Electron capture in spin-trap capped peptides. An experimental example of ergodic dissociation in peptide cation-radicals

Electron capture dissociation was studied with tetradecapeptides and pentadecapeptides that were capped at N-termini with a 2-(4'-carboxypyrid-2'-yl)-4-carboxamide group (pepy), e.g., pepy-AEQLLQEEQLLQEL-NH(2), pepy-AQEFGEQGQKALKQL-NH(2), and pepy-AQEGSEQAQKFFKQL-NH(2). Doubly and triply protonated peptide cations underwent efficient electron capture in the ion-cyclotron resonance cell to yield charge-reduced species. However, the electron capture was not accompanied by backbone dissociations. When the peptide ions were preheated by absorption of infrared photons close to the dissociation threshold, subsequent electron capture triggered ion dissociations near the remote C-terminus forming mainly (b(11-14) + 1)(+)* fragment ions that were analogous to those produced by infrared multiphoton dissociation alone. Ab initio calculations indicated that the N-1 and N-1' positions in the pepy moiety had topical gas-phase basicities (GB = 923 kJ mol(-1)) that were greater than those of backbone amide groups. Hence, pepy was a likely protonation site in the doubly and triply charged ions. Electron capture in the protonated pepy moiety produced the ground electronic state of the charge-reduced cation-radical with a topical recombination energy, RE = 5.43-5.46 eV, which was greater than that of protonated peptide residues. The hydrogen atom in the charge-reduced pepy moiety was bound by >160 kJ mol(-1), which exceeded the hydrogen atom affinity of the backbone amide groups (21-41 kJ mol(-1)). Thus, the pepy moiety functioned as a stable electron and hydrogen atom trap that did not trigger radical-type dissociations in the peptide backbone that are typical of ECD. Instead, the internal energy gained by electron capture was redistributed over the peptide moiety, and when combined with additional IR excitation, induced proton-driven ion dissociations which occurred at sites that were remote from the site of electron capture. This example of a spin-remote fragmentation provided the first clear-cut experimental example of an ergodic dissociation upon ECD.     

Backbone and side-chain specific dissociations of <Emphasis Type="Italic">z</Emphasis> ions from non-tryptic peptides

Backbone z-type fragment ions formed by electron-transfer dissociation (ETD) of doubly protonated peptides AAHAL, AHDAL, and AHADL were subjected to collisional activation and their dissociation products were studied by ETD-CID-MS³ and MS⁴. Electron structure theory calculations were performed to elucidate ion structures and reaction mechanisms. All z ions showed competitive eliminations of C₃H₇ and C₄H₈ from the C-terminal Leu side chain. The energetics and kinetics of these dissociations were studied computationally for the z₄ ion from AAHAL, and optimized structures are reported for several intermediates and transition states. RRKM calculations on the combined B3LYP and PMP2/6-311++G(2d,p) potential energy surface provided unimolecular rate constants that closely reproduced the experimental branching ratios for C₃H₇ and C₄H₈ eliminations. Mechanisms were also studied for the loss of CO₂ from z ions generated by ETD of AHDAL and AHADL and for a specific radical-induced Asp-C_α-CO backbone cleavage. CID of the z ions under study did not produce any fragment ions that would indicate cascade backbone dissociations triggered by the radical sites. In contrast, the majority of backbone dissociations occurred at bonds that were remote from the radical sites (spin-remote dissociations) and were triggered by proton migrations that were analogous to those considered for standard peptide ion fragmentations.     

Stereospecific control of peptide gas‐phase ion chemistry with cis and trans cyclo ornithine residues

We report non‐chiral amino acid residues cis‐ and trans‐1,4‐diaminocyclohexane‐1‐carboxylic acid (cyclo‐ornithine, cO) that exhibit unprecedented stereospecific control of backbone dissociations of singly charged peptide cations and hydrogen‐rich cation radicals produced by electron‐transfer dissociation. Upon collision‐induced dissociation (CID) in the slow heating regime, peptide cations containing trans‐cO residues undergo facile backbone cleavages of amide bonds C‐terminal to trans‐cO. By contrast, peptides with cis‐cO residues undergo dissociations at several amide bonds along the peptide ion backbone. Diastereoisomeric cO‐containing peptides thus provide remarkably distinct tandem mass spectra. The stereospecific effect in CID of the trans‐cO residue is explained by syn‐facially directed proton transfer from the 4‐ammonium group at cO to the C‐terminal amide followed by neighboring group participation in the cleavage of the CO―NH bond, analogous to the aspartic acid and ornithine effects. Backbone dissociations of diastereoisomeric cO‐containing peptide ions generate distinct [b_n]⁺‐type fragment ions that were characterized by CID‐MS³ spectra. Stereospecific control is also reported for electron‐transfer dissociation of cis‐ and trans‐cO containing doubly charged peptide ions. The stereospecific effect upon electron transfer is related to the different conformations of doubly charged peptide ions that affect the electron attachment sites and ensuing N―C_α bond dissociations.     

Where Does the Electron Go? Stable and Metastable Peptide Cation Radicals Formed by Electron Transfer

Electron transfer to doubly and triply charged heptapeptide ions containing polar residues Arg, Lys, and Asp in combination with nonpolar Gly, Ala, and Pro or Leu generates stable and metastable charge-reduced ions, (M + 2H)^+●, in addition to standard electron-transfer dissociation (ETD) fragment ions. The metastable (M + 2H)^+● ions spontaneously dissociate upon resonant ejection from the linear ion trap, giving irregularly shaped peaks with offset m/z values. The fractions of stable and metastable (M + 2H)^+● ions and their mass shifts depend on the presence of Pro-4 and Leu-4 residues in the peptides, with the Pro-4 sequences giving larger fractions of the stable ions while showing smaller mass shifts for the metastables. Conversion of the Asp and C-terminal carboxyl groups to methyl esters further lowers the charge-reduced ion stability. Collisional activation and photodissociation at 355 nm of mass-selected (M + 2H)^+● results in different dissociations that give sequence specific MS³ spectra. With a single exception of charge-reduced (LKGLADR + 2H)^+●, the MS³ spectra do not produce ETD sequence fragments of the c and z type. Hence, these (M + 2H)^+● ions are covalent radicals, not ion–molecule complexes, undergoing dramatically different dissociations in the ground and excited electronic states. The increased stability of the Pro-4 containing (M + 2H)^+● ions is attributed to radicals formed by opening of the Pro ring and undergoing further stabilization by hydrogen atom migrations. UV–VIS photodissociation action spectroscopy and time-dependent density functional theory calculations are used in a case in point study of the stable (LKGPADR + 2H)^+● ion produced by ETD. In contrast to singly-reduced peptide ions, doubly reduced (M + 3H)⁺ ions are stable only when formed from the Pro-4 precursors and show all characteristics of even electron ions regarding no photon absorption at 355 nm or ion-molecule reactions, and exhibiting proton driven collision induced dissociations.

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Graphical Abstract ?

     

Comparative dissociation of peptide polyanions by electron impact and photo-induced electron detachment

We compare product-ion mass spectra produced by electron detachment dissociation (EDD) and electron photodetachment dissociation (EPD) of multi-deprotonated peptides on a Fourier transform and a linear ion trap mass spectrometer, respectively. Both methods, EDD and EPD, involve the electron emission-induced formation of a radical oxidized species from a multi-deprotonated precursor peptide. Product-ion mass spectra display mainly fragment ions resulting from backbone cleavages of C_α-C bond ruptures yielding a and x ions. Fragment ions originating from N-C_α backbone bond cleavages are also observed, in particular by EPD. Although EDD and EPD methods involve the generation of a charge-reduced radical anion intermediate by electron emission, the product ion abundance distributions are drastically different. Both processes seem to be triggered by the location and the recombination of radicals (both neutral and cation radicals). Therefore, EPD product ions are predominantly formed near tryptophan and histidine residues, whereas in EDD the negative charge solvation sites on the backbone seem to be the most favorable for the nearby bond dissociation.     

Electron ionization mass spectrometry of systematically modified ligands in chromium(III) β-diketonates

The positive ion mass spectra of several chromium(III) β-diketonates with aliphatic α-substituents have been investigated. Metastable peaks in the spectra confirm that ions containing substituents with γ-H atoms undergo propene loss. This implies a McLafferty rearrangement of an open-chain ligand structure. Ethyl radicals are lost from n-butyl substituents; methyl groups are cleaved from the molecular ions of complexes formed from methyl-substituted ligands. The main fragment is, as expected, [ML₂]⁺; however, its further fragmentation is different from that of [ML₃]⁺. Electron donating substituents stabilize doubly charged molecular ions.     

The arginine anomaly: arginine radicals are poor hydrogen atom donors in electron transfer induced dissociations

Arginine amide radicals are generated by femtosecond electron transfer to protonated arginine amide cations in the gas phase. A fraction of the arginine radicals formed (2-amino-5-dihydroguanid-1'-yl-pentanamide, 1H) is stable on the 6.7 micros time scale and is detected after collisional reionization. The main dissociation of 1H is loss of a guanidine molecule from the side chain followed by consecutive dissociations of the 2-aminopentanamid-5-yl radical intermediate. Intramolecular hydrogen atom transfer from the guanidinium group onto the amide group is not observed. These results are explained by ab initio and density functional theory calculations of dissociation and transition state energies. Loss of guanidine from 1H is calculated to require a transition state energy of 68 kJ mol(-)(1), which is substantially lower than that for hydrogen atom migration from the guanidine group. The loss of guanidine competes with the reverse migration of the arginine alpha-hydrogen atom onto the guanidyl radical. RRKM calculations of dissociation kinetics predict the loss of guanidine to account for >95% of 1H dissociations. The anomalous behavior of protonated arginine amide upon electron transfer provides an insight into electron capture and transfer dissociations of peptide cations containing arginine residues as charge carriers. The absence of efficient hydrogen atom transfer from charge-reduced arginine onto sterically proximate amide group blocks one of the current mechanisms for electron capture dissociation. Conversely, charge-reduced guanidine groups in arginine residues may function as radical traps and induce side-chain dissociations. In light of the current findings, backbone dissociations in arginine-containing peptides are predicted to involve excited electronic states and proceed by the amide superbase mechanism that involves electron capture in an amide pi* orbital, which is stabilized by through-space coulomb interaction with the remote charge carriers.     

Inverse hydrogen migration in arginine-containing peptide ions upon electron transfer

Collisional electron transfer from gaseous Cs atoms was studied for singly and doubly protonated peptides Gly-Arg (GR) and Ala-Arg (AR) at 50- and 100-keV kinetic energies. Singly protonated GR and AR were discharged to radicals that in part rearranged by migration of a C_α hydrogen atom onto the guanidine group. The C_α-radical isomers formed were detected as stable anions following transfer of a second electron. In addition to the stabilizing rearrangements, the radicals underwent side-chain and backbone dissociations. The latter formed z fragments that were detected as the corresponding anions. Analysis of the (GR+H)^· radical potential energy surface using electronic structure theory in combination with Rice-Ramsperger-Kassel-Marcus calculations of rate constants indicated that the arginine C_α hydrogen atom was likely to be transferred to the arginine side-chain on the experimental timescale of ≤200 ns. Transfer of the Gly C_α-H was calculated to have a higher transition-state energy and was not kinetically competitive. Collisional electron transfer to doubly protonated GR and AR resulted in complete dissociation of (GR+2H)^+· and (AR+2H)^+· ions by loss of H, ammonia, and N-C_α bond cleavage. Electronic structure theory analysis of (GR+2H)^+· indicated the presence of multiple conformers and electronic states that differed in reactivity and steered the dissociations to distinct channels. Electron attachment to (GR+2H)²⁺ resulted in the formation of closely spaced electronic states of (GR+2H)^+· in which the electron density was delocalized over the guanidinium, ammonium, amide, and carboxyl groups. The different behavior of (GR+H)^· and (GR+2H)^+· is explained by the different timescales for dissociation and different internal energies acquired upon electron transfer.     

Electron capture and transfer dissociation: Peptide structure analysis at different ion internal energy levels

We decoupled electron-transfer dissociation (ETD) and collision-induced dissociation of charge-reduced species (CRCID) events to probe the lifetimes of intermediate radical species in ETD-based ion trap tandem mass spectrometry of peptides. Short-lived intermediates formed upon electron transfer require less energy for product ion formation and appear in regular ETD mass spectra, whereas long-lived intermediates require additional vibrational energy and yield product ions as a function of CRCID amplitude. The observed dependencies complement the results obtained by double-resonance electron-capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ECD in a cryogenic ICR trap. Compared with ECD FT-ICR MS, ion trap MS offers lower precursor ion internal energy conditions, leading to more abundant charge-reduced radical intermediates and larger variation of product ion abundance as a function of vibrational post-activation amplitude. In many cases decoupled CRCID after ETD exhibits abundant radical c-type and even-electron z-type ions, in striking contrast to predominantly even-electron c-type and radical z-type ions in ECD FT-ICR MS and especially activated ion-ECD, thus providing a new insight into the fundamentals of ECD/ETD.     

Effects of Electron-Transfer Coupled with Collision-Induced Dissociation (ET/CID) on Doubly Charged Peptides and Phosphopeptides

Electron-transfer dissociation (ETD) is a useful peptide fragmentation technique that can be applied to investigate post-translational modifications (PTMs), the sequencing of highly hydrophilic peptides, and the identification of large peptides and even intact proteins. In contrast to traditional fragmentation methods, such as collision-induced dissociation (CID), ETD produces c- and z^·-type product ions by randomly cleaving the N–Cα bonds. The disappointing fragmentation efficiency of ETD for doubly charged peptides and phosphopeptide ions has been improved by ETcaD (supplemental activation). However, the ETD data derived from most database search algorithms yield low confidence scores due to the presence of unreacted precursors and charge-reduced ions within MS/MS spectra. In this work, we demonstrate that eight out of ten standard doubly charged peptides and phosphopeptides can be effortlessly identified by electron-transfer coupled with collision-induced dissociation (ET/CID) using the SEQUEST algorithm without further spectral processing. ET/CID was performed with the further dissociation of the charge-reduced ions isolated from ETD ion/ion reactions. ET/CID had high fragmentation efficiency, which elevated the confidence scores of doubly charged peptide and phosphospeptide sequencing. ET/CID was found to be an effective fragmentation strategy in “bottom-up” proteomic analysis.     

Collisional energy dependence of peptide ion fragmentation

The energy dependence of fragmentation in a collision cell was measured for 2400 protonated peptide ions derived from the digestion of 24 proteins. The collision voltage at which the sum of the fragment ion abundances was equal to the remaining parent ion (V _1/2) was the principal measure of fragmentation effectiveness. Each class of peptides was characterized by a linear relation between V _1/2 and m/z whose slope depended on the peptide class and, with little adjustment, intersected the origin. Peptide ions where the number of protons is no greater than the number of arginine residues show the greatest slope, V _1/2/(m/z)=0.0472 (all slopes in units of V Da⁻¹ e). For peptides where the number of protons is greater than the number of arginines, but not greater than the total number of basic residues, the slope decreases to 0.0414 for singly charged ions, 0.0382 for doubly charged, 0.0346 for triply charged, and 0.0308 for more highly charged ions. With one mobile proton, the slope is about 0.029 for singly and doubly charged ions and slightly lower for more highly charged ions. With two or more mobile protons the slope is 0.0207. By removing m/z dependence, the deviation of V _1/2 from a line provides a relative measure of the ease of fragmentation of an ion in each class. This information can guide the selection of optimal conditions for tandem mass spectrometry studies in collision cells for selected peptide ions as well as aid in comparing the reactivity of ions differing in m/z and charge state.     

Electron capture in charge-tagged peptides. Evidence for the role of excited electronic states

Electron capture dissociation (ECD) was studied with doubly charged dipeptide ions that were tagged with fixed-charge tris-(2,4,6-trimethoxyphenyl)phosphonium-methylenecarboxamido (TMPP-ac) groups. Dipeptides GK, KG, AK, KA, and GR were each selectively tagged with one TMPP-ac group at the N-terminal amino group while the other charge was introduced by protonation at the lysine or arginine side-chain groups to give (TMPP-ac-peptide + H)(2+) ions by electrospray ionization. Doubly tagged peptide derivatives were also prepared from GK, KG, AK, and KA in which the fixed-charge TMPP-ac groups were attached to the N-terminal and lysine side-chain amino groups to give (TMPP-ac-peptide-ac-TMPP)(2+) dications by electrospray. ECD of (TMPP-ac-peptide + H)(2+) resulted in 72% to 84% conversion to singly charged dissociation products while no intact charge-reduced (TMPP-ac-dipeptide + H)(+) ions were detected. The dissociations involved loss of H, formation of (TMPP + H)(+), and N-C(alpha) bond cleavages giving TMPP-CH(2)CONH(2)(+) (c(0)) and c(1) fragments. In contrast, ECD of (TMPP-ac-peptide-ac-TMPP)(2+) resulted in 31% to 40% conversion to dissociation products due to loss of neutral TMPP molecules and 2,4,6-trimethoxyphenyl radicals. No peptide backbone cleavages were observed for the doubly tagged peptide ions. Ab initio and density functional theory calculations for (Ph(3)P-ac-GK + H)(2+) and (H(3)P-ac-GK + H)(2+) analogs indicated that the doubly charged ions contained the lysine side-chain NH(3)(+) group internally solvated by the COOH group. The distance between the charge-carrying phosphonium and ammonium atoms was calculated to be 13.1-13.2 A in the most stable dication conformers. The intrinsic recombination energies of the TMPP(+)-ac and (GK + H)(+) moieties, 2.7 and 3.15 eV, respectively, indicated that upon electron capture the ground electronic states of the (TMPP-ac-peptide + H)(+*) ions retained the charge in the TMPP group. Ground electronic state (TMPP-ac-GK + H)(+*) ions were calculated to spontaneously isomerize by lysine H-atom transfer to the COOH group to form dihydroxycarbinyl radical intermediates with the retention of the charged TMPP group. These can trigger cleavages of the adjacent N-C(alpha) bonds to give rise to the c(1) fragment ions. However, the calculated transition-state energies for GK and GGK models suggested that the ground-state potential energy surface was not favorable for the formation of the abundant c(0) fragment ions. This pointed to the involvement of excited electronic states according to the Utah-Washington mechanism of ECD.     

Natural structural motifs that suppress peptide ion fragmentation after electron capture

Series of doubly and triply protonated diarginated peptide molecules with different number of glutamic acid (E) and asparagine (N) residues were analyzed under ECD conditions. ECD spectra of doubly-protonated peptides show a strong dependence on the number of E and N residues. Both the backbone cleavages and hydrogen radical (H^•) loss from the charge-reduced precursor ions ([M+2H]^+•) were suppressed as the number of E and N residues increases. A strong inhibition of the backbone cleavages and H^• loss from [M+2H]^+• was found for peptides with 6E residues (or 4E + 2N residues). The results obtained using these model peptides were re-confirmed by analyzing N-arginated Fibrinopeptide-B (i.e., REGVNDNEEGFFSAR). In contrast to the N-arginated peptide, ECD of the doubly-protonated Fibrinopeptide-B and its analogues show extensive backbone cleavages leading to series of c- and z-ions (∼80% sequence coverage). Based on these results, it is believed that peptide ions with all surplus protons sequestered in arginine-residues would show enhanced stability under ECD conditions as the number of acid-residue increases. The suppression of backbone cleavages and H^• loss from [M+2H]^+• are presumably attributed to the low reactivity of the charge-reduced precursor ions. One of the possible hypothesis is that diarginated E-rich peptides may contain hydrogen bonds between carbonyl oxygen of E side chains and backbone amide hydrogen. These hydrogen bonds would provide extra stabilization for [M+2H]^+•. This is the first demonstration of natural structural motifs in peptides that would inhibit the backbone fragmentation of the charge-reduced peptide ions under ECD conditions.     

Electron transfer dissociation with supplemental activation to differentiate aspartic and isoaspartic residues in doubly charged peptide cations

Electron-transfer dissociation (ETD) with supplemental activation of the doubly charged deamidated tryptic digested peptide ions allows differentiation of isoaspartic acid and aspartic acid residues using the c + 57 or z ^• − 57 peaks. The diagnostic peak clearly localizes and characterizes the isoaspartic acid residue. Supplemental activation in ETD of the doubly charged peptide ions involves resonant excitation of the charge reduced precursor radical cations and leads to further dissociation, including extra backbone cleavages and secondary fragmentation. Supplemental activation is essential to obtain a high quality ETD spectrum (especially for doubly charged peptide ions) with sequence information. Unfortunately, the low-resolution of the ion trap mass spectrometer makes detection of the diagnostic peak, [M-60], for the aspartic acid residue difficult due to interference with side-chain loss from arginine and glutamic acid residues.     

The Early Life of a Peptide Cation-Radical. Ground and Excited-State Trajectories of Electron-Based Peptide Dissociations During the First 330 Femtoseconds

We report a new approach to investigating the mechanisms of fast peptide cation-radical dissociations based on an analysis of time-resolved reaction progress by Ehrenfest dynamics, as applied to an Ala-Arg cation-radical model system. Calculations of stationary points on the ground electronic state that were carried out with effective CCSD(T)/6-311++G(3df,2p) could not explain the experimental branching ratios for loss of a hydrogen atom, ammonia, and N–C_α bond dissociation in (AR + 2H)^+●. The Ehrenfest dynamics results indicate that the ground and low-lying excited electronic states of (AR + 2H)^+● follow different reaction courses in the first 330 femtoseconds after electron attachment. The ground (X) state undergoes competing loss of N-terminal ammonia and isomerization to an aminoketyl radical intermediate that depend on the vibrational energy of the charge-reduced ion. The A and B excited states involve electron capture in the Arg guanidine and carboxyl groups and are non-reactive on the short time scale. The C state is dissociative and progresses to a fast loss of an H atom from the Arg guanidine group. Analogous results were obtained by using the B3LYP and CAM-B3LYP density functionals for the excited state dynamics and including the universal M06-2X functional for ground electronic state calculations. The results of this Ehrenfest dynamics study indicate that reaction pathway branching into the various dissociation channels occurs in the early stages of electron attachment and is primarily determined by the electronic states being accessed. This represents a new paradigm for the discussion of peptide dissociations in electron based methods of mass spectrometry.     

Transition metals as electron traps. II. Structures,energetics and electron transfer dissociations of ternary Co,Ni and Zn–peptide complexes in the gas phase

Transition metal cations Co²⁺, Ni²⁺ and Zn²⁺ form 1 : 1 : 1 ternary complexes with 2,2′‐bipyridine (bpy) and peptides in aqueous methanol solutions that have been studied for tripeptides GGG and GGL. Electrospray ionization of these solutions produced singly charged [Metal(bpy)(peptide ? H)]⁺ and doubly charged [Metal(bpy)(peptide)]²⁺ ions (Metal = metal ion) that underwent charge reduction by glancing collisions with Cs atoms at 50 and 100 keV collision energies. Electron transfer to [Metal(bpy)(peptide)]²⁺ ions was less than 4.2 eV exoergic and formed abundant fractions of non‐dissociated charge‐reduced intermediates. Charge‐reduced [Metal(bpy)(peptide)]⁺ ions dissociated by the loss of a hydrogen atom, ammonia, water and ligands that depended on the metal ion. The Ni and Co complexes mainly dissociated by the elimination of ammonia, water, and the peptide ligand. The Zn complex dissociated by the elimination of ammonia and bpy. A sequence‐specific fragment was observed only for the Co complex. Electron transfer to [Metal(bpy)(peptide ? H)]⁺ was 0.6–1.6 eV exoergic and formed intermediate radicals that were detected as stable anions after a second electron transfer from Cs. [Metal(bpy)(peptide ? H)] neutrals and their anions dissociated by the loss of bpy and peptide ligands with branching ratios that depended on the metal ion. Optimized structures for several spin states, electron transfer and dissociation energies were addressed by combined density functional theory and Møller–Plesset perturbational calculations to aid interpretation of experimental data. The experimentally observed ligand loss and backbone cleavage in charge‐reduced [Metal(bpy)(peptide)]⁺ complexes correlated with the dissociation energies at the present level of theory. The ligand loss in ⁺CR^? spectra showed overlap of dissociations in charge‐reduced [Metal(bpy)(peptide ? H)] complexes and their anionic counterparts which complicated spectra interpretation and correlation with calculated dissociation energies. Copyright © 2009 John Wiley & Sons, Ltd.     

Fragmentation of Singly,Doubly, and Triply Charged Hydrogen Deficient Peptide Radical Cations in Infrared Multiphoton Dissociation and Electron Induced Dissociation

Gas phase fragmentation of hydrogen deficient peptide radical cations continues to be an active area of research. While collision induced dissociation (CID) of singly charged species is widely examined, dissociation channels of singly and multiply charged radical cations in infrared multiphoton dissociation (IRMPD) and electron induced dissociation (EID) have not been, so far, investigated. Here, we report on the gas phase dissociation of singly, doubly and triply charged hydrogen deficient peptide radicals, [M + nH]^(n+1)+· (n = 0, 1, 2), in MS³ IRMPD and EID and compare the observed fragmentation pathways to those obtained in MS³ CID. Backbone fragmentation in MS³ IRMPD and EID was highly dependent on the charge state of the radical precursor ions, whereas amino acid side chain cleavages were largely independent of the charge state selected for fragmentation. Cleavages at aromatic amino acids, either through side chain loss or backbone fragmentation, were significantly enhanced over other dissociation channels. For singly charged species, the MS³ IRMPD and EID spectra were mainly governed by radical-driven dissociation. Fragmentation of doubly and triply charged radical cations proceeded through both radical- and charge-driven processes, resulting in the formation of a wide range of backbone product ions including, a-, b-, c-, y-, x-, and z-type. While similarities existed between MS³ CID, IRMPD, and EID of the same species, several backbone product ions and side chain losses were unique for each activation method. Furthermore, dominant dissociation pathways in each spectrum were dependent on ion activation method, amino acid composition, and charge state selected for fragmentation.     

Formation and decay of doubly charged ions in n-alkanes from methane to triacontane

Coincidence techniques were used to study dissociative double ionization of selected n-alkanes from methane to triacontane (C₃₀H₆₂) and of the hexane isomers. Following photoionization at 40.8 eV, both covalent and coulombic dissociations of the molecular dications take place. The main decay route of doubly charged alkanes larger than butane is fast charge separation followed by secondary dissociation of energetic singly charged primary ions. A simulation based on quasi-equilibrium theory and the spectra of the isomers confirm this breakdown mechanism for hexane.     

Mass spectrometric study on 2-amino-as-triazino[6,5-c]quinoline and some of its derivatives

Electron impact induced fragmentations of 2-amino-as-triazino[6,5-c]quinoline and its 2-methylamino, 2-dimethylamino and 2-benzylamino analogues have been investigated. The main primary decomposition route of both the singly and the doubly charged molecular ions is the N₂ loss. For the singly charged ions the critical energy of this reaction is 110±10 kJ mol^?1 and the kinetic energy release is 61±4 kJ mol^?1. For the doubly charged ions these values are 90±10 kJ mol^?1 and 5±2 kJ mol^?1, respectively, indicating a significantly different reaction profile. The further fragmentation of [M? N₂]⁺˙ ions consists of radical eliminations from the 2-amino group with cleavages of the α- and β-bonds. Here a significant substituent effect is eliminations found suggesting an intramolecular cyclization reaction with a substituent migration. D and ¹⁵N labelling experiments have shown a minor extent of randomization of the labelled atoms and the occurrence of other hidden skeletal rearrangements during the fragmentation.     

Rearrangements in aryl substituted α, β-unsaturated nitriles. A collisional activation study

The rearrangement reactions following electron ionization in a number of aryl substituted conjugated nitriles have been studied using labelled compounds and collisional activation (CA) spectroscopy. The results indicate that α-phenyl cinnamonitriles and 9,10-dihydro-9-cyanophenanthrene rearrange to a common intermediate which loses CH₃˙ or CH₂CN˙ to give the ions at m/z 190 and 165. The CA spectrum of the deuterated analogue (compound 2) shows that there is a complete hydrogen scrambling prior to the loss of the CH₃˙ radical. The fluoroderivatives (compounds 5 and 6) behave similarly to the parent nitrile. The introduction of chlorine or bromine into the aromatic ring alters the fragmentation pattern and the only favoured decomposition pathway is the loss of a halogen radical. The CA spectra of the doubly charged ions at m/z 102 and 88 are also discussed. The CA spectrum of the M ⁺˙ ion 1,1-dicyano-2-phenyl ethylene is characterized by the presence of a rearrangement ion atm/z 103 (PhCN^{+ ˙}).