Supercharged protein and peptide lone formed by electrospray ionization

The multiple charging of large molecules in electrospray ionization provide s key advantages for obtaining accurate molecular weights by mass spectrome try and for obtaining structural information by tandem mass spectrometry an d MSn experiments. Addition of glycerol or m-nitrobenzyl alcohol into the e lectrospray solutions dramatically increases both the maximum observed char ge state and the abundances of the high charge states of protein and peptid e ions. Adding glycerol to acidified aqueous solutions of cytochrome c shif ts the most abundant charge state from 17+ to 21+, shifts the maximum charg e 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 myoglo bin 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 b asic 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 i ons should be useful for improving the performance of mass measurements on mass spectrometers with performances that decrease with increasing m/z, Thi s should also be particularly useful for tandem mass spectrometry experimen ts, such as electron capture dissociation, for which highly charged ions ar e desired.