In electrospray ionization, how much pull does an ion need to escape its droplet prison?

This article assumes that (1) that solute ions from a charged droplet are formed by field-assisted desorption, (2) that the charges on such a droplet are equidistantly spaced in a uniform array on the droplet surface, and (3) that the distance between charges on an evaporated ion cannot be less than the distance between charges on the droplet surface when that ion desorbed. These assumptions, together with mass spectral data for poly(ethylene glycols) and available information on the structure of their electrospray ions, allow us to determine that intercharge distance and thus the magnitude of the droplet surface field when those ions desorbed. Values of this desorption field for methanol-water droplets are 2.66, 1.55, and 1.46 V/nm for the smallest oligomer ions having respectively 2, 3 and 6 charges (Na⁺).     

Multi-charged oligonucleotide ion formation in sonic spray ionization.

An oligonucleotide tends to release hydrogen atoms from a phosphoric acid group and to form negative ions that can be detected by mass spectrometry. Usually, with a solution-spray based ionization technique, the negative ions are present in different charge states. Ion formation for the nucleotide is quite complicated and is easily influenced by matrix and other constituents in a sample solution, as well as by the operating parameters for a mass spectrometer. In this work, we studied oligonucleotide ion formation by using an ion trap mass spectrometer combined with a sonic spray ionization (SSI) source. An oligonucleotide with 20 bases was measured. Effects from contaminants and parameters affecting the ion production, such as a high voltage applied to the ionization source and sample solution-flow rate, were investigated. Our results showed that an ion with about one charge for every three bases was most abundant. However, the signal intensity and the mass spectrum pattern were sensitive to the matrix and operating parameters. One of the reasons for such sensitivity is that there are various ion states for an oligonucleotide. Any change in the matrix or an operating parameter may shift the balances between the ion states. Adding Tris, or (hydroxymethyl)aminomethane, enhanced the signal intensity of the oligonucleotide and promoted formation of the oligonucleotide ion with higher charges, while adding acetic acid favored the ions with lower charges, compared with that obtained in the medium without adding Tris and acetic acid. The effects on charged droplets and chemical enhancement were investigated. The mechanism for oligonucleotide ion formation is discussed.     

Solvent effect on analyte charge state,signal intensity,and stability in negative ion electrospray mass spectrometry; implications for the mechanism of negative ion formation

Department of Chemistry, University of New Orleans, New Orleans, Louisiana, USA The effect of solvent composition on negative ion electrospray ionization (ESI) mass spectrometry was examined. The onset potentials for ES1 of a series of chlorinated solvents and methanol were found to be within the range predicted by D. P. H. Smith, based on differences in the surface tension of the solvents used. The tendency toward electric discharge decreased with increasing percent weight of chlorine in the solvent. This effect has been attributed to an increasing propensity for electron capture for more highly chlorinated solvents. Addition of the electron scavenger gas SF, was even more effective at suppressing corona discharge phenomena. In a comparison of ultimate signal intensity obtainable for a test analyte in 10% methanol, the highest signal, which was stable over the widest range of temperatures, was exhibited by chloroform compared to dichloromethane, 1,2-dichloroethane, carbon tetrachloride, and methanol (100%). Chloroform, thus, is a recommended solvent for negative ion electrospray mass spectrometry (ES/MS) when solubility is not a limiting issue. Solvent polarity was shown to exhibit a profound influence on the distribution of charge states in negative ion ES/MS. For both chlorinated and nonchlorinated organic solvents, the higher the solution dielectric constant, the more the charge-state distribution is shifted toward higher charge states. These observations build on the “electrophoretic” mechanism of droplet charging. Solvents with high solution dielectric constants are considered to be most effective at stabilizing multiply charged ions (where charge separation is greatest), and they are likely to increase the level of droplet charging. Solvents with high basicities (gas phase and solution phase) and high proton affinities, yet low dielectric constants, favor lower charge states in ES mass spectra of lipid A and cardiolipin from Escherichia coli. This indicates that gas-phase processes and solvent basicity contribute much less toward ion formation than solution-phase solvation via preferred orientation of the solvent dipole.     

Further studies on the origins of asymmetric charge partitioning in protein homodimers

Dissociation of gas-phase protonated protein dimers into their constituent monomers can result in either symmetric or asymmetric charge partitioning. Dissociation of alpha-lactalbumin homodimers with 15+ charges results in a symmetric, but broad, distribution of protein monomers with charge states centered around 8+/7+. In contrast, dissociation of the 15+ heterodimer consisting of one molecule in the oxidized form and one in the reduced form results in highly asymmetric charge partitioning in which the reduced species carries away predominantly 11+ charges, and the oxidized molecule carries away 4+ charges. This result cannot be adequately explained by differential charging occurring either in solution or in the electrospray process, but appears to be best explained by the reduced species unfolding upon activation in the gas phase with subsequent separation and proton transfer to the unfolding species in the dissociation complex to minimize Coulomb repulsion. For dimers of cytochrome c formed directly from solution, the 17+ charge state undergoes symmetric charge partitioning whereas dissociation of the 13+ is asymmetric. Reduction of the charge state of dimers with 17+ charges to 13+ via gas-phase proton transfer and subsequent dissociation of the mass selected 13+ ions results in a symmetric charge partitioning. This result clearly shows that the structure of the dimer ions with 13+ charges depends on the method of ion formation and that the structural difference is responsible for the symmetric versus asymmetric charge partitioning observed. This indicates that the asymmetry observed when these ions are formed directly from solution must come about due either to differences in the monomer conformations in the dimer that exist in solution or that occur during the electrospray ionization process. These results provide additional evidence for the origin of charge asymmetry that occurs in the dissociation of multiply charged protein complexes and indicate that some solution-phase information can be obtained from these gas-phase dissociation experiments.     

Mechanism of charging and supercharging molecules in electrospray ionization

The origin of the extent of charging and the mechanism by which multiply charged ions are formed in electrospray ionization have been hotly debated for over a decade. Many factors can affect the number of charges on an analyte ion. Here, we investigate the extent of charging of poly(propyleneimine) dendrimers (generations 3.0 and 5.0), cytochrome c, poly(ethylene glycol)s, and 1,n-diaminoalkanes formed from solutions of different composition. We demonstrate that in the absence of other factors, the surface tension of the electrospray droplet late in the desolvation process is a significant factor in determining the overall analyte charge. For poly(ethylene glycol)s, 1,n-diaminoalkanes, and poly(propyleneimine) dendrimers electrosprayed from single-component solutions, there is a clear relationship between the analyte charge and the solvent surface tension. Addition of m-nitrobenzyl alcohol (m-NBA) into electrospray solutions increases the charging when the original solution has a lower surface tension than m-NBA, but the degree of charging decreases when this compound is added to water, which has a higher surface tension. Similarly, the charging of cytochrome c ions formed from acidified denaturing solutions generally increases with increasing surface tension of the least volatile solvent. For the dendrimers investigated, there is a strong correlation between the average charge state of the dendrimer and the Rayleigh limiting charge calculated for a droplet of the same size as the analyte molecule and with the surface tension of the electrospray solvent. A bimodal charge distribution is observed for larger dendrimers formed from water/m-NBA solutions, suggesting the presence of more than one conformation in solution. A similar correlation is found between the extent of charging for 1,n-diaminoalkanes and the calculated Rayleigh limiting charge. These results provide strong evidence that multiply charged organic ions are formed by the charged residue mechanism. A significantly smaller extent of charging for both dendrimers and 1,n-diaminoalkanes would be expected if the ion evaporation mechanism played a significant role.     

Origin of asymmetric charge partitioning in the dissociation of gas-phase protein homodimers

The origin of asymmetric charge and mass partitioning observed for gas-phase dissociation of multiply charged macromolecular complexes has been hotly debated. These experiments hold the potential to provide detailed information about the interactions between the macromolecules within the complex. Here, this unusual phenomenon of asymmetric charge partitioning is investigated for several protein homodimers. Asymmetric charge partitioning in these ions depends on a number of factors, including the internal energy, charge state, and gas-phase conformation of the complex, as well as the conformational flexibility of the protein monomer in the complex. High charge states of both cytochrome c and disulfide-reduced alpha-lactalbumin homodimers dissociate by a symmetrical charge partitioning process in which both fragment monomers carry away roughly an equal number of charges. In contrast, highly asymmetric charge partitioning dominates for the lower charge states. Cytochrome c dimer ions with eleven charges formed by electrospray ionization from two solutions in which the solution-phase conformation differs dissociate with dramatically different charge partitioning. These results demonstrate that these gas-phase complexes retain a clear "memory" of the solution from which they are formed, and that information about their solution-phase conformation can be obtained from these gas-phase dissociation experiments. Cytochrome c dimer ions formed from solutions in which the conformation of the protein is native show greater asymmetric charge partitioning with increasing ion internal energy. Cytochrome c dimers that are conformationally constrained with intramolecular cross-linkers undergo predominantly symmetric charge partitioning under conditions where asymmetric charge partitioning is observed for cytochrome c dimers without cross-links. Similar results are observed for alpha-lactalbumin homodimers. These results provide convincing evidence that the origin of asymmetric charge partitioning in these homodimers is the result of one of the protein monomers unfolding in the dissociation transition state. A mechanism that accounts for these observations is proposed.     

The role of conformation on electron capture dissociation of ubiquitin

Effects of protein conformation on electron capture dissociation (ECD) were investigated using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and Fourier-transform ion cyclotron resonance mass spectrometry. Under the conditions of these experiments, the electron capture efficiency of ubiquitin 6+ formed from three different solution compositions differs significantly, ranging from 51 +/- 7% for ions formed from an acidified water/methanol solution to 88 +/- 2% for ions formed from a buffered aqueous solution. This result clearly indicates that these protein ions retain a memory of their solution-phase structure and that conformational differences can be probed in an ECD experiment. Multiple conformers for the 7+ and 8+ charge states of ubiquitin were separated using FAIMS. ECD spectra of conformer selected ions of the same charge states differ both in electron capture efficiency and in the fragment ion intensities. Conformers of a given charge state that have smaller collisional cross sections can have either a larger or smaller electron capture efficiency. A greater electron capture efficiency was observed for ubiquitin 6+ that has the same collisional cross section as one ubiquitin 7+ conformer, despite the lower charge state. These results indicate that the shape of the molecule can have a greater effect on electron capture efficiency than either collisional cross section or charge state alone. The cleavage locations of different conformers of a given charge state were the same indicating that the presence of different conformers in the gas phase is not due to difference in where charges are located, but rather reflect conformational differences most likely originating from solution. Small neutral losses observed from the singly- and doubly-reduced ubiquitin 6+ do not show a temperature dependence to their formation, consistent with these ions being formed by nonergodic processes.     

Charge assisted laser desorption/ionization mass spectrometry of droplets

We propose and evaluate a new mechanism to account for analyte ion signal enhancement in ultraviolet-laser desorption mass spectrometry of droplets in the presence of corona ions. Our new insights are based on timing control of corona ion production, laser desorption, and peptide ion extraction achieved by a novel pulsed corona apparatus. We demonstrate that droplet charging rather than gas-phase ion-neutral reactions is the major contributor to analyte ion generation from an electrically isolated droplet. Implications of the new mechanism, termed charge assisted laser desorption/ionization (CALDI), are discussed and contrasted with those of the laser desorption atmospheric pressure chemical ionization method (LD-APCI). It is also demonstrated that analyte ion generation in CALDI occurs with external electric fields about one order of magnitude lower than those needed for atmospheric pressure matrix assisted laser desorption/ionization or electrospray ionization of droplets.     

Charge carrier field emission determines the number of charges on native state proteins in electrospray ionization

Although multiple charging in electrospray ionization (ESI) is essential to protein mass spectrometry, the underlying mechanism of multiple charging has not been explicated. Here, we present a new theory to describe ESI of native-state proteins and predict the number of excess charges on proteins in ESI. The theory proposes that proteins are ionized as charged residues in ESI, as they retain residual excess charges after solvent evaporation and do not desorb from charged ESI droplets. However, their charge state is not determined by the Rayleigh limit of a droplet of similar size to the protein; rather, their final charge state is determined by the electric field-induced emission of small charged solute ions and clusters from protein-containing ESI droplets. This theory predicts that the number of charges on a protein in ESI should be directly proportional to the square of the gas-phase protein diameter and to E*, the critical electric field strength at which ion emission from droplets occurs. This critical field strength is determined by the properties of the excess charge carriers (i.e., the solute) in droplets. Charge-state measurements of native-state proteins with molecular masses in the 5-76 kDa range in ammonium acetate and triethylammonium bicarbonate are in excellent agreement with theoretical predictions and strongly support the mechanism of protein ESI proposed here.     

Investigation on the role of pneumatic aspects in electrospray, desorption electrospray surface ionization and surface activated chemical ionization

This review reports the results of some studies carried out by us on the role of pneumatic aspects in electrospray and desorption electrospray surface ionization, with the aim to propose some relevant aspects of the mechanisms involved in these ionization methods. Electrospray ion sources, with the exception of the nano- electrospray source, operate with the concurrent action of a strong electrical field and a supplementary coaxial gas flow. The electrical field is responsible for electrospraying of the analyte solution but the use of a coaxial gas flow leads to a significant increase of the analyte signal and allows the use of higher solution flows. However, by employing capillary voltages much lower than those necessary to activate the electrospray phenomenon, analyte ions are still observed and this indicates that different mechanisms must be operative for ion production. Under these conditions, ion generation could take place from the neutral pneumatically sprayed droplet by field-induced droplet ionization. Also in the case of desorption electrospray ionization (DESI), and without any voltage on the spraying capillary as well as on the surface of interest, ions of analytes present on the surface become detectable and this shows that desorption/ionization of analytes occurs by neutral droplets impinging the surface. Consequently, the pneumatic effect of the impinging droplets plays a relevant role, and for these reasons the method has been called pneumatic assisted desorption (PAD). Some analogies existing between PAD and surface activated chemical ionization (SACI), based on the insertion of a metallic surface inside an atmospheric pressure chemical ionization source operating without corona discharge, are discussed.     

An ion trap-ion mobility-time of flight mass spectrometer with three ion sources for ion/ion reactions

This instrument combines the capabilities of ion/ion reactions with ion mobility (IM) and time-of-flight (TOF) measurements for conformation studies and top-down analysis of large biomolecules. Ubiquitin ions from either of two electrospray ionization (ESI) sources are stored in a three dimensional (3D) ion trap (IT) and reacted with negative ions from atmospheric sampling glow discharge ionization (ASGDI). The proton transfer reaction products are then separated by IM and analyzed via a TOF mass analyzer. In this way, ubiquitin +7 ions are converted to lower charge states down to +1; the ions in lower charge states tend to be in compact conformations with cross sections down to ～880 Ų. The duration and magnitude of the ion ejection pulse on the IT exit and the entrance voltage on the IM drift tube can affect the measured distribution of conformers for ubiquitin +7 and +6. Alternatively, protein ions are fragmented by collision-induced dissociation (CID) in the IT, followed by ion/ion reactions to reduce the charge states of the CID product ions, thus simplifying assignment of charge states and fragments using the mobility-resolved tandem mass spectrum. Instrument characteristics and the use of a new ion trap controller and software modifications to control the entire instrument are described.     

Hydrophobic and ionic interactions in nanosized water droplets

A number of situations such as protein folding in confined spaces, lubrication in tight spaces, and chemical reactions in confined spaces require an understanding of water-mediated interactions. As an illustration of the profound effects of confinement on hydrophobic and ionic interactions, we investigate the solvation of methane and methane decorated with charges in spherically confined water droplets. Free energy profiles for a single methane molecule in droplets, ranging in diameter (D) from 1 to 4 nm, show that the droplet surfaces are strongly favorable as compared to the interior. From the temperature dependence of the free energy in D = 3 nm, we show that this effect is entropically driven. The potentials of mean force (PMFs) between two methane molecules show that the solvent separated minimum in the bulk is completely absent in confined water, independent of the droplet size since the solute particles are primarily associated with the droplet surface. The tendency of methanes with charges (M(q+) and M(q-) with q(+) = |q(-)| = 0.4e, where e is the electronic charge) to be pinned at the surface depends dramatically on the size of the water droplet. When D = 4 nm, the ions prefer the interior whereas for D < 4 nm the ions are localized at the surface, but with much less tendency than for methanes. Increasing the ion charge to e makes the surface strongly unfavorable. Reflecting the charge asymmetry of the water molecule, negative ions have a stronger preference for the surface compared to positive ions of the same charge magnitude. With increasing droplet size, the PMFs between M(q+) and M(q-) show decreasing influence of the boundary owing to the reduced tendency for surface solvation. We also show that as the solute charge density decreases the surface becomes less unfavorable. The implications of our results for the folding of proteins in confined spaces are outlined.     

Study of the desorption/ionization mechanism in electrospray droplet impact secondary ion mass spectrometry

Electrospray droplet impact (EDI) secondary ion mass spectrometry (SIMS) is a desorption/ionization technique for mass spectrometry in which highly charged water clusters produced from an atmospheric-pressure electrospray are accelerated in vacuum by several kV and impact on the sample deposited on the metal substrate. The abundances of the secondary ions for C(60) and amino acids are measured as a function of the acceleration voltage of the primary charged water droplets. Two desorption/ionization mechanisms are suggested in the EDI ionization processes: low-energy and high-energy regimes. In the low-energy regime, the excess charges in the primary droplets play a role in the formation of secondary ions. In the high-energy regime, samples are ionized by the supersonic collision of the primary droplets with the sample. The yield of secondary ions increases by about three orders of magnitude with increase in the acceleration voltage of the primary droplets from 1.75 kV to 10 kV.     

Role of opposite charges in protein electrospray ionization mass spectrometry

The conformation dependence of protein spectra recorded by electrospray ionization mass spectrometry (ESI-MS) is an interesting and useful phenomenon, whose origin is still the object of debate. Different mechanisms have been invoked in the attempt to explain the lower charge state of folded versus unfolded protein ions in ESI-MS, such as electrostatic repulsions, solvent accessibility, charge availability, and native-like interactions. In this work we try to subject to direct experimental test the hypothesis that conformation-dependent neutralization of charges with polarity opposite to the net charge of the protein ion could play a critical role in such an effect. We present results of time-of-flight nano-ESI-MS on the peptide angiotensin II, indicating that negative charges of carboxylate groups can contribute to spectra recorded in positive-ion mode when stabilized by favorable electrostatic interactions, which is the central assumption of our hypothesis. Comparison of horse and spermwhale myoglobin (Mb) shows that changing the total number of basic residues within a given three-dimensional structure shifts the charge-state distribution (CSD) of the folded protein in positive-ion mode. This result appears to be in contrast to models in which electrostatic repulsions or availability of charges in the ESI droplets represent the limiting factor for the ionization of folded protein ions in ESI-MS. At the same time, it suggests a role of acidic residues in conformational effects in positive-ion mode. Furthermore, an attempt is made to rationalize those cases in which, in contrast, the main charge state observed in ESI-MS under non-denaturing conditions deviates considerably from the net charge expected on the basis of the amino-acid composition. These cases usually correspond to proteins with quite balanced content in basic and acidic residues, suggesting that this might be a factor influencing their charging behavior in ESI-MS. Experiments on mutants of ribonuclease Sa (RNase Sa) reveal that progressively reducing the excess of acidic residues, replacing them by lysine, causes almost no shift in the spectrum of the folded protein in negative-ion mode. Analogously, variants with an excess of three or five basic residues give similar spectra in positive-ion mode. These results indicate a lower limit to the extent of ionization observable by ESI-MS (6- or 8+ in the case of RNase Sa in water). Below such limit of net charge, changes in the relative amount of ionizable side chains do not affect the qualitative features of the observed CSDs. A progressive loss of signal intensity caused by the mutations in negative-ion mode suggests that low charge states might also be counterselected, even within the m/z range theoretically accessible to the instrument.     

Proton transfer reaction studies of multiply charged proteins in a high mass-to-charge ratio quadrupole mass spectrometer

Proton transfer reaction of multiply charged ions at high mass-to-charge ratios were explored with a low frequency quadrupole mass spectrometer. This instrument enabled a qualitative comparison of proton transfer reaction rates at low charge states for ions generated by electrospray ionization (ESI) from different solution conformations and for disulfide-linked versus disulfide-reduced protein ions. Proton transfer reactions that efficiently reduced the number of charges for ESI-generated ions to approximately the number of arginines in the polypeptide sequence were observed. No significant differences in gas-phase reaction rates were noted between different solution conformers. Differences in reaction rates between “native” and disulfide-reduced proteins were much smaller than those observed below m/z 2000 with lower proton affinity reagents or by using lower reagent concentrations. These smaller differences in reaction rates are thought to reflect the reduced electrostatic contributions from widely spaced charge sites and thus, the reduced sensitivity to an ion's three-dimensional structure or “compactness.”     

Fragmentation of organic ions bearing fixed multiple charges observed in MALDI MS

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI TOF MS) was used to analyze a series of synthetic organic ions bearing fixed multiple charges. Despite the multiple intrinsic charges, only singly charged ions were recorded in each case. In addition to the pseudo‐molecular ions formed by counterion adduction, deprotonation and electron capture, a number of fragment ions were also observed. Charge splitting by fragmentation was found to be a viable route for charge reduction leading to the formation of the observed singly charged fragment ions. Unlike multivalent metal ions, organic ions can rearrange and/or fragment during charge reduction. This fragmentation process will evidently complicate the interpretation of the MALDI MS spectrum. Because MALDI MS is usually considered as a soft ionization technique, the fragment ion peaks can easily be erroneously interpreted as impurities. Therefore, the awareness and understanding of the underlying MALDI‐induced fragmentation pathways is essential for a proper interpretation of the corresponding mass spectra. Due to the fragment ions generated during charge reduction, special care should be taken in the MALDI MS analysis of multiply charged ions. In this work, the possible mechanisms by which the organic ions bearing fixed multiple charges fragment are investigated. With an improved understanding of the fragmentation mechanisms, MALDI TOF MS should still be a useful technique for the characterization of organic ions with fixed multiple charges.     

On the relation between core electron binding energies of ions in solution and their solvation energies

Experimental and computational results from the study of positive and negative ions in solution are presented. The importance of short-range interactions between ion and solvent is studied with regard to core ionization of the ion. Exchange repulsion is found to be a significant factor in the interpretation of data for both cations and anions. Experimental results are presented for the core ionization of the OH^? ion in solution. The data show a strong similarity with corresponding data for the F^? ion, resulting in a large negative solvation energy for the final core hole state. The Be²⁺ ion shows large solvation energies for both ground- and core-ionized states, which is interpreted as due to charge transfer effects between solvent and ion.     

Effects of solvent and counterion on ion pairing and observed charge states of diquaternary ammonium salts in electrospray ionization mass spectrometry

Two diquaternary ammonium chloride salts have been used to examine the roles of solvent and counterion in determination of the degree of ion pairing in solution and the resultant charge state distributions in electrospray ionization mass spectrometry (ESI-MS). Three series of solvents, that is, alcohol, polar aprotic, and chlorinated solvents, have been employed to test the influence of solvent polarity and other parameters on the desorption behavior of diquaternary ammonium ions observed in ESI-MS. Solvents of higher polarity were found to yield gas-phase ions of higher charge states, in accordance with their reduced tendency toward ion pairing in solution. Counterion effects were investigated via the following approaches: (1) increase the diquaternary ammonium salt concentration; (2) increase the concentration of an external electrolyte that contained the common counterion Cl^?; (3) replace Cl^? with trifluoroacetate (TFA_c ^?); (4) increase the concentration of an external electrolyte that contained TFAc^?. These experiments indicate that variation of the specific counterion employed alters the degree of influence that the counterion exerts (via ion pairing) on electrospray ionization mass spectra. Increasing amounts of trifluoroacetate ions in a variety of solvent systems invariably led to a progressive shift of the observed ESI-MS charge states of diquaternary ammonium ions toward lower values.     

Communication: the formation of helium cluster cations following the ionization of helium nanodroplets: influence of droplet size and dopant

The He(n)(+)/He(2)(+) (n ≥ 3) signal ratios in the mass spectra derived from electron impact ionization of pure helium nanodroplets are shown to increase with droplet size, reaching an asymptotic limit at an average droplet size of approximately 50,000 helium atoms. This is explained in terms of a charge hopping model, where on average the positive charge is able to penetrate more deeply into the liquid helium as the droplet size increases. The deeper the point where the charge localizes to form He(2)(+), the greater the likelihood of collisions with the surrounding helium as the ion begins to leave the droplet, thus increasing the probability that helium will be ejected in the form of He(n)(+) (n ≥ 3) cluster ions rather than He(2)(+). The addition of a dopant alters the He(n)(+)/He(2)(+) ratio for small helium droplets, an observation attributed to the potential energy gradient created by the cation-dopant interaction and its effect in drawing the positive charge towards the dopant in the interior of the droplet.     

Evidence for unfolding and refolding of gas-phase cytochrome <Emphasis Type="Italic">c</Emphasis> ions in a Paul trap

The folding pathways of gas-phase cytochrome c ions produced by electrospray ionization have been studied by an ion trapping/ion mobility technique that allows conformations to be examined over extended timescales (10 ms to 10 s). The results show that the +9 charge state emerges from solution as a compact structure and then rapidly unfolds into several substantially more open structures, a transition that requires 30-60 ms; over substantially longer timescales (250 ms to 10 s) elongated states appear to refold into an array of folded structures. The new folded states are less compact than those that are apparent during the initial unfolding. Apparently, unfolding to highly open conformations is a key step that must occur before +9 ions can sample more compact states that are stable at longer times.