstrate that ion emission can affect macromolecular charging and that conformational changes to macromolecular structure can occur in nanodrops prior to the formation of naked gaseous ions.
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