Peptide backbone fragmentation initiated by side‐chain loss at cysteine residue in matrix‐assisted laser desorption/ionization in‐source decay mass spectrometry

Matrix‐assisted laser desorption/ionization in‐source decay (MALDI‐ISD) is initiated by hydrogen transfer from matrix molecules to the carbonyl oxygen of peptide backbone with subsequent radical‐induced cleavage leading to c′/z? fragments pair. MALDI‐ISD is a very powerful method to obtain long sequence tags from proteins or to do de novo sequencing of peptides. Besides classical fragmentation, MALDI‐ISD also shows specific fragments for which the mechanism of formation enlightened the MALDI‐ISD process. In this study, the MALDI‐ISD mechanism is reviewed, and a specific mechanism is studied in details: the N‐terminal side of Cys residue (Xxx‐Cys) is described to promote the generation of c′ and w fragments in MALDI‐ISD. Our data suggest that for sequences containing Xxx‐Cys motifs, the N–C_α bond cleavage occurs following the hydrogen attachment to the thiol group of Cys side‐chain. The c?/w fragments pair is formed by side‐chain loss of the Cys residue with subsequent radical‐induced cleavage at the N–C_α bond located at the left side (N‐terminal direction) of the Cys residue. This fragmentation pathway preferentially occurs at free Cys residue and is suppressed when the cysteines are involved in disulfide bonds. Hydrogen attachment to alkylated Cys residues using iodoacetamide gives free Cys residue by the loss of ?CH₂CONH₂ radical. The presence of alkylated Cys residue also suppress the formation of c?/w fragments pair via the (C_β)‐centered radical, whereas w fragment is still observed as intense signal. In this case, the z? fragment formed by hydrogen attachment of carbonyl oxygen followed side‐chain loss at alkylated Cys leads to a w fragment. Hydrogen attachment on peptide backbone and side‐chain of Cys residue occurs therefore competitively during MALDI‐ISD process. Copyright © 2013 John Wiley & Sons, Ltd.     

213 nm Ultraviolet Photodissociation on Peptide Anions: Radical-Directed Fragmentation Patterns

Characterization of acidic peptides and proteins is greatly hindered due to lack of suitable analytical techniques. Here we present the implementation of 213 nm ultraviolet photodissociation (UVPD) in high-resolution quadrupole-Orbitrap mass spectrometer in negative polarity for peptide anions. Radical-driven backbone fragmentation provides 22 distinctive fragment ion types, achieving the complete sequence coverage for all reported peptides. Hydrogen-deficient radical anion not only promotes the cleavage of C_α–C bond but also stimulates the breaking of N–C_α and C–N bonds. Radical-directed loss of small molecules and specific side chain of amino acids are detected in these experiments. Radical containing side chain of amino acids (Tyr, Ser, Thr, and Asp) may possibly support the N–C_α backbone fragmentation. Proline comprising peptides exhibit the unusual fragment ions similar to reported earlier. Interestingly, basic amino acids such as Arg and Lys also stimulated the formation of abundant b and y ions of the related peptide anions. Loss of hydrogen atom from the charge-reduced radical anion and fragment ions are rationalized by time-dependent density functional theory (TDDFT) calculation, locating the potential energy surface (PES) of ππ* and repulsive πσ* excited states of a model amide system.

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Investigations of the Mechanism of the “Proline Effect” in Tandem Mass Spectrometry Experiments: The “Pipecolic Acid Effect”

The fragmentation behavior of a set of model peptides containing proline, its four-membered ring analog azetidine-2-carboxylic acid (Aze), its six-membered ring analog pipecolic acid (Pip), an acyclic secondary amine residue N-methyl-alanine (NMeA), and the D stereoisomers of Pro and Pip has been determined using collision-induced dissociation in ESI-tandem mass spectrometers. Experimental results for AAXAA, AVXLG, AAAXA, AGXGA, and AXPAA peptides are presented, where X represents Pro, Aze, Pip, or NMeA. Aze- and Pro-containing peptides fragment according to the well-established “proline effect” through selective cleavage of the amide bond N-terminal to the Aze/Pro residue to give y_n ⁺ ions. In contrast, Pip- and NMA-fragment through a different mechanism, the “pipecolic acid effect,” selectively at the amide bond C-terminal to the Pip/NMA residue to give b_n ⁺ ions. Calculations of the relative basicities of various sites in model peptide molecules containing Aze, Pro, Pip, or NMeA indicate that whereas the “proline effect’ can in part be rationalized by the increased basicity of the prolyl-amide site, the “pipecolic acid effect” cannot be justified through the basicity of the residue. Rather, the increased flexibility of the Pip and NMeA residues allow for conformations of the peptide for which transfer of the mobile proton to the amide site C-terminal to the Pip/NMeA becomes energetically favorable. This argument is supported by the differing results obtained for AAPAA versus AA(D-Pro)AA, a result that can best be explained by steric effects. Fragmentation of pentapeptides containing both Pro and Pip indicate that the “pipecolic acid effect” is stronger than the “proline effect.” Figure

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Isomer Information from Ion Mobility Separation of High-Mannose Glycan Fragments

Extracted arrival time distributions of negative ion CID-derived fragments produced prior to traveling-wave ion mobility separation were evaluated for their ability to provide structural information on N-linked glycans. Fragmentation of high-mannose glycans released from several glycoproteins, including those from viral sources, provided over 50 fragments, many of which gave unique collisional cross-sections and provided additional information used to assign structural isomers. For example, cross-ring fragments arising from cleavage of the reducing terminal GlcNAc residue on Man₈GlcNAc₂ isomers have unique collision cross-sections enabling isomers to be differentiated in mixtures. Specific fragment collision cross-sections enabled identification of glycans, the antennae of which terminated in the antigenic α-galactose residue, and ions defining the composition of the 6-antenna of several of the glycans were also found to have different cross-sections from isomeric ions produced in the same spectra. Potential mechanisms for the formation of the various ions are discussed and the estimated collisional cross-sections are tabulated.

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Decarboxylative Coupling Reaction in ESI(−)-MS/MS of 4-Nitrobenzyl 4-Hydroxybenzoates: Triplet Ion–Neutral Complex-Mediated 4-Nitrobenzyl Transfer

In negative electrospray ionization mass spectrometry of 4-nitrobenzyl 4-hydroxybenzoates, a decarboxylation reaction, which was significantly promoted by the presence of a nitro group on the benzyl group, competed with radical elimination reactions. Density functional theory calculations indicated that decarboxylation of deprotonated 4-nitrobenzyl vanillate occurred via a radical route involving homolytic cleavage of the C_benzyl–O bond to give a triplet ion–neutral complex, followed by decarboxylative coupling.

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Difference of Electron Capture and Transfer Dissociation Mass Spectrometry on Ni<Superscript>2+</Superscript>-, Cu<Superscript>2+</Superscript>-, and Zn<Superscript>2+</Superscript>-Polyhistidine Complexes in the Absence of Remote Protons

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) in metal-peptide complexes are dependent on the metal cation in the complex. The divalent transition metals Ni²⁺, Cu²⁺, and Zn²⁺ were used as charge carriers to produce metal-polyhistidine complexes in the absence of remote protons, since these metal cations strongly bind to neutral histidine residues in peptides. In the case of the ECD and ETD of Cu²⁺-polyhistidine complexes, the metal cation in the complex was reduced and the recombination energy was redistributed throughout the peptide to lead a zwitterionic peptide form having a protonated histidine residue and a deprotonated amide nitrogen. The zwitterion then underwent peptide bond cleavage, producing a and b fragment ions. In contrast, ECD and ETD induced different fragmentation processes in Zn²⁺-polyhistidine complexes. Although the N–C_α bond in the Zn²⁺-polyhistidine complex was cleaved by ETD, ECD of Zn²⁺-polyhistidine induced peptide bond cleavage accompanied with hydrogen atom release. The different fragmentation modes by ECD and ETD originated from the different electronic states of the charge-reduced complexes resulting from these processes. The details of the fragmentation processes were investigated by density functional theory.

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Estimation of peptide N–Cα bond cleavage efficiency during MALDI‐ISD using a cyclic peptide

Matrix‐assisted laser desorption/ionization in‐source decay (MALDI‐ISD) induces N–C_α bond cleavage via hydrogen transfer from the matrix to the peptide backbone, which produces a c′/z? fragment pair. Subsequently, the z? generates z′ and [z + matrix] fragments via further radical reactions because of the low stability of the z?. In the present study, we investigated MALDI‐ISD of a cyclic peptide. The N–C_α bond cleavage in the cyclic peptide by MALDI‐ISD produced the hydrogen‐abundant peptide radical [M + 2H]⁺? with a radical site on the α‐carbon atom, which then reacted with the matrix to give [M + 3H]⁺ and [M + H + matrix]⁺. For 1,5‐diaminonaphthalene (1,5‐DAN) adducts with z fragments, post‐source decay of [M + H + 1,5‐DAN]⁺ generated from the cyclic peptide showed predominant loss of an amino acid with 1,5‐DAN. Additionally, MALDI‐ISD with Fourier transform‐ion cyclotron resonance mass spectrometry allowed for the detection of both [M + 3H]⁺ and [M + H]⁺ with two ¹³C atoms. These results strongly suggested that [M + 3H]⁺ and [M + H + 1,5‐DAN]⁺ were formed by N–C_α bond cleavage with further radical reactions. As a consequence, the cleavage efficiency of the N–C_α bond during MALDI‐ISD could be estimated by the ratio of the intensity of [M + H]⁺ and [M + 3H]⁺ in the Fourier transform‐ion cyclotron resonance spectrum. Because the reduction efficiency of a matrix for the cyclic peptide cyclo(Arg‐Gly‐Asp‐D‐Phe‐Val) was correlated to its tendency to cleave the N–C_α bond in linear peptides, the present method could allow the evaluation of the efficiency of N–C_α bond cleavage for MALDI matrix development. Copyright © 2016 John Wiley & Sons, Ltd.     

Top‐down mass spectrometry of intact phosphorylated β‐casein: Correlation between the precursor charge state and internal fragments

Phosphorylated proteins play essential roles in many cellular processes, and identification and characterization of the relevant phosphoproteins can help to understand underlying mechanisms. Herein, we report a collision‐induced dissociation top‐down approach for characterizing phosphoproteins on a quadrupole time‐of‐flight mass spectrometer. β‐casein, a protein with two major isoforms and five phosphorylatable serine residues, was used as a model. Peaks corresponding to intact β‐casein ions with charged states up to 36⁺ were detected. Tandem mass spectrometry was performed on β‐casein ions of different charge states (12⁺, and 15⁺ to 28⁺) in order to determine the effects of charge state on dissociation of this protein. Most of the abundant fragments corresponded to y, b ions, and internal fragments caused by cleavage of the N‐terminal amide bond adjacent to proline residues (Xxx‐Pro). The abundance of internal fragments increased with the charge state of the protein precursor ion; these internal fragments predominantly arose from one or two Xxx‐Pro cleavage events and were difficult to accurately assign. The presence of abundant sodium adducts of β‐casein further complicated the spectra. Our results suggest that when interpreting top‐down mass spectra of phosphoproteins and other proteins, researchers should consider the potential formation of internal fragments and sodium adducts for reliable characterization.     

Sequence Ion Structures and Dissociation Chemistry of Deprotonated Sucrose Anions

We investigate the tandem mass spectrometry of regiospecifically labeled, deprotonated sucrose analytes. We utilize density functional theory calculations to model the pertinent gas-phase fragmentation chemistry of the prevalent glycosidic bond cleavages (B₁-Y₁ and C₁-Z₁ reactions) and compare these predictions to infrared spectroscopy experiments on the resulting B₁ and C₁ product anions. For the C₁ anions, barriers to interconversion of the pyranose [α-glucose-H]^?, C₁ anions to entropically favorable ring-open aldehyde-terminated forms were modest (41 kJ mol^?1) consistent with the observation of a band assigned to a carbonyl stretch at ~?1680–1720 cm^?1. For the B₁ anions, our transition structure calculations predict the presence of both deprotonated 1,6-anhydroglucose and carbon 2-ketone ((4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)dihydro-2H-pyran-3(4H)-one) anion structures, with the latter predominating. This hypothesis is supported by our spectroscopic data which show diagnostic bands at 1600, 1674, and 1699 cm^?1 (deprotonated carbon 2-ketone structures), and at ~?1541 cm^?1 (both types of structure) and RRKM rate calculations. The deprotonated carbon 2-ketone structures are also the lowest energy product B₁ anions.

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Spectroscopic Identification of Cyclic Imide b2-Ions from Peptides Containing Gln and Asn Residues

In mass-spectrometry based peptide sequencing, formation of b- and y-type fragments by cleavage of the amide C–N bond constitutes the main dissociation pathway of protonated peptides under low-energy collision induced dissociation (CID). The structure of the b ₂ fragment ion from peptides containing glutamine (Gln) and asparagine (Asn) residues is investigated here by infrared ion spectroscopy using the free electron laser FELIX. The spectra are compared with theoretical spectra calculated using density functional theory for different possible isomeric structures as well as to experimental spectra of synthesized model systems. The spectra unambiguously show that the b₂-ions do not possess the common oxazolone structure, nor do they possess the alternative diketopiperazine structure. Instead, cyclic imide structures are formed through nucleophilic attack by the amide nitrogen atom of the Gln and Asn side chains. The alternative pathway involving nucleophilic attack from the side-chain amide oxygen atom leading to cyclic isoimide structures, which had been suggested by several authors, can clearly be excluded based on the present IR spectra. This mechanism is perhaps surprising as the amide oxygen atom is considered to be the better nucleophile; however, computations show that the products formed via attack by the amide nitrogen are considerably lower in energy. Hence, b₂-ions with Asn or Gln in the second position form structures with a five-membered succinimide or a six-membered glutarimide ring, respectively. b₂-Ions formed from peptides with Asn in the first position are spectroscopically shown to possess the classical oxazolone structure.      

Molecular structure and barriers to internal rotation of α-naphthalenesulfonyl chloride: a study by gas-phase electron diffraction and quantum chemical calculations

α-Naphthalenesulfonyl chloride, α-NaphSC, was studied by gas-phase electron diffraction (GED) and quantum chemical calculations (HF/6-311 + G**, HF/aug-cc-pVDZ, B3LYP/cc-pVDZ, B3LYP/cc-pVTZ, B3LYP/aug-cc-pVDZ, B3LYP/aug-cc-pVTZ, MP2/cc-pVDZ, and MP2/cc-pVTZ). The calculations predict the existence of two conformers with C ₁ (I) and C _s (II) symmetries. The most stable conformer I has an enantiomer. The experimental data of α-NaphSC obtained at 370(5) K could be best fitted by a C ₁ symmetry model indicating that only this form exists in the gas-phase. In this model the C_α–S–Cl plane deviates from the perpendicular orientation relative to the plane of the naphthalene skeleton. Under the applied experimental conditions, the mole fraction of a second less stable conformer II of α-NaphSC predicted by calculations is no more than 1 %. The following geometrical parameters of conformer I were obtained from the experiment (Å and °; uncertainties are in parentheses): r _h1(C–H) = 1.082(6), r _h1(C–C)_cp = 1.407(3), r _h1(C–S) = 1.764(5), r _h1(S–O)_av = 1.425(3), r _h1(S–Cl) = 2.051(5), ∠C–C_α–C = 122.5(1), ∠C_α–S–Cl = 101.5(10); C9–C1–S–Cl = 71.4(21). The calculated barriers to internal rotation of the sulfonyl chloride group exceed considerably the thermal energy values corresponding to the temperatures of the GED experiments. Natural bond orbitals analysis of the electron density distribution was carried out to explain the peculiarities of the molecular structure of the studied compound and the deviation from the structures of β-NaphSHal molecules and their benzene analogs.     

Near-UV Photodissociation of Tryptic Peptide Cation Radicals. Scope and Effects of Amino Acid Residues and Radical Sites

Peptide cation-radical fragment ions of the z-type, [^●AXAR⁺], [^●AXAK⁺], and [^●XAR⁺], where X = A, C, D, E, F, G, H, K, L, M, N, P, Y, and W, were generated by electron transfer dissociation of peptide dications and investigated by MS³-near-ultraviolet photodissociation (UVPD) at 355 nm. Laser-pulse dependence measurements indicated that the ion populations were homogeneous for most X residues except phenylalanine. UVPD resulted in dissociations of backbone CO─NH bonds that were accompanied by hydrogen atom transfer, producing fragment ions of the [y_n]⁺ type. Compared with collision-induced dissociation, UVPD yielded less side-chain dissociations even for residues that are sensitive to radical-induced side-chain bond cleavages. The backbone dissociations are triggered by transitions to second (B) excited electronic states in the peptide ion R-CH^●-CONH- chromophores that are resonant with the 355-nm photon energy. Electron promotion increases the polarity of the B excited states, R-CH⁺-C^●(O^–)NH-, and steers the reaction to proceed by transfer of protons from proximate acidic C_α and amide nitrogen positions.

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Spontaneous Isomerization of Peptide Cation Radicals Following Electron Transfer Dissociation Revealed by UV-Vis Photodissociation Action Spectroscopy

Peptide cation radicals of the z-type were produced by electron transfer dissociation (ETD) of peptide dications and studied by UV-Vis photodissociation (UVPD) action spectroscopy. Cation radicals containing the Asp (D), Asn (N), Glu (E), and Gln (Q) residues were found to spontaneously isomerize by hydrogen atom migrations upon ETD. Canonical N-terminal [z₄ + H]^+● fragment ion-radicals of the R-C^●H-CONH- type, initially formed by N?C_α bond cleavage, were found to be minor components of the stable ion fraction. Vibronically broadened UV-Vis absorption spectra were calculated by time-dependent density functional theory for several [^●DAAR + H]⁺ isomers and used to assign structures to the action spectra. The potential energy surface of [^●DAAR + H]⁺ isomers was mapped by ab initio and density functional theory calculations that revealed multiple isomerization pathways by hydrogen atom migrations. The transition-state energies for the isomerizations were found to be lower than the dissociation thresholds, accounting for the isomerization in non-dissociating ions. The facile isomerization in [^●XAAR + H]⁺ ions (X = D, N, E, and Q) was attributed to low-energy intermediates having the radical defect in the side chain that can promote hydrogen migration along backbone C_α positions. A similar side-chain mediated mechanism is suggested for the facile intermolecular hydrogen migration between the c- and [z + H]^●-ETD fragments containing Asp, Asn, Glu, and Gln residues.

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Gas-Phase Structure of Amyloid-β (12 – 28) Peptide Investigated by Infrared Spectroscopy, Electron Capture Dissociation and Ion Mobility Mass Spectrometry

The gas-phase structures of doubly and triply protonated Amyloid-β_12-28 peptides have been investigated through the combination of ion mobility (IM), electron capture dissociation (ECD) mass spectrometry, and infrared multi-photon dissociation (IRMPD) spectroscopy together with theoretical modeling. Replica-exchange molecular dynamics simulations were conducted to explore the conformational space of these protonated peptides, from which several classes of structures were found. Among the low-lying conformers, those with predicted diffusion cross-sections consistent with the ion mobility experiment were further selected and their IR spectra simulated using a hybrid quantum mechanical/semiempirical method at the ONIOM DFT/B3LYP/6-31 g(d)/AM1 level. In ECD mass spectrometry, the c/z product ion abundance (PIA) has been analyzed for the two charge states and revealed drastic differences. For the doubly protonated species, N – C_α bond cleavage occurs only on the N and C terminal parts, while a periodic distribution of PIA is clearly observed for the triply charged peptides. These PIA distributions have been rationalized by comparison with the inverse of the distances from the protonated sites to the carbonyl oxygens for the conformations suggested from IR and IM experiments. Structural assignment for the amyloid peptide is then made possible by the combination of these three experimental techniques that provide complementary information on the possible secondary structure adopted by peptides. Although globular conformations are favored for the doubly protonated peptide, incrementing the charge state leads to a conformational transition towards extended structures with 3₁₀- and α-helix motifs.      

Charge Transfer Dissociation (CTD) Mass Spectrometry of Peptide Cations: Study of Charge State Effects and Side-Chain Losses

1+, 2+, and 3+ precursors of substance P and bradykinin were subjected to helium cation irradiation in a 3D ion trap mass spectrometer. Charge exchange with the helium cations produces a variety of fragment ions, the number and type of which are dependent on the charge state of the precursor ions. For 1+ peptide precursors, fragmentation is generally restricted to C?CO backbone bonds (a and x ions), whereas for 2+ and 3+ peptide precursors, all three backbone bonds (C?CO, C?N, and N?Cα) are cleaved. The type of backbone bond cleavage is indicative of possible dissociation channels involved in CTD process, including high-energy, kinetic-based, and ETD-like pathways. In addition to backbone cleavages, amino acid side-chain cleavages are observed in CTD, which are consistent with other high-energy and radical-mediated techniques. The unique dissociation pattern and supplementary information available from side-chain cleavages make CTD a potentially useful activation method for the structural study of gas-phase biomolecules.

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Selective and Nonselective Cleavages in Positive and Negative CID of the Fragments Generated from In-Source Decay of Intact Proteins in MALDI-MS

Selective and nonselective cleavages in ion trap low-energy collision-induced dissociation (CID) experiments of the fragments generated from in-source decay (ISD) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of intact proteins are described in both positive and negative ion modes. The MALDI-ISD spectra of the proteins demonstrate common, discontinuous, abundant c- and z′-ions originating from cleavage at the N–Cα bond of Xxx-Asp/Asn and Gly-Xxx residues in both positive- and negative-ion modes. The positive ion CID of the c- and z′-ions resulted in product ions originating from selective cleavage at Asp-Xxx, Glu-Xxx and Cys-Xxx residues. Nonselective cleavage product ions rationalized by the mechanism of a “mobile proton” are also observed in positive ion CID spectra. Negative ion CID of the ISD fragments results in complex product ions accompanied by the loss of neutrals from b-, c-, and y-ions. The most characteristic feature of negative ion CID is selective cleavage of the peptide bonds of acidic residues, Xxx-Asp/Glu/Cys. A definite influence of α-helix on the CID product ions was not obtained. However, the results from positive ion and negative ion CID of the MALDI-ISD fragments that may have long α-helical domains suggest that acidic residues in helix-free regions tend to degrade more than those in helical regions.

Low Mass MS/MS Fragments of Protonated Amino Acids Used for Distinction of Their 13C- Isotopomers in Metabolic Studies

Glu, Gln, Pro, and Ala are the main amino acids involved in ammonia detoxification in mosquitoes. In order to develop a tandem mass spectrometry method (MS²) to monitor each carbon of the above isotopically-labeled ¹³C-amino acids for metabolic studies, the compositions and origins of atoms in fragments of the protonated amino acid should be first elucidated. Thus, various electrospray (ESI)-based MS² tools were employed to study the fragmentation of these unlabeled and isotopically-labeled amino acids and better understand their dissociation pathways. A broad range of fragments, including previously-undescribed low m/z fragments was revealed. The formulae of the fragments (from m/z 130 down to m/z 27) were confirmed by their accurate masses. The structures and conformations of the larger fragments of Glu were also explored by ion mobility mass spectrometry (IM-MS) and gas-phase hydrogen/deuterium exchange (HDX) experiments. It was found that some low m/z fragments (m/z 27–30) are common to Glu, Gln, Pro, and Ala. The origins of carbons in these small fragments are discussed and additional collision induced dissociation (CID) MS² fragmentation pathways are proposed for them. It was also found that small fragments (≤m/z 84) of protonated, methylated Glu, and methylated Gln are the same as those of the underivatized Glu and Gln. Taken together, the new approach of utilizing low m/z fragments can be applied to distinguish, identify, and quantify ¹³C-amino acids labeled at various positions, either in the backbone or side chain.      

Locating Pb2+ and Zn2+ in Zinc Finger-Like Peptides Using Mass Spectrometry

The binding preferences of Pb²⁺and Zn²⁺ in doubly charged complexes with zinc finger-like 12-residue peptides (Pep), [M_n(Pep-2(n-1)H)]²⁺ have been explored using tandem mass spectrometry. The peptides were synthesized strategically by blocking the N-terminus with an acetyl group and with four cysteine and/or histidine residues in positions 2, 5, 8, and 11, arranged in different motifs: CCHH, CHCH, and CCCC. The MS² spectra of the Pb²⁺ and Zn²⁺ complexes show multiple losses of water and a single methane loss and these provide a sensitive method for locating the metal dication and so elucidating its coordination. The elimination of a methane molecule indicated the position of the metal at the Cys2 residue. Whereas lead was observed to preferentially bind to cysteine residues, zinc was found to primarily bind to histidine residues and secondarily to cysteine residues. Preferential binding of lead to cysteine is preserved in the complexes with more than one Pb²⁺. Key to the mechanism of the loss of water and methane is the metal dication withdrawing electrons from the proximal amidic nitrogen. This acidic nitrogen loses its hydrogen to an amidic oxygen situated four atoms away leading to formation of a five-member ring and the elimination of water.      

Dissociative Electron Attachment to the Nitroamine HMX (Octahydro-1,3,5,7-Tetranitro-1,3,5,7-Tetrazocine)

In the present study, dissociative electron attachment (DEA) measurements with gas phase HMX, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine, C₄H₈N₈O₈, have been performed by means of a crossed electron-molecular beam experiment. The most intense signals are observed at 46 and 176 u and assigned to NO₂ ^? and C₃H₆N₅O₄ ^?, respectively. Anion efficiency curves for 15 negatively charged fragments have been measured in the electron energy region from about 0–20 eV with an energy resolution of ~0.7 eV. Product anions are observed mainly in the low energy region, near 0 eV, arising from surprisingly complex reactions associated with multiple bond cleavages and structural and electronic rearrangement. The remarkable instability of HMX towards electron attachment with virtually zero kinetic energy reflects the highly explosive nature of this compound. Substantially different intensity ratios of resonances for common fragment anions allow distinguishing the nitroamines HMX and royal demolition explosive molecule (RDX) in negative ion mass spectrometry based on free electron capture.