Protein fluorescence measurements within electrospray droplets

The conformation of cytochrome c molecules within electrospray droplets is investigated by monitoring the laser induced fluorescence of its single try ptophan residue (Trp-59). By increasing the alcohol concentration of the el ectrosprayed solutions, protein denaturation is induced, giving rise to sig nificant changes in the intensity of the detected fluorescence. Comparison with analogous denaturation experiments in solution provides information ab out the relative protein conformations and differences between the bulk-sol ution and droplet environments. Both electrospray-plume and bulk-solution f luorescence measurements using low methanol concentration solutions indicat e the presence of folded protein structures. At high methanol content, fluo rescence measurements are consistent with the presence of partly denatured or unfolded conformations. At intermediate methanol content, differences ar e observed between the extent of denaturation in solution and that within t he droplets, suggesting electrosprayed proteins have more compact structure s than those detected in bulk measurements using solutions of similar compo sition. This infers that some fraction of the proteins within the droplets have refolded relative to their bulk-solution conformation. Protein denatur ation experiments using the low vapor pressure solvent l-propanol indicate that differences between the droplet and solution measurements are not due to solvent evaporation effects. It is suggested that different droplet conf ormations are more likely the result of protein diffusion to the droplet su rface and effects of the droplet/air interface. To our knowledge, these are the first reported measurements of protein fluorescence within electrospra y droplets (J Am Soc Mass Spectrom 2001, 12, 716-725) (C) 2001 American Soc iety for Mass Spectrometry.