Gas-phase proton transfer reactions involving multiply charged cytochrome c ions and water under thermal conditions

Abstract

Investigations of gas-phase proton transfer reactions have been performed on protein molecular ions generated by electrospray ionization (ESI). Their reactions were studied in a heated capillary inlet/reactor prior to expansion into a quadrupole mass spectrometer. Results from investigations involving protonated horse heart cytochrome c and H, O suggest that Coulombit effects can lower reaction barriers as well as aid in entropically driven reactions. For example, the charge state distribution observed by a quadrupole mass spectrometer for multiply protonated cytochrome c without the addition of any reactive gas ranges from 9+ to 19+ , with the [M + 15H](15+) ion being the most intense peak. With the addition of H2O (proton affinity approximately 170.3±2 kcal/mol) to the capillary reactor at 120°C, the charge state distribution shifts to a lower charge, ranging from 13+ to less than 9+. Under the same conditions with argon (proton affinity approximately 100 kcal/mol) as the reactive gas, no shift in the charge state distribution is observed. The results demonstrate that proton transfer to water can occur for highly protonated molecular ions, a process that would be expected to be highly endothermic for singly protonated molecules (for which Coulombic destabilization is not significant). The results imply that the charge state distribution from ESI is somewhat dependent upon the mechanism and speed of the droplet evaporation/ion desolvation process, which may vary substantially with the ESI/mass spectrometry interface design.

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

“…Increasing the gas-phase concentration of solvent molecules does affect the maximum charge state and charge state distributions of multiply protonated ions [10]. This effect is illustrated for cytochrome c in Figure 6.…”

“…Steric effects will reduce this fraction even further. This, combined with the limited number of actual collisions, contributes to a relatively low probability for reactive gas-phase encounters between analyte ions and solvent molecules.Increasing the gas-phase concentration of solvent molecules does affect the maximum charge state and charge state distributions of multiply protonated ions [10]. This effect is illustrated for cytochrome c in Figure 6.…”

“…One outstanding challenge in ESI mass spectrometry is to accurately predict the observed charge state distribution of a large molecule, given its primary structure, the composition of the solvent system from which the ions are formed, instrumental conditions, etc. Several factors have been shown to influence the observed charge state distribution, including molecular conformation [4][5][6], acid-base chemistry both in solution and in the gas phase [7][8][9][10][11], solvent composition [8], instrumental factors, etc. Several models have been proposed to qualitatively account for some of these effects [12][13][14][15].…”

“…The authors noted, however, that the maximum charge state of the peptide (KAP) 10 , when electro-sprayed from water/isopropanol solution, resulted in mass spectra with a maximum charge state of 10+, whereas the maximum based on the calculated GB app and the GB of isopropanol is 8+. The authors stated that "the proton affinity of the most volatile solvent does not appear to determine the maximum charge state observed."…”

Investigation of deprotonation reactions on globular and denatured proteins at atmospheric pressure by ESSI-MS

“…High-pressure mass spectrometry (HPMS) experiments were designed by the group of Kebarle to study cluster formation from small organic or inorganic ions, and water in particular [9,10]. This apparatus was then modified by the group of Smith [11,12], who developed a new Y shaped capillary interface/ flow reactor connected to a quadrupole mass spectrometer. One inlet was fitted with an ESI source while the second inlet was used to introduce the basic reactant (volatile amines or water vapor) at a known flow rate.…”