Negative electrospray droplet exposure to gaseous bases for the manipulation of protein charge state distributions.

The exposure of electrospray droplets to vapors of reagents of various base strengths affects protein negative charge state distributions independent of initial solution conditions. Volatile bases are introduced into the counter-current nitrogen drying gas of an electrospray interface to interact with charged droplets as they undergo desolvation/disintegration, shifting charge state distributions of proteins to higher, more negative, charge states. Alterations of charge state distributions can implicate protein folding/unfolding phenomena. Species bound by relatively weak interactions can be preserved, at least to some extent, allowing for the observation of high charge states of protein-ligand complexes, such as high negative charge states of holomyoglobin. The binding of carbonic anhydrase with its Zn(2+) cofactor is apparently preserved when the holo-form of the protein is exposed to basic vapors (i.e., the Zn(2+) ion remains associated with the protein), but this prevents the appearance of charge states higher than -17. Charge state distributions of proteins containing disulfide bonds shift slightly with the leak-in of basic vapors, but when these disulfide bonds are reduced with dithiothreitol in solution, charge states higher than the number of acidic sites (Asp, Glu, and C-terminus) are observed. Since there is no observed change in the distributions of buffered proteins exposed to these reagent vapors, the charge state changes are attributed largely to a pH affect. High pK(a) and highly volatile reagents have been found to be the most effective in terms of observing the maximum negative charge state of the biomolecule of interest.