Nanoelectrospray ionization of protein mixtures: solution pH and protein pI

Solutions consisting of single proteins and mixtures of proteins at different pH values have been subjected to both positive ion and negative ion nanoelectrospray ionization to study the influence of solvent pH and protein pI on the ionization responses of proteins. As has been noted previously, it is possible to form protein ions of one polarity despite the fact that the proteins are present as the opposite polarity in solution. However, total response under this condition tends to be at least an order of magnitude less than the condition in which the nanoelectrospray ionization polarity is the same as the net charge of the proteins in solution. Furthermore, maximum signals in positive ion mode were noted when the pH value of the solution was 4-5 units lower than the protein pI. In the negative ion mode, maximum protein anion signals were observed when the pH was roughly 5 units higher than the protein pI. While only small changes in the abundance-weighted average charge were noted as a function of solution conditions, the extent of sodium ion incorporation was seen to depend strongly on the relationship between net protein charge in solution and gas-phase ion polarity. Sodium ion incorporation was minimized under conditions of maximum signal (i.e., low pH positive ion mode and high pH negative ion mode). Sodium ion incorporation was highest when the protein ion polarities in solution and the gas phase were opposite. These observations are consistent with the charged residue model for electrospray ionization and suggest that a degree of selectivity for electrospray ionization applied to protein mixtures can be realized via judicious selection of solution pH and ionization polarity. Furthermore, the relative extent of sodium ion incorporation under a given set of conditions appears to correlate, at least qualitatively, with protein pI.