Solvent Leveling Explains Supercharging in Electrospray Ionization Mass Spectrometry

Supplementing standard electrospray ionization (ESI) solvents with specific low-volatility organic compounds (e.g., sulfolane or any positional isomer of nitrobenzyl alcohol) increases biomolecular analyte charge for mass spectrometry in the phenomenon known as supercharging. Controversial mechanisms responsible for increasing charge are considered, and the data is found to correlate highly to solvent leveling; i.e., protonated solvent is the strongest acid in a solution because any stronger acid simply dissociates to protonate more solvent. Hence, the recipe for increasing charge in positive ion mode is to make the protonated solvent into a stronger acid (equivalent to reducing the neutral solvent's basicity). That change is accomplished by adding involatile, weak bases to the solvent. A secondary effect of weak base additives is to suppress the solution-phase ionization of weak acid residues; e.g., reducing opposite charging. Here the abilities of analogous compounds to increase or decrease charging in positive ion mode ESI are predicted from experimentally measured basicities. Consistently, amides, nitriles, and pyrazoles more basic than water reduced the average charge of protein analytes electrosprayed from denaturing solutions, while analogues less basic than water increased the average charge, establishing the veracity of solvent leveling as a supercharging mechanism. In other words, reducing the charge departing on solvent leaves more charge for the protein analyte.