Electrospray ionization mass spectrometry (ESI-MS) is often used to monitor protein conformational dynamics in solution, for example acid unfolding, by following the changes in positive-ion charge-state distributions in response to changes of ambient conditions, for example solution pH. Deconvolution of these charge-state distributions often reveals the presence of multiple protein conformers coexisting in solution in equilibrium. The ion signal corresponding to each conformer depends on its size (which determines the average charge state of the protein ions) and heterogeneity (which determines the spread of the ion signal). In the present work, we seek to explore how the ion signal of individual protein conformers can be influenced by other factors not related to protein shape, with particular attention being paid to contributions from solution acid-base chemistry. The composition of the buffer was found to exert a significant influence on the ion signal by inducing apparent charge reduction of the protein ions. This effect was ascribed to protein-base (anion) complex formation in solution followed by dissociation of the neutral conjugated acid from the complex in the gas-phase. The resulting shift in the charge-state distribution occurs in the pH range from pK_a to approximately (pK_a – 1.5) and is induced by the elevated concentration of the anion in solution. On the other hand, intrinsic charges on the protein in solution have been shown to have no effect on the appearance of the charge-state distributions, lending further credibility to the notion that protein shape is the only structural determinant of the ion signal in ESI-MS.

Keywords: electrospray ionization, mass spectrometry, protein conformation, charge-state distribution, chemometrics