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.

Gas-phase reactions of hydrated alkaline earth metal ions, M2+ (H2O)n (M = Mg, Ca, Sr, Ba and n = 4-7), with benzene.

Gas-phase reactions of hydrated divalent alkaline earth metal ions and benzene were investigated by electrospray ionization Fourier-transform mass spectrometry. Rate constants for solvent-exchange reactions were determined as a function of hydration extent for Mg2+, Ca2+, Sr2+, and Ba2+ clusters containing four to seven water molecules each. All of the strontium and barium clusters react quickly with benzene. Barium reacts slightly faster than the corresponding strontium cluster with the same number of water molecules attached. For calcium, clusters with four and five water molecules react quickly, whereas those with six and seven water molecules do not. Magnesium with four water molecules reacts quickly, but not when five through seven water molecules are attached. The slow reactivity observed for some of these clusters indicates that the cation-pi interaction between the metal ion and benzene is partially screened by the surrounding water molecules. The reactivity of magnesium with seven water molecules is intermediate that of the hexa- and pentahydrate and the tetrahydrate. This result is consistent with the seventh water molecule being in the outer shell and much more weakly bound. The unusual trend in reactivity observed for magnesium may be due to the presence of mixed shell structures observed previously. These results are the first to provide information about the relative importance of cation-pi interactions in divalent metal ions as a function of metal hydration extent. Such studies should also provide a model and some insight into the relative binding affinities of divalent metal ions to aromatic residues on peptides and proteins.

Hydration of gas-phase ions formed by electrospray ionization.

The hydration of gas-phase ions produced by electrospray ionization was investigated. Evidence that the hydrated ions are formed by two mechanisms is presented. First, solvent condensation during the expansion inside the electrospray source clearly occurs. Second, some solvent evaporation from more extensively solvated ions or droplets is apparent. To the extent that these highly solvated ions have solution-phase structures, then the final isolated gas-phase structure of the ion will be determined by the solvent evaporation process. This process was investigated for hydrated gramicidin S in a Fourier-transform mass spectrometer. Unimolecular dissociation rate constants of isolated gramicidin S ions with between 2 and 14 associated water molecules were measured. These rate constants increased from 16 to 230 s-1 with increasing hydration, with smaller values corresponding to magic numbers.

Hydration energies and structures of alkaline earth metal ions, M2+(H2O)n, n = 5-7, M = Mg, Ca, Sr, and Ba.

The evaporation of water from hydrated alkaline earth metal ions, produced by electrospray ionization, was studied in a Fourier transform mass spectrometer. Zero-pressure-limit dissociation rate constants for loss of a single water molecule from the hydrated divalent metal ions, M(2+)(H(2)O)(n) (M = Mg, Ca, and Sr for n = 5-7, and M = Ba for n = 4-7), are measured as a function of temperature using blackbody infrared radiative dissociation. From these values, zero-pressure-limit Arrhenius parameters are obtained. By modeling the dissociation kinetics using a master equation formalism, threshold dissociation energies (E(o)) are determined. These reactions should have a negligible reverse activation barrier; therefore, E(o) values should be approximately equal to the binding energy or hydration enthalpy at 0 K. For the hepta- and hexahydrated ions at low temperature, binding energies follow the trend expected on the basis of ionic radii: Mg > Ca > Sr > Ba. For the hexahydrated ions at high temperature, binding energies follow the order Ca > Mg > Sr > Ba. The same order is observed for the pentahydrated ions. Collisional dissociation experiments on the tetrahydrated species result in relative dissociation rates that directly correlate with the size of the metals. These results indicate the presence of two isomers for hexahydrated magnesium ions: a low-temperature isomer in which the six water molecules are located in the first solvation shell, and a high-temperature isomer with the most likely structure corresponding to four water molecules in the inner shell and two water molecules in the second shell. These results also indicate that the pentahydrated magnesium ions have a structure with four water molecules in the first solvation shell and one in the outer shell. The dissociation kinetics for the hexa- and pentahydrated clusters of Ca(2+), Sr(2+), and Ba(2+) are consistent with structures in which all the water molecules are located in the first solvation shell.

Separation and characterization of amines from individual atrial gland vesicles of Aplysia californica.

Several amine-containing components of individual vesicles from the atrial gland of Aplysia californica were identified with capillary electrophoresis (CE). On-line derivatization with naphthalene-2,3-dicarboxaldehyde was performed, and the derivatized amine-containing components were detected with laser-induced fluorescence (LIF). Amino acids, including taurine, that had not been determined previously in atrial gland vesicles were observed by using CE-LIF, and their identities were confirmed with CE, HPLC, NMR, and electrospray ionization mass spectrometry. The finding that taurine is packaged and stored into secretory vesicles supports the hypothesis that taurine may exhibit neuromodulatory activity. The bioactive peptides, well-known to be in atrial gland vesicles, were detected in lysed vesicle samples fractionated with HPLC and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These peptides were also observed in single-vesicle runs with CE-LIF. The atrial gland vesicles (ranging from 0.5 to 2 microns diameter and 65 aL to 4 fL volume, respectively) studied in this work represent the smallest biological entities to be analyzed chemically on an individual basis.

Probing single secretory vesicles with capillary electrophoresis.

Secretory vesicles obtained from the atrial gland of the gastropod mollusk Aplysia californica were chemically analyzed individually with a combination of optical trapping, capillary electrophoresis separation, and a laser-induced fluorescence detection. With the use of optical trapping, a single vesicle that had attoliters (10(-18) liters) of volume was introduced into the tapered inlet of a separation capillary. Once the vesicle was injected, it was lysed, and its components were fluorescently labeled with naphthalene-2, 3-dicarboxaldehyde before separation. The resultant electropherograms indicated distinct variations in the contents of single vesicles.

Conformations and folding of lysozyme ions in vacuo.

Proton transfer reactivity of isolated charge states of the protein hen egg-white lysozyme shows that multiple distinct conformations of this protein are stable in the gas phase. The reactivities of the 9+ and 10+ charge state ions, formed by electrospray ionization of "native" (disulfide-intact) and "denatured" (disulfide-reduced) solutions, are consistent with values calculated for ions in their crystal structure and fully denatured conformations, respectively. Charge states below 8+ of both forms, formed by proton stripping, have similar or indistinguishable reactivities, indicating that the disulfide-reduced ions fold in the gas phase to a more compact conformation.