Structure of electron-capture dissociation fragments from charge-tagged peptides probed by tunable infrared multiple photon dissociation

In this study, we propose the first spectroscopic structural characterization of c-type ions produced by ECD of a peptide. The structure of c-type ions formed by electron capture dissociation and the overall mechanism leading to their formation are still a question of debate. Depending on the mechanism, c-type ions have been proposed to have either an enol-imine structure (-C(OH)NH) or an amide one (-C(O)-NH2). Since these ions are isomeric, mass spectrometry only cannot discriminate between them, but infrared spectroscopy can bring experimental evidence and help determine which scheme is operative. Using the coupling between a tunable free electron laser and a FT-ICR mass spectrometer, we show that c-type ions have an amide structure, characterized by an IR signature of the C=O stretch at 1731 cm(-1). This result is particularly interesting from the perspective of the elucidation of the ECD mechanism.     

Hydrogen rearrangement to and from radical z fragments in electron capture dissociation of peptides

Hydrogen rearrangement is an important process in radical chemistry. A high degree of H. rearrangement to and from z. ionic fragments (combined occurrence frequency 47% compared with that of z.) is confirmed in analysis of 15,000 tandem mass spectra of tryptic peptides obtained with electron capture dissociation (ECD), including previously unreported double H. losses. Consistent with the radical character of H. abstraction, the residue determining the formation rate of z' = z. + H. species is found to be the N-terminal residue in z. species. The size of the complementary c(m)' fragment turned out to be another important factor, with z' species dominating over z. ions for m < or = 6. The H. atom was found to be abstracted from the side chains as well as from alpha-carbon groups of residues composing the c' species, with Gln and His in the c' fragment promoting H. donation and Asp and Ala opposing it. Ab initio calculations of formation energies of .A radicals (A is an amino acid) confirmed that the main driving force for H. abstraction by z. is the process exothermicity. No valid correlation was found between the NC(alpha) bond strength and the frequency of this bond cleavage, indicating that other factors than thermochemistry are responsible for directing the site of ECD cleavage. Understanding hydrogen attachment to and loss from ECD fragments should facilitate automatic interpretation ECD mass spectra in protein identification and characterization, including de novo sequencing.     

On the mechanism of electron-capture-induced dissociation of peptide dications from 15n-labeling and crown-ether complexation

15N-labeling of di- and tripeptides reveals that electron capture to doubly protonated peptides results almost exclusively in ammonia loss from the N-terminal end, which clearly shows that a significant fraction of electron capture occurs at this end. In accordance with this finding, the competing channel of N-Calpha bond breakage leads to z+* ions and neutral c fragments after electron capture to small dications. In larger peptides that live long enough for internal proton exchanges to occur, c+ ions are also formed and in some cases in dominant yield. Attachment of one or two crown ethers to ammonium groups is likely to reduce the probability of proton transfer, which enhances the formation of z+* relative to c+. The total yield of z+* and c+ is, however, more or less unchanged, which indicates that proton transfer or hydrogen transfer from a NH3 group to the amide group is not required for the N-Calpha bond breakage.     

Investigation of c ions formed by N‐terminally charged peptides upon collision‐induced dissociation

Peptide fragments such as b and y sequence ions generated upon low‐energy collision‐induced dissociation have been routinely used for tandem mass spectrometry (MS/MS)‐based peptide/protein identification. The underlying formation mechanisms have been studied extensively and described within the literature. As a result, the ‘mobile proton model’ and ‘pathways in competition model’ have been built to interpret a majority of peptide fragmentation behavior. However, unusual peptide fragments which involve unfamiliar fragmentation pathways or various rearrangement reactions occasionally appear in MS/MS spectra, resulting in confused MS/MS interpretations. In this work, a series of unfamiliar c ions are detected in MS/MS spectra of the model peptides having an N‐terminal Arg or deuterohemin group upon low‐energy collision‐induced dissociation process. Both the protonated Arg and deuterohemin group play an important role in retention of a positive charge at the N‐terminus that is remote from the cleavage sites. According to previous reports and our studies involving amino acid substitutions and hydrogen–deuterium exchange, we propose a McLafferty‐type rearrangement via charge‐remote fragmentation as the potential mechanism to explain the formation of c ions from precursor peptide ions or unconventional b ions. Density functional theory calculations are also employed in order to elucidate the proposed fragmentation mechanisms. Copyright © 2016 John Wiley & Sons, Ltd.     

Bond dissociation of the dipeptide dialanine and its derivative alanine anhydride induced by low energy electrons

Dissociative electron attachment to dialanine and alanine anhydride has been studied in the gas phase utilizing a double focusing two sector field mass spectrometer. We show that low-energy electrons (i.e., electrons with kinetic energies from near zero up to 13 eV) attach to these molecules and subsequently dissociate to form a number of anionic fragments. Anion efficiency curves are recorded for the most abundant anions by measuring the ion yield as a function of the incident electron energy. The present experiments show that as for single amino acids (M), e.g., glycine, alanine, valine, and proline, the dehydrogenated closed shell anion (M-H)(-) is the most dominant reaction product. The interpretation of the experiments is aided by quantum chemical calculations based on density functional theory, by which the electrostatic potential and molecular orbitals are calculated and the initial electron attachment process prior to dissociation is investigated.     

Electron capture dissociation of complexes of diacylglycerophosphocholine and divalent metal ions: Competition between charge reduction and radical induced phospholipid fragmentation

Divalent metal complexes of phosphocholines, [Metal(II)(L)(n)](2+) (where Metal=Cu(2+), Co(2+), Mg(2+), and Ca(2+), L=1,2-dihexanoyl-sn-glycero-3-phosphocholine [6:0/6:0GPCho] and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine [16:0/18:1GPCho] and n=2-5), were formed upon electrospray ionization mass spectrometry (ESI/MS) of 8 mM solution of phosphocholine (L) with 4 mM metal salt (Metal). The electron capture dissociation (ECD) reactions of these [Metal(II)(L)(n)](2+) complexes were examined via Fourier-transform ion-cyclotron resonance mass spectrometry. A rich and complex chemistry was observed, including charge reduction and fragmentation involving losses of a methyl radical, trimethylamine, and the acyl chains. The predominant reaction channel was dependent on the size (n) of the complex, the metal and ligand used, and the size of the acyl chain. Thus charge reduction dominates the ECD spectra of the larger phosphocholine, 16:0/18:1GPCho, but is largely absent in the smaller 6:0/6:0GPCho. For complexes of 16:0/18:1GPCho, n=4-5, fragmentation from the head group mainly occurs via loss of the methyl radical and trimethylamine. At n=3, the relative abundance of fragments due to loss of acyl chain radicals increases. The abundances of ions arising from these radical losses increase further for the n=2 complexes, thereby providing information on the composition and position of the 16:0 and 18:1 acyl groups. Thus ECD of metal complexes provides structurally useful information on the phosphocholine, including the nature of the head group, the acyl chains, and the positions of the acyl chains.     

On the conformation of bipyridine dications and cation radicals

Ab initio STO-3G calculations with partial geometry optimization were performed to determine the conformational structure of 2,2′- and 4,4′-bipyridine dications and their cation radicals. The results suggest a considerably twisted conformation for the dications, whereas the corresponding radical cations are nearly planar. Reasons for the structure differences are discussed and related to the herbicidal action of these compounds.     

Solitonic waves in polyene dications and principles of charge carrier localization in π‐conjugated organic materials

The quantum‐chemical investigations by ab initio method (restricted Hartree–Fock/6‐31G**) have been performed for a series of unsubstituted, monosubstituted, and disubstituted neutral polyenes and their double charged cations. The waves of charge alternation (characterized by the difference in the electron densities at the nearest carbon atoms or Δq function) and bond length alternation (characterized by the lengths difference of the nearest carbon–carbon bonds or Δl function) are reported. Comparisons are made with the corresponding monocationic polymethine molecules. We found that ionization by two electrons results in formation of two solitonic waves of charge alternation, rather than superposition of two overlapping solitonic waves into one. These waves behave similar to two independent elastic particles, which do not penetrate into each other despite the special confinement by the length of chromophore π‐system. In monosubstituted polyene dication, Δq and Δl functions contain two waves each; however, only one wave is mobile and sensitive to a change of the chemical nature of the terminal group, whereas the second wave remains practically unchanged. The introduction of one oxymethyl or phenyl terminal groups leads to a relatively small shift of the mobile wave from the center to a direction of the terminal group. The effect of the amino or tropilium terminal groups is much more pronounced and leads to a shift of the mobile wave to the end of the molecule. In disubstituted polyene dication, both solitonic waves become mobile and shift symmetrically to both ends. The general principles of the charge localization described in this study may be used in molecular design and fine‐tuning of the charge transport properties in plastic photovoltaics and other organic semiconducting materials. © 2012 Wiley Periodicals, Inc.     

Electron capture induced dissociation of doubly protonated pentapeptides: dependence on molecular structure and charge separation

We have studied electron capture induced dissociation of a set of doubly protonated pentapeptides, all composed of one lysine (K) and either four glycine (G) or four alanine (A) residues, as a function of the sequence of these building blocks. Thereby the separation of the two charges, sequestered on the N-terminal amino group and the lysine side chain, is varied. The characteristic cleavage of N-C(α) bonds is observed for all peptides over the whole backbone length, with the charge carrying fragments always containing K. The resulting fragmentation patterns are very similar if G is replaced by A. In the case of [XKXXX+2H](2+) (X=A or G), a distinct feature is observed in the distribution of backbone cleavage fragments and the probability for ammonia loss is drastically reduced. This may be due to an isomer with an amide oxygen as protonation site giving rise to the observed increase in breakage at a specific site in the molecule. For the other peptides, a correlation with the distance between amide oxygen and the charge at the lysine side chain has been found. This may be an indication that it is only the contribution from this site to the charge stabilization of the amide π(*) orbitals which determines relative fragment intensities. For comparison, complexes with two crown ether molecules have been studied as well. The crown ether provides a shielding of the charge and prevents the peptide from folding and internal hydrogen bonding, which leads to a more uniform fragmentation behavior.     

The effect of charge state and the localization of charge on the collision-induced dissociation of peptide ions

The effect that charge state has on the collision-induced dissociation (CID) of peptide ions is examined in detail for several representative peptides under high-energy collision conditions. The CID spectra of singly and doubly charged precursor ions (generated by fast-atom bombardment and electrospray ionization, respectively) are compared for several peptides with similar primary structure. It is shown that for peptides that contain highly basic amino acids, the dissociation of doubly charged ions is strongly influenced by the position of these residues within the peptide and the general observations reported concerning the dissociation of singly charged ions can be extended to precursors with higher charge states. Based on the dissociation behavior of the doubly charged ions of these peptides, it is demonstrated that two charges can reside in close proximity in the precursor ions, overcoming possible repulsion effects, when favored by a high concentration of basic sites. In addition)’ this work illustrates that in the case of doubly charged ions..the charge state of some fragment ions can be determined directly from the mass-to-charge ratio assignments of the CID spectrum.     

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.     

Influence of charge state and sodium cationization on the electron detachment dissociation and infrared multiphoton dissociation of glycosaminoglycan oligosaccharides

Electron detachment dissociation (EDD) Fourier transform mass spectrometry has recently been shown to be a useful method for tandem mass spectrometry analysis of sulfated glycosaminoglycans (GAGs). EDD produces abundant glycosidic and cross-ring fragmentations that are useful for localizing sites of sulfation in GAG oligosaccharides. Although EDD fragmentation can be used to characterize GAGs in a single tandem mass spectrometry experiment, SO3 loss accompanies many peaks and complicates the resulting mass spectra. In this work we demonstrate the ability to significantly decrease SO3 loss by selection of the proper ionized state of GAG precursor ions. When the degree of ionization is greater than the number of sulfate groups in an oligosaccharide, a significant reduction in SO3 loss is observed in the EDD mass spectra. These data suggested that SO3 loss is reduced when an electron is detached from carboxylate groups instead of sulfate. Electron detachment occurs preferentially from carboxylate versus sulfate for thermodynamic reasons, provided that carboxylate is in its ionized state. Ionization of the carboxylate group is achieved by selecting the appropriate precursor ion charge state, or by the replacement of protons with sodium cations. Increasing the ionization state by sodium cation addition decreases, but does not eliminate, SO3 loss from infrared multiphoton dissociation of the same GAG precursor ions.     

Comparative study of the hydration systems formed during interactions of hydrogen phosphate dianions with putrescine, nor-putrescine and magnesium dications

A comparative study of hydration systems, formed as a result of the interaction between hydrogen phosphate dianions and three naturally occurring cations (putrescine (Put), its nor-homologue (nPut) and magnesium), is presented. On the basis of X-ray data and IR, NMR and calorimetric measurements, we have determined how the structure and physicochemical properties of the cations influence the system of phosphate residue hydration. Our study demonstrates that the stability of the hydration systems depends not only on the character of the bonds used by water to link with other salt components (coordinate or hydrogen bonds), but also on the location of the water molecules in the crystal lattice. In addition, contrary to magnesium salts, the dehydration of diamine (Put and nPut) hydrogen phosphates is reversible. Both dehydration and rehydration processes take place in the solid state. During rehydration, the crystalline anhydrous salt absorbs water molecules from the atmosphere. This leads to the reconstruction of the hydrated salt structure; this means that the salt which is the product of rehydration is identical with that obtained by crystallization from water solution.     

Thermochemistry of some π-π charge transfer complexes formed between methylbenzene donors and π-electron acceptors

The heats of formation for- ^* charge transfer interactions have been computed from the charge transfer spectra of molecular complexes formed between methylbenzene donors and-electron acceptors.Miss Rajni Sachdeva and Anita Singla are grateful to I.C.A.R. (India) and C. S. I. R. (India), respectively, for financial assistance.     

On the HSAB based estimate of charge transfer between adsorbates and metal surfaces

The applicability of the HSAB based electron charge transfer parameter, ΔN, is analyzed for molecular and atomic adsorbates on metal surfaces by means of explicit DFT calculations. For molecular adsorbates ΔN gives reasonable trends of charge transfer if work function is used for electronegativity of metal surface. For this reason, calculated work functions of low Miller index surfaces for 11 different metals are reported. As for reactive atomic adsorbates, e.g., N, O, and Cl, the charge transfer is proportional to the adatom valence times the electronegativity difference between the metal surface and the adatom, where the electronegativity of metal is represented by a linear combination of atomic Mulliken electronegativity and the work function of metal surface. It is further shown that the adatom-metal bond strength is linearly proportional to the metal-to-adatom charge transfer thus making the ΔN parameter a useful indicator to anticipate the corresponding adsorption energy trends.     

A comparative study of the collision induced dissociation and the electron capture dissociation of model peptides using ESI-FTMS

Tandem mass spectra of several model peptides, including KG3WG3K, NG3WG3N, RG7R, RG3WG3R, RG3DG3R, RG3EG3R, RG3FG3R and RG5WG5R were studied using both SORI-CID and ECD methods. By cross comparing the fragmentation pattern of these peptides using the same dissociation method and the same peptide using different dissociation methods, interesting spectral features that are related to the mechanisms of dissociation under SORI-CID and ECD conditions were extracted. Both dissociation methods were believed to be charge-directed. Due presumably to the stepwise ion activation, peptide ion dissociation under SORI-CID conditions was influenced mainly by "localized" hydrogen bonds. Consistent with previous literature findings, mobility proton model could be used to account for the spectral features observed. Substantial changes in the fragmentation patterns of these peptides were observed by using ECD methods. By postulating that the initial tertiary structures of the peptide ions were retained prior to electron capture process, the changes in fragmentation pattern could be attributed to the directing effect of the "global" hydrogen-bonding network. From the present results, no special preference was observed for cleavage at the backbone linkages adjacent to tryptophan residue over other inter-residual linkages. The previous reported nine-times cleavage preference at the C-terminal side of the tryptophan residue should therefore be attributed to some sequence specific phenomena.     

UV-vis, IR spectra and thermal studies of charge transfer complex formed between poly(amidoamine) dendrimers and iodine

The intermolecular charge-transfer (CT) complexes formed between two poly(amidoamine) dendrimers (PAMAM) from zero (D1) and second generation (D2) as donor and iodine as sigma-acceptor have been studied spectrophotometrically in the chloroform medium. The suggested structures of the solid iodine charge-transfer complexes investigated by several techniques using elemental analysis, mid infrared spectra, and thermal analysis (TGA and DTG) of the solid CT-complexes along with the photometric titration curves for the reactions. The results indicate the formation of two CT-complexes [(D1)]-I(2) and [(D2)]-2I(2) with acceptor:donor molar ratios of 1:1 and 1:2, respectively. The kinetic parameters (non-isothermal method) for their decomposition have been evaluated by graphical methods using the equations of Horowitz-Metzger (HM) and Coats-Redfern (CR).