Surface-Induced Protein Unfolding in Submicron Electrospray Emitters.

The charging of protein ions formed by nanoelectrospray ionization (nanoESI) with tips that are between 1.5 μm and 250 nm in outer diameter is compared. More charging is obtained with the smaller tip sizes for proteins that have a net positive charge in solution, and additional high-charge-state distributions are often observed. A single charge-state distribution of holo-myoglobin ions is produced by nanoESI from a slightly acidified aqueous solution with the micron outer diameter tips, but some apo-myoglobin ions are produced with the submicron tips. In contrast, the charge-state distributions for proteins with a net negative charge in solution do not depend on tip size. Both the formation of high charge states and the appearance of higher-charge-state distributions, as well as the loss of the heme group from myoglobin, indicate that a fraction of the protein population is unfolding with the smaller tips. The increased charging with the smaller tip sizes for proteins with a net positive charge but not for proteins with a net negative charge indicates that the unfolding occurs prior to nanoelectrospray ionization as a result of Coulombic attraction between positively charged protein molecules in solution and the glass surfaces of the emitter tips that are negatively charged. These results demonstrate a novel method for producing highly charged protein ions that does not require exposing the proteins to additional chemicals either in solution or in the gas phase.