Modeling the maximum charge state of arginine-containing peptide ions formed by electrospray ionization

A model for the gas-phase proton transfer reactivity of multiply protonated molecules is used to quantitatively account for the maximum charge states of a series of arginine-containing peptide ions measured by Downard and Biemann (Int. J. Mass Spectrom. Ion Processes 1995, 148, 191-202). We find that our calculations account exactly for the maximum charge state for 7 of the 10 peptides and are off by one charge for the remaining 3. These calculations clearly predict the trend in maximum charge states for these peptides and provide further evidence that the maximum charge state of ions formed by electrospray ionization is determined by their gas-phase proton transfer reactivity.     

Protein structural effects in gas phase ion/molecule reactions with diethylamine.

The relationship between gas-phase protein structure and ion/molecule reactivity is explored in comparisons between native and disulfide-reduced aprotinin, lysozyme, and albumin. Reactions are performed in the atmospheric-pressure inlet to a quadrupole mass spectrometer employing a novel capillary interface-reactor. In reactions with equal concentrations of diethylamine, multiply protonated molecules generated by electrospray ionization (ESI) of 'native' proteins shifted to lower charge states than did multiply protonated molecules from ESI of the disulfide-reduced counterparts, suggesting that the disulfide-reduced protein ions are less reactive than native protein ions of the same charge state. Differences in reactivity may arise from protonation of different amino acid residues and/or differences in the proximities of charge sites in the two molecules. These results suggest that the reactivity of multiply charged proteins can be significantly affected by their gas-phase structure.     

A New Model for Multiply Charged Adduct Formation Between Peptides and Anions in Electrospray Mass Spectrometry

A new model has been developed to account for adduct formation on multiply charged peptides observed in negative ion electrospray mass spectrometry. To obtain a stable adduct, the model necessitates an approximate matching of apparent gas-phase basicity (GB_app) of a given proton bearing site on the peptide with the gas-phase basicity (GB) of the anion attaching at that site. Evidence supporting the model is derived from the fact that for [Glu] Fibrinopeptide B, higher GB anions dominated in adducts observed at higher negative charge states, whereas lower GB anions appeared predominately in lower charge state adducts. Singly charged adducts were only observed for lower GB anions: HSO₄^–, I^–, CF₃COO^–. Ions that have medium GBs (NO₃^–, Br^–, H₂PO₄^–) only form adducts having −2 charge states, whereas Cl^– (higher GB) can form adducts having −3 charge states. The model portends that (1) carboxylate groups are much more basic than available amino groups; (2) apparent GBs of the various carboxylate groups on peptides do not vary substantially from one another; and (3) apparent GBs of the individual carboxylate and amino sites do not behave independently. This model was developed for negative ion attachment but an analogous mechanism is also proposed for the positive ion mode wherein (1) binding of a neutral at an amino site polarizes this amino group, but hardly affects apparent GBs of other sites; (2) proton addition (charge state augmentation) at one site can decrease the instrinsic GBs of other potential protonation sites and lower their apparent GBs.     

Gas-phase proton transfer reactions involving multiply charged cytochrome c ions and water under thermal conditions

Investigations of gas-phase proton transfer reactions have been performed on protein molecular ions generated by electrospray ionization (ESI). Their reactions were studied in a heated capillary inlet/reactor prior to expansion into a quadrupole mass spectrometer. Results from investigations involving protonated horse heart cytochrome c and H, O suggest that Coulombit effects can lower reaction barriers as well as aid in entropically driven reactions. For example, the charge state distribution observed by a quadrupole mass spectrometer for multiply protonated cytochrome c without the addition of any reactive gas ranges from 9+ to 19+ , with the [M + 15H]¹⁵⁺ ion being the most intense peak. With the addition of H₂O (proton affinity approximately 170.3±2 kcal/mol) to the capillary reactor at 120°C, the charge state distribution shifts to a lower charge, ranging from 13+ to less than 9+. Under the same conditions with argon (proton affinity approximately 100 kcal/mol) as the reactive gas, no shift in the charge state distribution is observed. The results demonstrate that proton transfer to water can occur for highly protonated molecular ions, a process that would be expected to be highly endothermic for singly protonated molecules (for which Coulombic destabilization is not significant). The results imply that the charge state distribution from ESI is somewhat dependent upon the mechanism and speed of the droplet evaporation/ion desolvation process, which may vary substantially with the ESI/mass spectrometry interface design.     

Proton transfer reactions of derivatized fullerene trications

The occurrence or absence of proton transfer from derivatized fullerene trications C₆₀H³⁺, to the parent neutral XH, is employed to obtain upper or lower limits to the apparent gas-phase acidity GA_app as well as to the estimated absolute gas-phase acidity. A comparison with the reactivity of analogous dicationic adducts indicates that C₆₀XH³⁺ is generally more acidic than C₆₀XH²⁺: for example, the difference in GA values for C₆₀NCCH ₃ ⁿ⁺ (n 5 2, 3; formed in the addition reaction of C ₆₀ ⁿ⁺ with CH₃CN) is estimated to be at least 42 kcal mol21. In almost all instances, estimated GA values for the tricationic adducts are far below the gas-phase basicities of the parent neutral XH, mirroring a trend seen from the proton-transfer reactivity of multiply protonated protein molecules: a qualitative difference between the fullerene adducts and the multiply protonated biomolecules routinely produced by electrospray ionization is that the fullerene adduct ions (with the exception of C₆₀NH ₃ ³⁺ ) appear to possess only one acidic proton.     

Surface-induced dissociation of singly and multiply protonated polypropylenamine dendrimers

The ease of fragmentation of various charge states of protonated polypropylenamine (POPAM) dendrimers is investigated by surface-induced dissociation. Investigated are the protonated diaminobutane propylenamines [DAB(PA)n] DAB(PA)8 (1+ and 2+), DAB(PA)16 (2+ and 3+), and DAB(PA)32 (3+ and 4+). These ions have been proposed to fragment by charge-directed intramolecular nucleophilic substitution (SNi) reactions. Differences in relative fragment ion abundances between charge states can be related to the occupation of different protonation sites. These positions can be rationalized based on estimates of Coulomb energies and gas-phase basicities of the protonation/fragmentation sites. The laboratory collision energies at which the fragment ion current is approximately 50% of the total ion current were found to increase with the size, but to be independent of charge state of the protonated POPAM dendrimers. It is suggested that intramolecular Coulomb repulsion within the multiply protonated POPAM dendrimers selected for activation does not readily result in easier fragmentation, which is in accordance with the proposed fragmentation mechanism.     

Gas-phase ion/ion reactions of multiply protonated polypeptides with metal containing anions

Gas-phase reactions of multiply protonated polypeptides and metal containing anions represent a new methodology for manipulating the cationizing agent composition of polypeptides. This approach affords greater flexibility in forming metal containing ions than commonly used methods, such as electrospray ionization of a metal salt/peptide mixture and matrix-assisted laser desorption. Here, the effects of properties of the polypeptide and anionic reactant on the nature of the reaction products are investigated. For a given metal, the identity of the ligand in the metal containing anion is the dominant factor in determining product distributions. For a given polypeptide ion, the difference between the metal ion affinity and the proton affinity of the negatively charged ligand in the anionic reactant is of predictive value in anticipating the relative contributions of proton transfer and metal ion transfer. Furthermore, the binding strength of the ligand anion to charge sites in the polypeptide correlates with the extent of observed cluster ion formation. Polypeptide composition, sequence, and charge state can also play a notable role in determining the distribution of products. In addition to their usefulness in gas-phase ion synthesis strategies, the reactions of protonated polypeptides and metal containing anions represent an example of a gas-phase ion/ion reaction that is sensitive to polypeptide structure. These observations are noteworthy in that they allude to the possibility of obtaining information, without requiring fragmentation of the peptide backbone, about ion structure as well as the relative ion affinities associated with the reactants.     

Protonation in electrospray mass spectrometry: Wrong-way-round or right-way-round?

The term “wrong-way-round ionization” has been used in studies of electrospray ionization to describe the observation of protonated or deprotonated ions when sampling strongly basic or acidic solutions (respectively) where such ions are not expected to exist in appreciable concentrations in solution. Study of the dependence of ionization of the weak base caffeine on the electrospray capillary potential reveals three distinct contributors to wrong-way-round ionization. At near-neutral pH in solutions of low ionic strength, protonation of caffeine results from the surface enrichment of electrolytically produced protons in the surface layer of the droplets from which ions are desorbed. For solutions made strongly basic with ammonia, gas-phase proton transfer from ammonium ions can create protonated caffeine. These two mechanisms have been discussed previously elsewhere. For solutions of high ionic strength at neutral or high pH, the data suggest that discharge-induced ionization is responsible for the production of protonated caffeine. This mechanism probably accounts for some of the wrong-way-round ionization reported elsewhere.     

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.     

Study of the efficiency for ion transfer through bent capillaries

Discontinuous atmospheric pressure interfaces (DAPIs) with bent capillaries represent a highly simplified and flexible means for introducing ions into a vacuum manifold for mass analysis or gas phase ion reactions. In this work, a series of capillaries of different radians and curvatures were used with DAPI for studying the impact of the capillary bending on the ion transfer. The variation of transfer efficiency was systematically characterized for dry and solvated ions. The efficiency loss for dry ions was less than one order of magnitude, even with a three‐turn bent capillary. The transfer of solvated ions generated by electrospray was found to be minimally impacted by the bending of the transfer capillary. For multiply protonated ions, the transfer efficiency for ions at lower charge states could be relatively well retained, presumably due to the lower reactivity associated with proton transfer reaction and the compensation in intensity by conversion of ions at higher charge states. Copyright © 2012 John Wiley & Sons, Ltd.     

Proton mobility and main fragmentation pathways of protonated lysylglycine

Theoretical model calculations were performed to validate the 'mobile proton' model for protonated lysylglycine (KG). Detailed scans carried out at various quantum chemical levels of the potential energy surface (PES) of protonated KG resulted in a large number of minima belonging to various protonation sites and conformers. Transition structures corresponding to proton transfer reactions between different protonation sites were determined, to obtain some energetic and structural insight into the atomic details of these processes. The rate coefficients of the proton transfer reactions between the isomers were calculated using the Rice-Ramsperger-Kassel-Marcus (RRKM) method in order to obtain a quantitative measure of the time-scale of these processes. Our results clearly indicate that the added proton is less mobile for protonated KG than for peptides lacking a basic amino acid residue. However, the energy needed to reach the energetically less favorable but-from the point of view of backbone fragmentation-critical amide nitrogen protonation sites is available in tandem mass spectrometers operated under low-energy collision conditions. Using the results of our scan of the PES of protonated KG, the dissociation pathways corresponding to the main fragmentation channels for protonated KG were also determined. Such pathways include loss of ammonia and formation of a protonated alpha-amino-epsilon-caprolactam. The results of our theoretical modeling, which revealed all the atomic details of these processes, are in agreement with the available experimental results.     

Effects of cation charge-site identity and position on electron-transfer dissociation of polypeptide cations

The effect of cation charge site on gas-phase ion/ion reactions between multiply protonated model peptides and singly charged anions has been examined. Insights are drawn from the quantitative examination of the product partitioning into competing channels, such as proton transfer (PT) versus electron transfer (ET), electron transfer followed by dissociation (ETD) versus electron transfer without dissociation (ET, no D), and fragmentation of backbone bonds versus fragmentation of side chains. Peptide cations containing protonated lysine, arginine, and histidine showed similar degrees of electron transfer, which were much higher than the peptide having fixed-charge sites, that is, trimethyl ammonium groups. Among the four types of cation charge sites, protonated histidine showed the highest degree of ET, no D, while no apparent intact electron-transfer products were observed for peptides with protonated lysine or arginine. All cation types showed side chain losses with arginine yielding the greatest fraction and lysine the smallest. The above trends were observed for each electron-transfer reagent. However, proton transfer was consistently higher with 1,3-dinitrobeznene anions, as was the fraction of side-chain losses. The partitioning of products among the various electron-transfer channels provides evidence for several of the mechanisms that have been proposed to account for electron-transfer dissociation and electron-capture dissociation. The simplest picture to account for all of the observations recognizes that several mechanisms can contribute to the observed products. Furthermore, the identity of the anionic reagent and the positions of the charge sites can affect the relative contributions of the competing mechanisms.     

“Best Match” Model and Effect of Na+/H+ Exchange on Anion Attachment to Peptides and Stability of Formed Adducts in Negative Ion Electrospray Mass Spectrometry

The “Best Match” model has been extended to account for the role that Na⁺/H⁺ exchange plays on anion attachment in negative ion electrospray. Without any Na⁺/H⁺ exchange on (Glu) fibrinopeptide B, the higher basicity anions F^? and CH₃COO^? can hardly form observable adducts; however, after multiple Na⁺/H⁺ exchanges, adduct formation is enabled. Moreover, dissociation pathways of CF₃COO^? adducts with singly deprotonated peptides that have undergone 0 to 3 Na⁺/H⁺ exchanges exhibit a shift in CID product ions from losing predominately CF₃COOH (case of 0 Na⁺/H⁺ exchanges) to losing predominately CF₃COO^? (case of 3 Na⁺/H⁺ exchanges). These phenomena can be rationalized by considering that Na⁺ cations exchange at, and serve to “block”, the most acidic sites, thereby forcing implicated anions to attach to lower acidity protons. In addition to forming ion pairs with carboxylate groups, Na⁺ also participates in formation of tri-atomic ions of the form ANaA^? during adduct dissociation. The fact that low gas-phase basicity (GB) anions preferentially form ANaA^? species, even though high GB anions form more stable tri-atomic species, indicates that the monatomic ions were not in close contact in the initial adduct. The propensity for formation of stable anionic adducts is dependent on the degree of matching between anion GBs and GB_app of deprotonated sites on the peptide. The GB_app is raised dramatically as the charge state of the peptide increases via a through-space effect. The presence of Na⁺ on carboxylate sites substantially decreases the GB_app by neutralizing these sites, while slightly increasing the intrinsic GBs by an inductive effect.

Gas-phase peptide fragmentation: how understanding the fundamentals provides a springboard to developing new chemistry and novel proteomic tools

This tutorial provides an overview of the evolution of some of the key concepts in the gas-phase fragmentation of different classes of peptide ions under various conditions [e.g. collision-induced dissociation (CID) and electron transfer dissociation (ETD)], and then demonstrates how these concepts can be used to develop new methods. For example, an understanding of the role of the mobile proton and neighboring group interactions in the fragmentation reactions of protonated peptides has led to the design of the 'SELECT' method. For ETD, a model based on the Landau-Zener theory reveals the role of both thermodynamic and geometric effects in the electron transfer from polyatomic reagent anions to multiply protonated peptides, and this predictive model has facilitated the design of a new strategy to form ETD reagent anions from precursors generated via ESI. Finally, two promising, emerging areas of gas-phase ion chemistry of peptides are also described: (1) the design of new gas-phase radical chemistry to probe peptide structure, and (2) selective cleavage of disulfide bonds of peptides in the gas phase via various physicochemical approaches.     

Role of the site of protonation in the low-energy decompositions of gas-phase peptide ions

The dissociation of singly or multiply protonated peptide ions by using low-energy collisional activation (CA) is highly dependent on the sites of protonation. The presence of strongly basic amino acid residues in the peptide primary structure dictates the sites of protonation, which generates a precursor ion population that is largely homogeneous with respect to charge sites. Attempts to dissociate this type of precursor ion population by low-energy CA result in poor fragmentation via few pathways. The work described here represents a systematic investigation of the effects of charge heterogeneity in the precursor ion population of a series of model peptides in low-energy CA experiments. Incorporation of acidic residues in the peptide RLC*IFSC*FR (where C* indicates a cysteic acid residue), for example, balances the charge on the basic arginine residues, which enables the ionizing protons to reside on a number of less basic sites along the peptide backbone. This results in a precursor ion population that is heterogeneous with respect to charge site. Low-energy CA of these ions results in diverse and efficient fragmentation. Molecular modeling has been utilized to demonstrate that energetically preferred conformations incorporate an intraionic interaction between arginine and cysteic acid residues.     

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.”     

Investigation of deprotonation reactions on globular and denatured proteins at atmospheric pressure by ESSI-MS

Deprotonation reactions of multiply charged protein ions have been studied by introducing volatile reference bases at atmospheric pressure between an electrosonic spray ionization (ESSI) source and the inlet of a mass spectrometer. Apparent gas-phase basicities (GB(app)) of different charge states of protein ions were determined by a bracketing approach. The results obtained depend on the conformation of the protein ions in the gas phase, which is linked to the type of buffer used (denaturing or nondenaturing). In nondenaturing buffer, the GB(app) values are consistent with values predicted by the group of Kebarle using an electrostatic model (J. Mass Spectrom.2002, 38, 618) based on the crystal structures, but taking into account salt bridges between ionized basic and acidic sites on the protein surface. A new basicity order for the most basic sites was therefore obtained. An excellent agreement with the charge residue model (CRM) is obtained when comparing the observed and calculated maximum charge state. Decharging of the proteins in the electrosonic spray process could be also useful in the study on noncovalent complexes, by decreasing repulsive electrostatic interactions. A unified mechanism of the ESSI process is proposed.     

A new approach for the study of gas-phase ion-ion reactions using electrospray ionization

A simple flow reactor which facilitates the study and application of ion-ion and ion-molecule reactions at near atmospheric pressures is reported. Reactant ions were generated by electrospray ionization and discharge ionization methods, although any ionization sources amenable to atmospheric pressure may be used. Ions of opposite charge are generated in spatially separate ion sources and are swept into capillary inlets where the flows are merged and where reaction(s) can occur. Among the reactions investigated were the partial neutralization of multiply protonated polypeptides and proteins such as melittin, bradykinin, cytochrome c, and myoglobin by reaction with discharge-generated anions, the partial neutralization of multiply charged anions of oligodeoxyadenylic acid (d(pA)₃) by reaction with discharge-generated cations, the partial neutralization of bovine A-chain insulin anions by reaction with myoglobin [M+nH]ⁿ⁺ ions, and the reaction of multiply protonated melittin with discharge-generated cations. The cation-anion reactions generally resulted in a shift to lower charge (higher mass-to-charge ratio) in the products’ charge state distributions and the transfer of solvent molecules to the macromolecule products. Multiply protonated melittin was detected in a less highly solvated state with the positive discharge in operation.     

Substituent effects on the gas-phase fragmentation reactions of sulfonium ion containing peptides

The multistage mass spectrometric (MS/MS and MS3) gas-phase fragmentation reactions of methionine side-chain sulfonium ion containing peptides formed by reaction with a series of para-substituted phenacyl bromide (XBr where X=CH2COC6H4R, and R=--COOH, --COOCH3, --H, --CH3 and --CH2CH3) alkylating reagents have been examined in a linear quadrupole ion trap mass spectrometer. MS/MS of the singly (M+) and multiply ([M++nH](n+1)+) charged precursor ions results in exclusive dissociation at the fixed charge containing side chain, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility). However, loss of the methylphenacyl sulfide side-chain fragment as a neutral versus charged (protonated) species was observed to be highly dependent on the proton mobility of the precursor ion, and the identity of the phenacyl group para-substituent. Molecular orbital calculations were performed at the B3LYP/6-31+G** level of theory to calculate the theoretical proton affinities of the neutral side-chain fragments. The log of the ratio of neutral versus protonated side-chain fragment losses from the derivatized side chain were found to exhibit a linear dependence on the proton affinity of the side-chain fragmentation product, as well as the proton affinities of the peptide product ions. Finally, MS3 dissociation of the nominally identical neutral and protonated loss product ions formed by MS/MS of the [M++H]2+ and [M++2H]3+ precursor ions, respectively, from the peptide GAILM(X)GAILK revealed significant differences in the abundances of the resultant product ions. These results suggest that the protonated peptide product ions formed by gas-phase fragmentation of sulfonium ion containing precursors in an ion trap mass spectrometer do not necessarily undergo intramolecular proton 'scrambling' prior to their further dissociation, in contrast to that previously demonstrated for peptide ions introduced by external ionization sources.     

Selective transport of amino acids into the gas phase: driving forces for amino acid solubilization in gas-phase reverse micelles

We report a study on encapsulation of various amino acids into gas-phase sodium bis(2-ethylhexyl) sulfosuccinate (NaAOT) reverse micelles, using electrospray ionization guided-ion-beam tandem mass spectrometry. Collision-induced dissociation of mass-selected reverse micellar ions with Xe was performed to probe structures of gas-phase micellar assemblies, identify solute-surfactant interactions, and determine preferential incorporation sites of amino acids. Integration into gas-phase reverse micelles depends upon amino acid hydrophobicity and charge state. For examples, glycine and protonated amino acids (such as protonated tryptophan) are encapsulated within the micellar core via electrostatic interactions; while neutral tryptophan is adsorbed in the surfactant layer. As verified using model polar hydrophobic compounds, the hydrophobic effect and solute-interface hydrogen-bonding do not provide sufficient driving force needed for interfacial solubilization of neutral tryptophan. Neutral tryptophan, with a zwitterionic structure, is intercalated at the micellar interface between surfactant molecules through complementary effects of electrostatic interactions between tryptophan backbone and AOT polar heads, and hydrophobic interactions between tryptophan side chain and AOT alkyl tails. Protonation of tryptophan could significantly improve its incorporation capacity into gas-phase reverse micelles, and displace its incorporation site from the micellar interfacial zone to the core; protonation of glycine, on the other hand, has little effect on its encapsulation capacity. Another interesting observation is that amino acids of different isoelectric points could be selectively encapsulated into, and transported by, reverse micelles from solution to the gas phase, based upon their competition for protonation and subsequent encapsulation within the micellar core.