Disparity between solution‐phase equilibria and charge state distributions in positive‐ion electrospray mass spectrometry

Two peptides, bradykinin and gramicidin S, were used to investigate the relationship between protonation in the solution phase and charge state distribution observed in electrospray ionization (ES) mass spectra. The degree of protonation in solution was estimated using acid‐base equilibrium calculations where possible. Protonation in solution was varied by adjusting pH, solvent composition and peptide concentration. Major disparities were observed between calculated solution‐phase peptide protonation and the charge state distributions observed in ES mass spectra. The [(M + 2H) 2+ ]/[(M + H) + ] ratio calculated in solution was larger than the abundance ratio (M + 2H) 2+ /(M + H) + in the ES mass spectra of all acidic aqueous (pH < 6.5) and non‐aqueous solutions; in basic aqueous solutions (pH > 9.5) the opposite was true. At high pH, electrophoretic droplet charging may reduce the activity of OH − in positively charged droplets. The results at low pH imply the existence of supplementary factors in the ES ionization process which largely attenuate the degree of charging in the gas phase as compared with solution. Factors such as the increasing intra‐ and intermolecular coulombic repulsion between charge carriers (protons) and increasing attractive forces between protonated sites and counterions at progressively later stages of charged droplet evaporation were hypothesized to be chiefly responsible for this effect. Non‐aqueous solvents of high basicity compete with analytes to some extent for available protons, forming protonated solvent molecules while decreasing the sensitivity and the degree of multiple charging of peptides.