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 tryptophan residue (Trp-59). By increasing the alcohol concentration of the electrosprayed solutions, protein denaturation is induced, giving rise to significant changes in the intensity of the detected fluorescence. Comparison with analogous denaturation experiments in solution provides information about the relative protein conformations and differences between the bulk-solution and droplet environments. Both electrospray-plume and bulk-solution fluorescence measurements using low methanol concentration solutions indicate the presence of folded protein structures. At high methanol content, fluorescence measurements are consistent with the presence of partly denatured or unfolded conformations. At intermediate methanol content, differences are observed between the extent of denaturation in solution and that within the droplets, suggesting electrosprayed proteins have more compact structures than those detected in bulk measurements using solutions of similar composition. This infers that some fraction of the proteins within the droplets have refolded relative to their bulk-solution conformation. Protein denaturation experiments using the low vapor pressure solvent 1-propanol indicate that differences between the droplet and solution measurements are not due to solvent evaporation effects. It is suggested that different droplet conformations are more likely the result of protein diffusion to the droplet surface and effects of the droplet/air interface. To our knowledge, these are the first reported measurements of protein fluorescence within electrospray droplets.
Rodriguez-Cruz
S E
SE
The Rowland Institute for Science, Cambridge, Massachusetts 02142, USA.
Khoury
J T
JT
Parks
J H
JH
D000438:Alcohols / Q000737:chemistry; D000465:Algorithms; D003574:Cytochrome c Group / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012997:Solvents; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
D000438:Alcohols; D003574:Cytochrome c Group; D011506:Proteins; D012997:Solvents