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Solution additives for supercharging proteins beyond the theoretical maximum proton-transfer limit in electrospray ionization mass spectrometry.

Electrospray ionization (ESI) can be used to form multiply charged ions, which is beneficial for obtaining accurate molecular weight values and sequence information for proteins in many types of mass spectrometry (MS) measurements. The effects of surface tension, dipole moment, and dielectric constant (properties that have been implicated in the enhancement of protein charging in ESI) of 14 solution additives on the protein charge-state distributions that are formed in ESI for three proteins (ubiquitin, cytochrome c, and carbonic anhydrase II) were investigated. We discovered that two solution additives, propylene carbonate and ethylene carbonate, which have high surface tension and dipole moment values, can be used to form significantly higher protonation states of these three common test proteins than have been reported previously by use of other methods and additives, such as that obtained with benchmark "supercharging" additives (m-nitrobenzyl alcohol and sulfolane). By use of ethylene carbonate and propylene carbonate, nearly the entire charge state distributions of protonated ubiquitin and cytochrome c ions can be shifted to higher charge states than the theoretical maximum protein charging protonation limit in ESI that is predicted on the basis of proton-transfer reactivity. Ethylene carbonate, propylene carbonate, o-nitroanisole, m-nitrobenzyl alcohol, and sulfolane are all effective at increasing the extent of charging of deprotonated protein ions in negative ionization mode ESI-MS experiments. This indicates that physicochemical properties that are independent of polarity (e.g., surface tension) can be responsible for supercharging and should not be excluded without additional information. In addition, these data indicate that nonvolatile additives with high dipole moments are not necessarily sufficient to supercharge proteins. Overall, these results suggest that it should be possible to discover new additives that increase protein charging even further.