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.     

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.     

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.     

On the maximum charge state and proton transfer reactivity of peptide and protein ions formed by electrospray ionization

A relatively simple model for calculation of the energetics of gas-phase proton transfer reactions and the maximum charge state of multiply protonated ions formed by electrospray ionization is presented. This model is based on estimates of the intrinsic proton transfer reactivity of sites of protonation and point charge Coulomb interactions. From this model, apparent gas-phase basicities (GB^app) of multiply protonated ions are calculated. Comparison of this value to the gas-phase basicity of the solvent from which an ion is formed enables a maximum charge state to be calculated. For 13 commonly electrosprayed proteins, our calculated maximum charge states are within an average of 6% of the experimental values reported in the literature. This indicates that the maximum charge state for proteins is determined by their gas-phase reactivity. Similar results are observed for peptides with many basic residues. For peptides with few basic residues, we find that the maximum charge state is better correlated to the charge state in solution. For low charge state ions, we find that the most basic sites Arg, Lys, and His are preferentially protonated. A significant fraction of the less basic residues Pro, Trp, and Gln are protonated in high charge state ions. The calculated GB^app of individual protonation sites varies dramatically in the high charge state ions. From these values, we calculate a reduced cross section for proton transfer reactivity that is significantly lower than the Langevin collision frequency when the GB^app of the ion is approximately equal to the GB of the neutral base.     

Dissociation of tetrameric ions of noncovalent streptavidin complexes formed by electrospray ionization

The noncovalent tetrameric association of the protein streptavidin formed by electrospray ionization (ESI) mass spectrometry has been observed intact and dissociated in the gas phase. An extended mass-to-charge ratio range quadrupole mass spectrometer was employed to examine the effects of harsher conditions in the ESI atmosphere-vacuum interface region on the streptavidin tetramer. Thermally induced dissociation caused the mass spectra to exhibit a series of complementary monomer and trimer ions that correspond to decomposition of the tetrameric species. Similar results were obtained with tandem mass spectrometric experiments on a Fourier transform ion cyclotron resonance mass spectrometer by application of sustained off-resonance irradiation (SORI) on a selected tetrameric charge state. The technique of single-frequency quadrupole excitation was used to accomplish selected-ion accumulation of the 14 + charge state of the tetramer during ion injection. Subsequent low energy SORI combined with broadband quadrupole cooling produced the 7 + monomer and 7 + trimer species, as well as the 6 + monomer and 8 + trimer complementary ions. The observed asymmetric breakup of the tetramer is qualitatively explained by using physical models.     

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]+.     

Interaction of angiotensin peptides and zinc metal ions probed by electrospray ionization mass spectrometry

Electrospray ionization-tandem mass spectrometry experiments were used to provide evidence regarding the sites of interactions between zinc metal ions and angiotensin peptides. The electrospray ionization mass spectra of histidine-containing human angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) and angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) in the presence of zinc show abundant multiply charged ions for the zinc-attached peptide [M + aZn²⁺ +(c ? 2a)H⁺]^c+, where a = 1, 2 and c is charge. From collisionally activated dissociation experiments, with both low energy (triple quadrupole mass spectrometry) and high energy collisions (linked scan at constant B/E with a double focusing instrument) of the [M + Zn]²⁺ and [M + Zn + H]³⁺ ions for angiotensin II, a [b ₆ + Zn]²⁺ species is produced as the most abundant product ion, suggesting that the zinc interaction site is in the vicinity of the His⁶ residue. Additionally, tandem mass spectra from the zinc-attached ions for angiotensin I show abundant [b ₆ + Zn]²⁺ and [b ₉ + Zn]²⁺ products, providing evidence that both His⁶ and His⁹ are involved in zinc coordination.     

Effects of solvent on the maximum charge state and charge state distribution of protein ions produced by electrospray ionization

The effects of solvent composition on both the maximum charge states and charge state distributions of analyte ions formed by electrospray ionization were investigated using a quadrupole mass spectrometer. The charge state distributions of cytochrome c and myoglobin, formed from 47%/50%/3% water/solvent/acetic acid solutions, shift to lower charge (higher m/z) when the 50% solvent fraction is changed from water to methanol, to acetonitrile, to isopropanol. This is also the order of increasing gas-phase basicities of these solvents, although other physical properties of these solvents may also play a role. The effect is relatively small for these solvents, possibly due to their limited concentration inside the electrospray interface. In contrast, the addition of even small amounts of diethylamine (<0.4%) results in dramatic shifts to lower charge, presumably due to preferential proton transfer from the higher charge state ions to diethylamine. These results clearly show that the maximum charge states and charge state distributions of ions formed by electrospray ionization are influenced by solvents that are more volatile than water. Addition of even small amounts of two solvents that are less volatile than water, ethylene glycol and 2-methoxyethanol, also results in preferential deprotonation of higher charge state ions of small peptides, but these solvents actually produce an enhancement in the higher charge state ions for both cytochrome c and myoglobin. For instruments that have capabilities that improve with lower m/z, this effect could be taken advantage of to improve the performance of an analysis.     

Translational energy spectroscopy of NO+ ions formed by charge transfer from Ar+

Translational energy spectroscopy (TES) of NO⁺ ions formed by Ar⁺ charge exchange has been studied. The two features observed in the spectrum are assigned to transitions from the υ″ = 0 and possibly υ″ = 1 and 2 levels of the a³Σ⁺ state to the low vibrational levels of the w³Δ and b′³Σ⁻ states. Comparison with previous TES spectra of NO⁺ formed by electron impact is reported and demonstrates the high selectivity of the charge transfer reaction in populating the first excited state of NO⁺.     

The importance of charge-separation reactions in tandem mass spectrometry of doubly protonated angiotensin II formed by electrospray ionization: Experimental considerations and structural implications

The occurrence of charge-separation reactions in tandem mass spectrometry of doubly protonated angiotensin II is demonstrated by the use of mass-analyzed ion kinetic energy spectrometry (MIKES) and kinetic energy release distributions (KERDs). Linked scans at a constant B/E severely discriminate against product ions formed by charge-separation reactions. Although the products are significantly more abundant in MIKES experiments, instrumental discrimination still makes quantitation of relative product ion abundances highly inaccurate. The most probable KERs (T _{m. p.}) and the average KERs (T _ave.) of the reactions are determined from the KERDs, and these values are compared to the KERs determined from the peak widths at half-height (T _{0. 5}). The measurement of T _{0. 5} is a poor approximation to T _{m. p.} and T _ave.. The T _{m. p.} is used to calculate a most probable intercharge distance, which is compared to results from molecular dynamics calculations. The results provide evidence with regard to the mechanisms of fragmentation of multiply charged ions and the location of the charge site in relation to the decomposition reactions.     

Differences in the keto–enol equilibrium of [M + H]+ ions of bupropion formed by electron impact and fast atom bombardment ionization

The fragmentation of the protonated molecular ion of bupropion produced by collisionally induced decomposition is shown to depend on the ionization method used to form the [M + H]⁺ ion. The daughter ion products do not depend on the energy of decomposition, i.e. high- or low-energy collisions, but on the ratio of the keto-enol equilibrium as influenced by the ionization process.     

Gas-phase H/D exchange reactions of polyamine complexes: (M + H)+, (M + alkali metal+), and (M + 2H)2+

Gas-phase hydrogen/deuterium exchange reactions between noncovalent polyamine complexes and D2O, CH3OD, or ND3 are undertaken in a quadrupole ion trap mass spectrometer. Structural features of the protonated polyamines can be differentiated by the rates and overall extent of exchange, specifically the presence of propylene units and/or a cyclic structure noticeably decreases exchange compared to the exchange observed for acyclic polyamines with only ethylene bridges between amino groups. Significant differences are observed for singly protonated vs. doubly protonated complexes, where the doubly protonated complexes undergo more efficient exchange at a higher rate than the analogous singly protonated complexes. Molecular modeling calculations suggest that more diffuse conformations may exist for the higher charge states, thus facilitating H/D exchange. In addition, H/D exchange reactions between the alkali metal cationized complexes and ND3 are nearly quenched, compared to the significant exchange seen for singly protonated complexes. A conformational change or the loss of a low energy reaction pathway may explain the limited exchange reactions seen when a bulky cation replaces a proton in the complex.     

Multiply charged ions of ruthenium(II), rhodium(III) and cobalt(III) complexes in electrospray ionization mass spectrometry

Multiply charged ions from electrospray ionization (ESI) were observed for ruthenium-bidentate ligand complexes, such as [RuL₂B]X₂ and [(RuL₂)₂B]X₄, where L is 2,2′-bipyridine, B are tetradentate ligands of 2,2′-bis(2′-pyridyl)bibenzimidazole and 2,6-bis(2′-pyridyl)benzodiimidazole, bidentate ligand of 2-(2′-pyridyl)benzimidazole and related compounds and X is CIO₄^- or CI^-. ESI mass spectra showed a simple mass pattern for easy structural assignment and detecting impurities. The mass spectra for binuclear complexes provide a charge state distribution ranging from 4+ to 2+ for Ru(II)—Ru(II) compounds and 5+ to 2+ for Ru(II)—Rh(III) compounds. It was found that different multiply charged ions are generated by loss of counterions and by protonation/deprotonation at the proton site of ligands B. The abundances of these ions are qualitatively explained in terms of the acidity of metal complexes depending on the bridging ligand structures and the charge of the metal ions. Ions produced by removal of ligands were hardly observed.     

Generation of gas-phase VO2+, VOOH+, and VO2+-nitrile complex ions by electrospray ionization and collision-induced dissociation

Cationic metal species normally function as Lewis acids, accepting electron density from bound electron-donating ligands, but they can be induced to function as electron donors relative to dioxygen by careful control of the oxidation state and ligand field. In this study, cationic vanadium(IV) oxohydroxy complexes were induced to function as Lewis bases, as demonstrated by addition of O2 to an undercoordinated metal center. Gas-phase complex ions containing the vanadyl (VO2+), vanadyl hydroxide (VOOH+), or vanadium(V) dioxo (VO2+) cation and nitrile (acetonitrile, propionitrile, butyronitrile, or benzonitrile) ligands were generated by electrospray ionization (ESI) for study by multiple-stage tandem mass spectrometry. The principal species generated by ESI were complexes with the formula [VO(L)n]2+, where L represents the respective nitrile ligands and n=4 and 5. Collision-induced dissociation (CID) of [VO(L)5]2+ eliminated a single nitrile ligand to produce [VO(L)4]2+. Two distinct fragmentation pathways were observed for the subsequent dissociation of [VO(L)4]2+. The first involved the elimination of a second nitrile ligand to generate [VO(L)3]2+, which then added neutral H2O via an association reaction that occurred for all undercoordinated vanadium complexes. The second [UO(L)4]2+ fragmentation pathway led instead to the formation of [VOOH(L)2]+ through collisions with gas-phase H2O and concomitant losses of L and [L+H]+. CID of [VOOH(L)2]+ caused the elimination of a single nitrile ligand to generate [VOOH(L)]+, which rapidly added O2 (in addition to H2O) by a gas-phase association reaction. CID of [VONO3(L)2]+, generated from spray solutions created by mixing VOSO4 and Ba(NO3)2 (and precipitation of BaSO4), caused elimination of NO2 to produce [VO2(L)2]+. CID of [VO2(L)2]+ produced elimination of a single nitrile ligand to form [VO2(L)]+, a V(V) analogue to the O2-reactive V(IV) species [VOOH(L)]+; however, this V(V) complex was unreactive with O2, which indicates the requirement for an unpaired electron in the metal valence shell for O2 addition. In general, the [VO2(L)2]+ species required higher collisions energies to liberate the nitrile ligand, suggesting that they are more strongly bound than the [VOOH(L)2]+ counterparts.     

Binding sites of [Ru(bpy)2(H2O)2](BF4)2 with sulfur- and histidine-containing peptides studied by electrospray ionization mass spectrometry and tandem mass spectrometry

The composition and binding sites of cis-[Ru(II)(bpy)2]2+-bound sulfur-containing peptides of Met-Arg-Phe-Ala, glutathione and oxidized glutathione, and also histidine-containing peptide of oxidized insulin B chain, were investigated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). The composition of Ru(II)-containing peptides was precisely determined by ESI-MS, zoom scan and simulation of isotope distribution patterns. MS/MS analysis shows that, in sulfur-containing peptides, the Ru(II) complex prefers to anchor to a carboxyl group, although some other potential binding sites of thiol, thioether and N-terminal amino groups present in these peptides, and in oxidized insulin B chain, Ru(II) first anchors to His10, then either to the hydroxyl group of Thr27 or to the carboxyl group of Ala30. Its secondary structure and microenvironment surrounding the potential binding sites may affect the binding ability of cis-[Ru(II)(bpy)2]2+ to oxidized insulin B chain.     

Reduction of copper(II) complexes by electron capture in an electrospray ionization source

The relative proportion of 1:1 Cu(I)– and Cu(II)–peptide complexes PeptCu(I)⁺ and [Pept?H+Cu(II)]⁺ yielded by electrospray ionization of copper sulfate and GlyHisLys solutions in water/methanol was examined under different source conditions. Two factors leading to an increase in Cu(I) complex ratio were found. (1) Increase of nozzle–skimmer voltages caused collision-induced dissociation of Cu(II) complexes, and most probably favor ligand-to-metal electron transfers that result in the decoordination of oxydated ligands to form PeptCu⁺. (2) Independent of these “innersphere” processes that involve only electron exchange inside the coordination sphere around the metal cation, an increase in source voltages with a concomitant increase of current and, supposedly, electron counterflow between the counterelectrode and the capillary caused an increase in PeptCu⁺ relative proportion. The hypothesis that an “outersphere” electron capture might happen in these conditions was verified by using discharge supressing SF₆ gas as nebulizing gas. The electronegative gas reduced the current brought on by high voltages and inhibited the PeptCu⁺ increase phenomenon.     

Structure and fragmentation of [C3H7O]+ ions formed by chemical ionization

The unimolecular metastable and collision-induced fragmentation reactions of [C₃H₇O]⁺ ions produced by gas-phase protonation of acetone, propanal, propylene oxide, oxetan and allyl alcohol have been studied. The CID studies show that protonation of acetone and allyl alcohol yield different stable ions with distinct structures while protonation of propanal or propylene oxide yield [C₃H₇O]⁺ ions of the same structure. Protonated oxetan rearranges less readily to give the same structure(s) as protonated propanal and propylene oxide. The [C₃H₇O]⁺ ions fragmenting as metastable ions after formation by CI have a higher internal energy than the same ions fragmenting after formation by EI. Deuteronation of the C₃H₆O isomers using CD₄ reagent gas shows that loss of C₂H₃D proceeds by a different mechanism than loss of C₂H₄. The results are discussed in terms of potential energy profile for the [C₃H₇O]⁺˙ system proposed earlier.     

Structure and fragmentation of [C6H13O]+ ions formed by chemical ionization

The structure and fragmentation of eight [C₆H₁₃O] ⁺ ions formed by protonation of C₆H₁₂O carbonyl compounds in the gas phase have been investigated using isotopic labeling and metastable ion studies to investigate the fragmentation reactions and collisional dissociation studies to probe ion structures. Protonated 3-methyl-2-pentanone and protonated 2-methyl-3-pentanone readily-interconvert by pinacolic-retro-pinacolic rearrangements; the remaining six ions represent stable ion structures, although in many cases fragmentation is preceded by pinacolic-type rearrangements. Unimolecular (metastable ion) fragmentation of the [C₆H₁₃O] ⁺ species occurs by elimination of H₂O, C₃H₆, C₄H₈ and C₂H₄O. The last three elimination reactions appear to occur through the intermediacy of a proton-bound complex of a carbonyl compound and an olefin, with the proton residing with the species of higher proton affinity on decomposition of the complex.