Conformational and Noncovalent Complexation Changes in Proteins during Electrospray Ionization

Abstract

Electrospray ion sources efficiently produce gas-phase ions from proteins and their noncovalent complexes. Charge-state distributions of these ions are increasingly used to gauge their conformations in the solution phase. Here we investigate how this correlation is affected by the spraying conditions at the early stage of droplet generation, prior to the ionization process. We followed the folding behavior of model proteins cytochrome c and ubiquitin and the dissociation of the noncovalent holomyoglobin complex. Spray current measurements, fast Taylor cone imaging, and mass analysis of the generated ions indicated that the protein structure experienced conformational or complexation changes upon variations in the spraying mode of the electrospray ionization source. These effects resulted in a departure from the original secondary, tertiary, and quaternary structure of proteins, possibly introducing artifacts in related studies. Therefore, if a particular gas-phase ion conformation is required or correlations with the liquid-phase conformations are studied, it is advantageous to maintain a particular spraying mode. Alternatively, spraying mode-induced changes can be utilized to alter the structure of proteins in, for example, time-resolved experiments for the study of protein folding dynamics.