Comparative study of H3O+ (aq) and NH4+ (aq) on electrophoresis, protonating ability, and sodiation of proteins

Abstract

Positive-mode nano-electrospray ionization (nESI) was applied to the analysis of a mixture solution of gramicidin S (G), ubiquitin (U), and cytochrome c (C) in H2O/MeOH (50/50) under acidic, neutral, and basic conditions, i.e., with the addition of acetic acid (AcOH), ammonium acetate (AcONH4), and ammonia (NH3). The ion signal intensities for the acidic AcOH solution were approximately one order of magnitude higher than those for the NH3 or AcONH4 solutions. This marked difference is attributed to electrophoresis, i.e., much higher mobility of H3O+ (Grotthuss mechanism) than that of NH4+ in aqueous organic solvent. For the H2O/MeOH solution without additives, [G + 2H]2+ was observed as a major ion but U and C were barely detected. In contrast, for the AcOH solution, unfolded ubiquitin and cytochrome c ions of [U + nH]n+ (n = 8∼13) and [C + nH]n+ (n = 11∼20) were detected with weaker [G + 2H]2+. This indicates that denatured [U + nH]n+ (n = 8∼13) and [C + nH]n+ (n = 11∼20) are more surface-active than [G + 2H]2+. For the nearly neutral AcONH4 solution, U and C were detected as the folded molecule ions. The non-occurrence of denaturation for the U and C in the AcONH4 solution is due to the fact that NH4+(aq) is thermochemically 12.8 kcal mol−1 more stable than H3O+(aq) and denaturation of proteins is totally suppressed. For the basic NH3 solution, however, both denatured and native U and C were detected with a bimodal charge distribution. This suggested that U and C are more susceptible to denaturation in the basic NH3 solution than in the nearly neutral AcONH4 solution. The formation of sodiated U, [U + 6H −xH + xNa]6+ (x = 0−8), was interpreted as the specific sodiation of 0–8 carboxyl groups in the amino acid residues. This study highlights the important effects of different additive chemistries on the fate of proteins during the ionization process.