Response surface study on protein charge–state distributions in electrospray ionization mass spectrometry related to multiple ion equilibria in solution

The reproducibility of protein charge–state distributions (CSDs) in electrospray ionization mass spectrometry (ESI-MS) is studied on the basis of the apoenzyme of myoglobin. Using PLS response surface methodology, the influence of the experimental factors protein and buffer concentrations, butanol solvent and flow rate of infusion on spectral intensities can be quantified and used for further treatment of CSD data. A direct relationship between CSDs and charged protein ion species can be deduced on the basis of a simultaneous multiple ion equilibrium model including up to 56 protolysis equilibria.