Supercharged protein and peptide ions formed by electrospray ionization.

The multiple charging of large molecules in electrospray ionization provides key advantages for obtaining accurate molecular weights by mass spectrometry and for obtaining structural information by tandem mass spectrometry and MS(n) experiments. Addition of glycerol or m-nitrobenzyl alcohol into the electrospray solutions dramatically increases both the maximum observed charge state and the abundances of the high charge states of protein and peptide ions. Adding glycerol to acidified aqueous solutions of cytochrome c shifts the most abundant charge state from 17+ to 21+, shifts the maximum charge state from 20+ to 23+, and shifts the average charge state from 16.6+ to 20.9+. Much less m-nitrobenzyl alcohol (<1%) is required to produce similar results. With just 0.7% m-nitrobenzyl alcohol, even the 24+ charge state of cytochrome c is readily observed. Similar results are obtained with myoglobin and (Lys)4. For the latter molecule, the 5+ charge state is observed in the electrospray mass spectrum obtained from solutions containing 6.7% m-nitrobenzyl alcohol. This charge state corresponds to protonation of all basic sites in this peptide. Although the mechanism for enhanced charging is unclear, it does not appear to be a consequence of conformational changes of the analyte molecules. This method of producing highly charged protein ions should be useful for improving the performance of mass measurements on mass spectrometers with performances that decrease with increasing m/z. This should also be particularly useful for tandem mass spectrometry experiments, such as electron capture dissociation, for which highly charged ions are desired.

Effects of solvent on the maximum charge state and charge state distribution of protein ions produced by electrospray ionization.

The effects of solvent composition on both the maximum charge states and charge state distributions of analyte ions formed by electrospray ionization were investigated using a quadrupole mass spectrometer. The charge state distributions of cytochrome c and myoglobin, formed from 47%/50%/3% water/solvent/acetic acid solutions, shift to lower charge (higher m/z) when the 50% solvent fraction is changed from water to methanol, to acetonitrile, to isopropanol. This is also the order of increasing gas-phase basicities of these solvents, although other physical properties of these solvents may also play a role. The effect is relatively small for these solvents, possibly due to their limited concentration inside the electrospray interface. In contrast, the addition of even small amounts of diethylamine (<0.4%) results in dramatic shifts to lower charge, presumably due to preferential proton transfer from the higher charge state ions to diethylamine. These results clearly show that the maximum charge states and charge state distributions of ions formed by electrospray ionization are influenced by solvents that are more volatile than water. Addition of even small amounts of two solvents that are less volatile than water, ethylene glycol and 2-methoxyethanol, also results in preferential deprotonation of higher charge state ions of small peptides, but these solvents actually produce an enhancement in the higher charge state ions for both cytochrome c and myoglobin. For instruments that have capabilities that improve with lower m/z, this effect could be taken advantage of to improve the performance of an analysis.