Hydrogen/deuterium exchange in parallel with acid/base induced protein conformational change in electrospray droplets.

The exposure of electrospray droplets to vapors of deuterating reagents during droplet desolvation in the interface of a mass spectrometer results in hydrogen/deuterium exchange (HDX) on the sub-millisecond time scale. Deuterated water is used to label ubiquitin and cytochrome c with minimal effect on the observed charge state distribution (CSD), suggesting that the protein conformation is not being altered. However, the introduction of deuterated versions of various acids (e.g., CD3COOD and DCl) and bases (ND3) induces unfolding or refolding of the protein while also labeling these newly formed conformations. The extent of HDX within a protein CSD associated with a particular conformation is essentially constant, whereas the extent of HDX can differ significantly for CSDs associated with different conformations from the same protein. In some cases, multiple HDX distributions can be observed within a given charge state (as is demonstrated with cytochrome c) suggesting that the extent of HDX and CSDs share a degree of complementarity in their sensitivities for protein conformation. The CSD is established late in the evolution of ions in electrospray whereas the HDX process presumably takes place in the bulk of the droplet throughout the electrospray process. Back exchange is also performed in which proteins are prepared in deuterated solvents prior to ionization and exposed to undeuterated vapors to exchange deuteriums for hydrogens. The degree of deuterium uptake is easily controlled by varying the identity and partial pressure of the reagent introduced into the interface. Since the exchange occurs on the sub-millisecond time scale, the use of deuterated acids or bases allows for transient species to be generated and labeled for subsequent mass analysis.