Hydration of gas-phase gramicidin S (M + 2H)2+ ions formed by electrospray: The transition from solution to gas-phase structure

The hydration of doubly protonated gas-phase ions of gramicidin S formed by electrospray ionization was investigated. Under “gentle” electrospray conditions, a near Gaussian distribution of (M + 2H + nH₂O)²⁺ ions with n up to 50 can be readily formed. These extensively hydrated gas-phase ions should have structures similar to those in solution. For intermediate extents of hydration, the “naked” or unsolvated ion is present in unusually high abundance. This is attributed to a competition between solvation of the charges by water vs intramolecular self-solvation via hydrogen bonding. In addition, “magic” numbers of attached water molecules are observed for n = 8, 11, and 14. These magic numbers are attributed to favorable arrangements of water molecules surrounding the charge and surface of the peptide in the gas phase. These results are indicative of a gentle stepwise transformation from the solution-phase structure of the ion to the preferred gas-phase structure as solvent evaporates from the hydrated ions.     

Electrospray mass spectrometry: Ethylene glycol as a solvent and its effects on ion desorption

Ethylene glycol solutions of gramicidin S, myoglobin and tetrabutylammonium bromide were analysed by means of electrospray mass spectrometry and their spectra were compared with those of aqueous solutions. The evaporation of water and ethylene glycol droplets, initially at room or elevated temperature, in air at room temperature was modelled. It was found that under conditions where a water droplet's radius would shrink by ～30%, an ethylene glycol droplet shrinks negligibly. Further, droplets that are initially hot (such as those that are ejected from a heated electrospray needle) cool very rapidly owing to evaporation and heat loss to ambient air, and subsequently evaporate much like droplets that are initially at room temperature. For gramicidin S, the ion abundances in ethylene glycol as solvent were ～200 times lower than those in water under room temperature operating conditions. In experiments where the spray probe was heated to ～100°C to reduce the viscosity of ethylene glycol, the gramicidin response difference between the solvents decreased to about a factor of 40. Similar trends were observed for myoglobin and the tetrabutylammonium ion. The gramicidin abundances in ethylene glycol, relative to those in water, are orders of magnitude too large to be accounted for using the conventional solvent evaporation model. It is speculated that decreasing the viscosity increases the velocity of ions drifting in ethylene glycol towards the solution/air interface and increases the total number of analyte ions desorbed at the Taylor cone during electrospray.     

Internal energy distributions in desorption electrospray ionization (DESI)

The internal energy distributions of typical ions generated by desorption electrospray ionization (DESI) were measured using the "survival yield" method, and compared with corresponding data for electrospray ionization (ESI) and electrosonic spray ionization (ESSI). The results show that the three ionization methods produce populations of ions having internal energy distributions of similar shapes and mean values (1.7-1.9 eV) suggesting similar phenomena, at least in the later stages of the process leading from solvated droplets to gas-phase ions. These data on energetics are consistent with the view that DESI involves "droplet pick-up" (liquid-liquid extraction) followed by ESI-like desolvation and gas-phase ion formation. The effects of various experimental parameters on the degree of fragmentation of p-methoxy-benzylpyridinium ions were compared between DESI and ESSI. The results show similar trends in the survival yields as a function of the nebulizing gas pressure, solvent flow rate, and distance from the sprayer tip to the MS inlet. These observations are consistent with the mechanism noted above and they also enable the user to exercise control over the energetics of the DESI ionization process, through manipulation of external and internal ion source parameters.     

Experimental observation of the transition between gas-phase and aqueous solution structures for acetylcholine, nicotine, and muscarine ions

Structural information on acetylcholine and its two agonists, nicotine, and muscarine has been obtained from the interpretation of infrared spectra recorded in the gas-phase or in low pH aqueous solutions. Simulated IR spectra have been obtained using explicit water molecules or a polarization continuum model. The conformational space of the very flexible acetylcholine ions is modified by the presence of the solvent. Distances between its pharmacophoric groups cover a lower range in hydrated species than in isolated species. A clear signature of the shift of protonation site in nicotine ions is provided by the striking change of their infrared spectrum induced by hydration. On the contrary, structures of muscarine ions are only slightly influenced by the presence of water.     

Comparison of thermospray and ion spray mass spectrometry in an atmospheric pressure ion source

Ion spray is an approach to liquid chromatography, mass spectrometry which includes features common to the electrospray and ion evaporation interfaces. Thermospray is a liquid chromatographic/mass spectrometric technique which utilizes heat and electrolytes in the mobile phase to generate sample ions. In this paper the operation of these two techniques at atmospheric pressure are compared with respect to the effects of solvent composition and electrolyte ion concentration for the production of ions from compounds that are ionized in solution (safranin orange, acid black 1 and testosterone sulfate) and un-ionized in solution (methyl red, adenosine and diethylstilbestrol). The results indicate that at atmospheric pressure ion spray produces ions by the ion evaporation mechanism while thermospray produces ions by both gas-phase chemical ionization and ion evaporation processes.     

Density functional study of intradimer proton transfers in hydrated adenine dimer ions, A2(+)(H2O)n (n = 0-2)

Structures of mono- and dihydrated adenine dimers and their cations were calculated using B3LYP density functional theory with the 6-31+G(d,p) basis set, in order to help understand photofragmentation experiments of hydrated adenine dimers from the energetics point of view. Several important pathways leading to the major fragmentation product, protonated adenine ion (AH(+)), thermodynamically at minimum costs were investigated at the ground-state electronic potential surface of hydrated adenine dimer cations. Our calculations suggest that the proton transfer from one adenine moiety to the other in hydrated dimer ions readily occurs with negligible barriers in normal hydration conditions. In asymmetrically hydrated ions, however, the proton transfer to more hydrated adenine moieties is kinetically hindered due to heightened transition-state barriers, while the other way is still barrierless. Such directional preference in proton transfer may be characterized as a unique dimer ion property, stemming from the difference in basicity of the two nitrogen atoms involved in the double hydrogen bond that would be equivalent without hydration. We also found that dimer cleavage requires about 4 times larger energy than evaporation of individual water molecules, so it is likely that most solvent molecules evaporate before the eventual dimer cleavage when available internal energy is limited.     

Comparison of intensities between doubly charged ions [M + 2H]2+ and singly charged ions [M + H]+ of gramicidin S by electrospray mass spectrometry.

The reason why the intensity of doubly charged ions [M + 2H]2+ of gramicidin S is higher than that of singly charged ions [M + H]+ in electrospray is investigated by ion evaporation theory. As a result of comparison between the total free energies of extracting [M + 2H]2+ and [M + H]+ from a charged droplet to infinity, it is found that the total free energy of [M + 2H]2+ is estimated to be lower than that of [M + H]+. This clearly supports the experimental result. In addition, the importance of the electrostatic contribution in electrospray is demonstrated by showing the result that the total free energy of [M + 2H]2+ without electrostatic contribution is higher than that of [M + H]+.     

Effect of buffer cations and of H3O+ on the charge states of native proteins. Significance to determinations of stability constants of protein complexes

The progressive reduction of charge in charge states of non-denatured proteins (lysozyme, ubiquitin, and cytochrome c), observed with nanospray in the positive ion mode, when the buffer salt ammonium acetate is replaced by ethylammonium acetates (EtNH(3)Ac, Et(2)NH(2)Ac and Et(3)NHAc) is rationalized on the basis of the charge residue model (CRM). The charge states of the multiply protonated protein are shown to be controlled by the increasing gas-phase basicities, GB(B), of the bases(B) NH(3), EtNH(2), Et(2)NH and Et(3)N. Charge states derived from evaluated apparent gas-phase basicities GB(app) of the basic side-chains of the protein and the known GB(B) of the above bases are found to be in agreement with the experimentally observed charge states. This is a requirement of the CRM, because in this model the small positive ions (the buffer cations in the present case) at the surface of the electrospray droplets are the excess ions that provide the charge of the final small droplet that contains the protein molecule and on evaporation of the solvent transfer the charge to the protein. The observed charge states in the absence of buffer salts, i.e. pure water, are attributed to excess H(3)O(+) ions produced by the electrolysis process that attends electrospray. A proposed extended mechanism provides predictions of factors that determine the sensitivity for detection of the multiply protonated proteins. Consideration of restraints imposed by the CRM lead to some simple predictions for conditions that should be present to obtain accurate determinations by electrospray and nanospray of stability constants for the protein-complex equilibrium in aqueous solution.     

Ejection of solvated ions from electrosprayed methanol/water nanodroplets studied by molecular dynamics simulations

The ejection of solvated small ions from nanometer-sized droplets plays a central role during electrospray ionization (ESI). Molecular dynamics (MD) simulations can provide insights into the nanodroplet behavior. Earlier MD studies have largely focused on aqueous systems, whereas most practical ESI applications involve the use of organic cosolvents. We conduct simulations on mixed water/methanol droplets that carry excess NH(4)(+) ions. Methanol is found to compromise the H-bonding network, resulting in greatly increased rates of ion ejection and solvent evaporation. Considerable differences in the water and methanol escape rates cause time-dependent changes in droplet composition. Segregation occurs at low methanol concentration, such that layered droplets with a methanol-enriched periphery are formed. This phenomenon will enhance the partitioning of analyte molecules, with possible implications for their ESI efficiencies. Solvated ions are ejected from the tip of surface protrusions. Solvent bridging prior to ion secession is more extensive for methanol/water droplets than for purely aqueous systems. The ejection of solvated NH(4)(+) is visualized as diffusion-mediated escape from a metastable basin. The process involves thermally activated crossing of a ~30 kJ mol(-1) free energy barrier, in close agreement with the predictions of the classical ion evaporation model.     

Is droplet evaporation crucial in the mechanism of electrospray mass spectrometry?

Evaporation of solvent from charged droplets was found not to be a prerequisite to ion desorption in electrospray mass spectrometry. Evidence of evaporation was absent in an examination of the electrospray mass spectral profiles of cytochrome c and myoglobin in 0.2% acetic and propionic acid solutions; the pHs of these two acid solutions are expected to change in opposite directions with evaporation. The results strongly suggest that ions, as observed in electrospray mass spectrometry, are desorbed from solutions that have undergone minimal evaporation, in other words, at the beginning rather than later parts of the electrospray process. It is speculated that ions are desorbed directly from the solution-air interface at the needle tip.     

Determination of affinity constants and response factors of the noncovalent dimer of gramicidin by electrospray ionization mass spectrometry and mathematical modeling

The dimerization of gramicidin, a 15-residue membrane peptide, in solution can be viewed as a model for protein-protein interactions. We reported previously that the dimer can be observed when electrosprayed from organic solvents and that the abundances of the dimer depends on the dielectric constant of the solvent. Here, we report an effort to determine an affinity constant for the dimerization of gramicidin by using gas-phase abundance. Two issues affecting the determination are the electrospray-induced dissociation of the dimer and discrimination in the electrospray of the dimer compared with the monomer. Other methods developed for the purpose of determining affinity from mass spectral abundance do not address the dissociation of the complex in the gas phase or can not be applied for cases of low affinity constant, K(a). We present a mathematical model that uses the ratio of the signal intensities of the dimer and the monomer during a titration. The model also incorporates the dissociation and an electrospray ionization-response factor of the dimer for extracting the affinity constant for the dimerization of gramicidin. The dimerization constants from the new method agree within a factor of two with values reported in the literature.     

Structure of the hydrated and dimethyl sulfoxide solvated rubidium ions in solution

The structure of the hydrated and the dimethyl sulfoxide solvated rubidium ions in solution has been determined by means of large-angle X-ray scattering (LAXS) and extended X-ray absorption fine structure (EXAFS) studies. The models of the hydrated and dimethyl sulfoxide solvated rubidium ions fitting the experimental data best are square antiprisms with Rb-O bond distances of 2.98(2) and 2.98(3) A, respectively. The EXAFS data show a significant asymmetry in the Rb-O bond distance distribution with C(3) values of 0.0076 and 0.015 A(3), respectively. No second hydration sphere is observed around the hydrated rubidium ion. The dimethyl sulfoxide solvated rubidium ion displays a Rb-O-S bond angle of ca. 130 degrees, which is typical for a medium hard electron acceptor such as rubidium.     

Importance of gas-phase proton affinities in determining the electrospray ionization response for analytes and solvents

The effect of gas-phase proton transfer reactions on the mass spectral response of solvents and analytes with known gas-phase proton affinities was evaluated. Methanol, ethanol, propanol and water mixtures were employed to probe the effect of gas-phase proton transfer reactions on the abundance of protonated solvent ions. Ion-molecule reactions were carried out either in an atmospheric pressure electrospray ionization source or in the central quadrupole of a triple-quadrupole mass spectrometer. The introduction of solvent vapor with higher gas-phase proton affinity than the solvent being electrosprayed caused protons to transfer to the gas-phase solvent molecules. In mixed solvents, protonated solvent clusters of the solvent with higher gas-phase proton affinity dominated the resulting mass spectra. The effect of solvent gas-phase proton affinity on analyte response was also investigated, and the analyte response was suppressed or eliminated in solvents with gas-phase proton affinities higher than that of the analyte.     

Solvation of copper(II) sulfate in binary water/N,N-dimethylformamide mixtures: from the solution to the gas phase

The solvation of copper(II) sulfate in binary mixtures of water and N,N-dimethylformamide (DMF) is studied by a combined approach using electrochemical studies in solution and a mass spectrometric assay of the solvated ions formed from these solutions upon electrospray ionization (ESI). In the condensed phase, the limiting transference numbers (t(+/-)(o)) and the apparent ion association constants (K(A)'s) of CuSO(4) have been determined in water/DMF solutions at 20 degrees C. The t(+)(o) values decrease with increasing DMF content, demonstrating a gradual solvation of Cu(2+) by DMF molecules. The association constants indicate that aggregation becomes more pronounced as the DMF content increases. In order to achieve complementary insight, the intrinsic interactions among the ions and solvent molecules are investigated in gas-phase experiments of the CuSO(4)/water/DMF system using ESI mass spectrometry. Under the conditions used, the dications [Cu(DMF)(n)](2+) (n = 3-6), [Cu(2)(DMF)(n)SO(4)](2+) (n = 2-7), and [Cu(3)(DMF)(n)(SO(4))(2)](2+) (n = 2-7), and the monocations [Cu(OH)(DMF)(n)](+), [Cu(DMF)(n)(HSO(4))](+) (both, n = 1-3), and [Cu(DMF)(n)](+) (n = 1, 2), are formed as the leading copper-containing cations. Likewise, polynuclear copper clusters observed in the anion ESI spectra support partial aggregation occurring in solution. The gas-phase studies clearly support the conclusions that (i) DMF is a highly preferred ligand for CuII in comparison to water and that (ii) DMF supports ion association for which the mass spectrometric data suggest the formation of polynuclear copper clusters.     

Evidence that water can reduce the kinetic stability of protein-hydrophobic ligand interactions

The first quantitative comparison of the thermal dissociation rate constants measured for protein-ligand complexes in their hydrated and dehydrated states is described. Rate constants, measured using surface plasmon resonance spectroscopy, are reported for the dissociation of the 1:1 complexes of bovine β-lactoglobulin (Lg) with the fatty acids (FA), palmitic acid (PA), and stearic acid (SA), in aqueous solution at pH 8 and at temperatures ranging from 5 to 45 °C. The rate constants are compared to values determined from time-resolved blackbody infrared radiative dissociation measurements for the gaseous deprotonated (Lg+FA)(n-) ions, where n = 6 and 7, at temperatures ranging from 25 to 66 °C. Notably, the hydrated (Lg+PA) complex is kinetically less stable than the corresponding gas phase (Lg+PA)(n-) ions at all temperatures investigated; the hydrated (Lg+SA) complex is kinetically less stable than the gaseous (Lg+SA)(n-) ions at temperatures <45 °C. The greater kinetic stability of the gaseous (Lg+FA)(n-) ions originates from significantly larger, by 11-12 kcal mol(-1), E(a) values. It is proposed that the differences in the dissociation E(a) values measured in solution and the gas phase reflect the differential hydration of the reactant and the dissociative transition state.     

Nanojets, electrospray, and ion field evaporation: molecular dynamics simulations and laboratory experiments

The energetics, interfacial properties, instabilities, and fragmentation patterns of electrosprays made from formamide salt solutions are investigated in a mass spectrometric vacuum electrospray experiment and using molecular dynamics (MD) simulations. The electrospray source is operated in a Taylor cone-jet mode, with the nanojet that forms being characterized by high surface-normal electric field strengths in the vicinity of 1 V/nm. Mass-to-charge ratios were determined for both positive and negative currents sprayed from NaI-formamide solutions with solute-solvent mole ratios of 1:8.4 and 1:36.9, and from KI-formamide solutions with mole ratios of 1:41 and 1:83. The molecular dynamics simulations were conducted on isolated 10 nm NaI-formamide droplets at mole ratios of 1:8 and 1:16. The droplet was subjected to a uniform electric field with strengths ranging between 0.5 and 1.5 V/nm. Both the experiments and simulations demonstrate a mixed charge emission regime where field-induced desorption of solvated ions and charged droplets occurs. The macroscopic parameters, such as average mass-to-charge ratio and maximum surface-normal field strengths deduced from the simulations are found to be in good agreement with the experimental work and consistent with electrohydrodynamic theory of cone-jets. The observed mass spectrometric Na (+) and I (-) solvated ion distributions are consistent with a thermal evaporation process, and are correctly reproduced by the simulation after incorporation of the different flight times and unimolecular ion dissociation rates in the analysis. Alignment of formamide dipoles and field-induced reorganization of the positive and negative ionic charges in the interfacial region are both found to contribute to the surface-normal field near the points of charge emission. In the simulations the majority of cluster ions are found to be emitted from the tip of the jet rather than from the neck region next to the Taylor cone. This finding is consistent with the experimental energy distributions of the solvated ions which demonstrate that indeed most ions are emitted closer to the jet region, that is, beyond the cone-neck region where ohmic losses occur. This observation is also consistent with continuum electrohydrodynamic predictions of cluster-ion evaporation at surface regions of high curvature and therefore maximum surface electric field strengths, which may be the cone-neck region, the breakup region of the jet (usually near the tip of the jet), or the emitted charged droplets. In the nanoscale jets observed in this study, the regions of highest spatial curvature are at the ends of the jets where nascent drops either are forming or have just detached.     

Evaporation rate from aerosol OT-water-n-heptane inverse micellar systems

The evaporation rate of aerosol OT-water-n-heptane inverse micellar systems was studied. The evaporation behavior of solutions was strongly affected when compared with pure heptane. The changes are related to the structure of the micelles and the properties of the water molecules dissolved in the micelles. The initial rate of evaporation reaches a maximum when the surfactant head groups are hydrated with three water molecules, a minimum when the first hydration shell of Na⁺ ions is completed, and then rises and stabilizes until the second hydration shell is completed. The presence of free water when the ratio water molecules to surfactant molecules exceeds 12 produces a new rise of the initial evaporation rate, which is always higher than that of puren-heptane.     

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.     

Solution structure of papain as studied by molecular mechanics and molecular dynamics techniques

In this paper, we report the molecular mechanics and molecular dynamics studies of the hydration of papain using the AMBER and CHARMm programs. We studied papain in an environment with minimal hydration involving only the X-ray waters and also in the hydrated environment by adding an extra 9 Å layer of water around the residues. The effect of nonbond cutoff was studied by performing minimizations with 8 Å and 15 Å nonbond cutoffs using the program AMBER. Two different solvent models—a constant dielectric and a distance-dependent dielectric—were considered. The AMBER-minimized structure and the average structure obtained from the CHARMm simulations for papain solvated with X-ray waters are presented and compared with the X-ray crystal structure results. Results of a similar comparison of the hydrated structures were also presented. The calculated root mean square deviation between the minimized structure and the X-ray structure is smaller for the hydrated system than for the system hydrated with only the X-ray waters. Results of the molecular mechanics and molecular dynamics simulations were presented for the various regions of papain. The hydration of the active site of papain and the effect of hydration on the torsional motion of the active site residues are presented. © 1996 by John Wiley & Sons, Inc.     

Probing the intrinsic features and environmental stabilization of multiply charged anions

Multiply charged anions (MCAs) represent exotic, highly energetic species in the gas-phase due to their propensity to undergo unimolecular decay via electron loss or ionic fragmentation. There is considerable fundamental interest in these systems since they display novel potential energy surfaces that are characterized by Coulomb barriers. Over recent years, considerable progress has been made in understanding the factors that affect the stability, decay pathways and reactivity of gas-phase MCAs, mainly as a result of the application of electrospray ionization as a generic technique for transferring solution-phase MCAs into the gas-phase for detailed characterization. We review contemporary work in this field, focusing on the factors that control the intrinsic stability of MCAs, both as isolated gas-phase ions, and on their complexation with solvent molecules and counter-ions. While studies of MCAs are primarily of fundamental interest, several classes of important biological ions are commonly observed as MCAs in the gas-phase (e.g. oligonucleotides, sugars). Recent results for biologically relevant ions are emphasised, since a fundamental understanding of the properties of gas-phase MCAs will be highly valuable for developing further analytical methods to study these important systems.