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.     

Gas‐Phase Unimolecular Dissociation Reveals Dominant Base Property of Protonated Homocysteine Sulfinyl Radical Ions

Homocysteine sulfinyl radical (^SO?Hcy) is a reactive intermediate involved during oxidative damage of DNA in the presence of high concentrations of homocysteine (Hcy). The short lifetime of ^SO?Hcy makes its preparation, isolation, and characterization challenging using traditional chemical measurement tools. Herein, we demonstrate the first study on mass‐selected protonated ^SO?Hcy ions in the gas phase by investigating its unimolecular dissociation pathways from low energy collision‐induced dissociation (CID). Tandem mass spectrometry (MS/MS), stable‐isotope labeling, and theoretical calculations were employed to rationalize the observed fragmentation pathways. The dominant dissociation channel of protonated ^SO?Hcy was a charge‐directed H₂O loss from the protonated sulfinyl radical (‐SO?) moiety, forming a thiyl radical (‐S?), which further triggered sequential radical‐directed ?SH loss through multiple pathways. Compared to cysteine sulfinyl radical (^SO?Cys), the small structural change induced by one additional methylene group in the side chain of ^SO?Hcy significantly promotes its base property while reducing the radical reactivity of sulfinyl radical. This observation provides new insight into studying reactions of ^SO?Hcy with biomolecules, which are critical in understanding toxicity induced by high levels of Hcy in biological conditions.     

C alpha-C backbone fragmentation dominates in electron detachment dissociation of gas-phase polypeptide polyanions

Fragmentation of peptide polyanions by electron detachment dissociation (EDD) has been induced by electron irradiation of deprotonated polypeptides [M-nH](n-) with >10 eV electrons. EDD has been found to lead preferentially to a* and x fragment ions (C(alpha)-C backbone cleavage) arising from the dissociation of oxidized radical anions [M-nH]((n-1)-*. We demonstrate that C(alpha)-C cleavages, which are otherwise rarely observed in tandem mass spectrometry, can account for most of the backbone fragmentation, with even-electron x fragments dominating over radical a* ions. Ab initio calculations at the B3 LYP level of theory with the 6-311+G(2 p,2 d)//6-31+G(d,p) basis set suggested a unidirectional mechanism for EDD (cleavage always N-terminal to the radical site), with a*, x formation being favored over a, x* fragmentation by 74.2 kJ mol(-1). Thus, backbone C(alpha)-C bonds N-terminal to proline residues should be immune to EDD, in agreement with the observations. EDD may find application in mass spectrometry for such tasks as peptide sequencing and localization of labile post-translational modifications, for example, those introduced by sulfation and phosphorylation. EDD can now be performed not only in Fourier transform mass spectrometry, but also in far more widely used quadrupole (Paul) ion traps.     

Gas‐phase fragmentations of N‐methylimidazolidin‐4‐one organocatalysts

N‐methylimidazolidin‐4‐one organocatalysts were studied in the gas phase. Protonated and sodium‐cationized (sodiated) molecules are conveniently accessible by electrospray mass spectrometry. Protonation enables three different closed‐shell paths of ring cleavage leading to iminium ions. The fragmentation pattern is largely unaffected by exocyclic substituents and thus is valuable to characterize the substance type as N‐methylimidazolidin‐4‐ones. Sodiated species show a distinctly different fragmentation that is less useful for characterization purposes: apart from signal loss due to dissociation of Na⁺, the observation of benzyl radical loss is by far predominant. Only in absence of a benzyl substituent, an analogue of the third ring cleavage (loss of [C₂H₅NO]) is observed. Copyright © 2017 John Wiley & Sons, Ltd.     

Fragmentation of oligoribonucleotides from gas-phase ion-electron reactions

We have recently demonstrated that both electron capture dissociation (ECD) and electron detachment dissociation (EDD) can provide complementary sequence-specific cleavage of DNA compared with collision activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD). However, EDD is preferred because of more extensive fragmentation at higher sensitivity (due to its negative ion mode operation). Here, we extend the radical ion chemistry of these two gas-phase ion-electron reaction techniques to the characterization of RNA. Compared with DNA, rather limited information is currently available on the gas-phase fragmentation of RNA. We found that the ECD fragmentation patterns of the oligoribonucleotides A6, C6, and CGGGGC are nucleobase dependent, suggesting that cleavage proceeds following electron capture at the nucleobases. Only limited backbone cleavage was observed in ECD. EDD, on the other hand, provided complete sequence coverage for the RNAs A6, C6, G6, U6, CGGGGC, and GCAUAC. The EDD fragmentation patterns were different from those observed with CAD and IRMPD in that the dominant product ions correspond to d- and w-type ions rather than c- and y-type ions. The minimum differences between oligoribonucleotides suggest that EDD proceeds following direct electron detachment from the phosphate backbone.     

Hexuronic Acid Stereochemistry Determination in Chondroitin Sulfate Glycosaminoglycan Oligosaccharides by Electron Detachment Dissociation

Electron detachment dissociation (EDD) has previously provided stereo-specific product ions that allow for the assignment of the acidic C-5stereochemistry in heparan sulfate glycosaminoglycans (GAGs), but application of the same methodology to an epimer pair in the chondroitin sulfate glycoform class does not provide the same result. A series of experiments have been conducted in which glycosaminoglycan precursor ions are independently activated by electron detachment dissociation (EDD), electron induced dissociation (EID), and negative electron transfer dissociation (NETD) to assign the stereochemistry in chondroitin sulfate (CS) epimers and investigate the mechanisms for product ion formation during EDD in CS glycoforms. This approach allows for the assignment of electronic excitation products formed by EID and detachment products to radical pathways in NETD, both of which occur simultaneously during EDD. The uronic acid stereochemistry in electron detachment spectra produces intensity differences when assigned glycosidic and cross-ring cleavages are compared. The variations in the intensities of the doubly deprotonated (0,2)X(3) and Y(3) ions have been shown to be indicative of CS-A/DS composition during the CID of binary mixtures. These ions can provide insight into the uronic acid composition of binary mixtures in EDD, but the relative abundances, although reproducible, are low compared with those in a CID spectrum acquired on an ion trap. The application of principal component analysis (PCA) presents a multivariate approach to determining the uronic acid stereochemistry spectra of these GAGs by taking advantage of the reproducible peak distributions produced by electron detachment.     

Dissociation mechanisms and implication for the presence of multiple conformations for peptide ions with arginine at the C‐terminus: time‐resolved photodissociation study

Time‐resolved photodissociation (PD) patterns of singly protonated peptides with arginine at the C‐terminus (C‐arg peptide ions) have been used to classify the dissociation channels into two categories, i.e. high‐energy channels generating v, w and x and low‐energy ones generating b, y and z. x + 1 formed by C_α? CO cleavage seems to be the intermediate ion in high‐energy channels just as a + 1 is for N‐arg peptide ions. Difference in time‐resolved pattern indicates that the two sets of channels, high‐ and low‐energy ones, are not in direct competition. Noncompetitive dissociation is also indicated by the observation of anomalous effect of matrix used in matrix‐assisted laser desorption ionization, a cooler matrix generating more high‐energy product ions both in spontaneous dissociation and in PD. Results from detailed investigation suggest that the two sets of channels start from two (or more) different conformations. Copyright © 2010 John Wiley & Sons, Ltd.     

Electron Capture Dissociation of Hydrogen-Deficient Peptide Radical Cations

Hydrogen-deficient peptide radical cations exhibit fascinating gas phase chemistry, which is governed by radical driven dissociation and, in many cases, by a combination of radical and charge driven fragmentation. Here we examine electron capture dissociation (ECD) of doubly, [M + H]^2+?, and triply, [M + 2H]^3+?, charged hydrogen-deficient species, aiming to investigate the effect of a hydrogen-deficient radical site on the ECD outcome and characterize the dissociation pathways of hydrogen-deficient species in ECD. ECD of [M + H]^2+? and [M + 2H]^3+? precursor ions resulted in efficient electron capture by the hydrogen-deficient species. However, the intensities of c- and z-type product ions were reduced, compared with those observed for the even electron species, indicating suppression of N?CC_?? backbone bond cleavages. We postulate that radical recombination occurs after the initial electron capture event leading to a stable even electron intermediate, which does not trigger N?CC_?? bond dissociations. Although the intensities of c- and z-type product ions were reduced, the number of backbone bond cleavages remained largely unaffected between the ECD spectra of the even electron and hydrogen-deficient species. We hypothesize that a small ion population exist as a biradical, which can trigger N?CC_?? bond cleavages. Alternatively, radical recombination and N?CC_?? bond cleavages can be in competition, with radical recombination being the dominant pathway and N?CC_?? cleavages occurring to a lesser degree. Formation of b- and y-type ions observed for two of the hydrogen-deficient peptides examined is also discussed.     

Characterization of nucleic acid higher order structure by high-resolution tandem mass spectrometry

Mass spectrometry (MS) is extensively used for the identification and sequencing of nucleic acids but has so far seen limited use for characterization of their higher order structures. Here, we have applied a range of different tandem mass spectrometry techniques, including electron detachment dissociation (EDD), infrared multiphoton dissociation (IRMPD), activated ion (AI) EDD, and EDD/IRMPD MS³, in a Fourier transform ion cyclotron resonance mass spectrometer to the characterization of three isomeric 15mer DNAs with different sequences and predicted solution-phase structures. Our goal was to explore whether their structural differences could be directly probed with these techniques. We found that all three 15mers had higher order structures in the gas phase, although preferred structures were predicted for only two of them in solution. Nevertheless, EDD, AI EDD, and EDD/IRMPD MS³ experiments yielded different cleavage patterns with less backbone fragmentation for the more stable solution-phase structure than for the other two 15mers. By contrast, no major differences were observed in IRMPD, although the extent of backbone cleavage was higher with that technique for all three 15mers. Thus, experiments utilizing the radical ion chemistry of EDD can provide complementary structural information compared to traditional slow heating methods, such as IRMPD, for structured nucleic acids.     

Radical Stability Directs Electron Capture and Transfer Dissociation of β‐Amino Acids in Peptides

We report on the characteristics of the radical‐ion‐driven dissociation of a diverse array of β‐amino acids incorporated into α‐peptides, as probed by tandem electron‐capture and electron‐transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for β‐amino acids than for their α‐form counterparts. In particular, only aromatic (e.g., β‐Phe), and to a substantially lower extent, carbonyl‐containing (e.g., β‐Glu and β‐Gln) amino acid side chains, lead to N? C_β bond cleavage in the corresponding β‐amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical‐driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from α‐amino acids, ECD of peptides composed mainly of β‐amino acids reveals a shift in cleavage priority from the N? C_β to the C_α? C bond. The incorporation of CH₂ groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical‐driven β‐amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research.     

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.     

Combining UV photodissociation action spectroscopy with electron transfer dissociation for structure analysis of gas‐phase peptide cation‐radicals

We report the first example of using ultraviolet (UV) photodissociation action spectroscopy for the investigation of gas‐phase peptide cation‐radicals produced by electron transfer dissociation. z ‐Type fragment ions ^●Gly‐Gly‐Lys⁺, coordinated to 18‐crown‐6‐ether (CE), are generated, selected by mass and photodissociated in the 200–400 nm region. The UVPD action spectra indicate the presence of valence‐bond isomers differing in the position of the C_α radical defect, (α‐Gly)‐Gly‐Lys⁺(CE), Gly‐(α‐Gly)‐Lys⁺(CE) and Gly‐Gly‐(α‐Lys⁺)(CE). The isomers are readily distinguishable by UV absorption spectra obtained by time‐dependent density functional theory (TD‐DFT) calculations. In contrast, conformational isomers of these radical types are calculated to have similar UV spectra. UV photodissociation action spectroscopy represents a new tool for the investigation of transient intermediates of ion‐electron reactions. Specifically, z ‐type cation radicals are shown to undergo spontaneous hydrogen atom migrations upon electron transfer dissociation. Copyright © 2015 John Wiley & Sons, Ltd.     

Reactions in Tirapazamine Induced by the Attachment of Low‐Energy Electrons: Dissociation Versus Roaming of OH

Tirapazamine (TPZ) has been tested in clinical trials on radio‐chemotherapy due to its potential highly selective toxicity towards hypoxic tumor cells. It was suggested that either the hydroxyl radical or benzotriazinyl radical may form as bioactive radical after the initial reduction of TPZ in solution. In the present work, we studied low‐energy electron attachment to TPZ in the gas phase and investigated the decomposition of the formed TPZ^? anion by mass spectrometry. We observed the formation of the (TPZ–OH)^? anion accompanied by the dissociation of the hydroxyl radical as by far the most abundant reaction pathway upon attachment of a low‐energy electron. Quantum chemical calculations suggest that NH₂ pyramidalization is the key reaction coordinate for the reaction dynamics upon electron attachment. We propose an OH roaming mechanism for other reaction channels observed, in competition with the OH dissociation.     

Contrasting Effects of Axial Ligands on Electron‐Transfer Versus Proton‐Coupled Electron‐Transfer Reactions of Nonheme Oxoiron(IV) Complexes

The effects of axial ligands on electron‐transfer and proton‐coupled electron‐transfer reactions of mononuclear nonheme oxoiron(IV) complexes were investigated by using [Fe^IV(O)(tmc)(X)]ⁿ⁺ ( 1 ‐X) with various axial ligands, in which tmc is 1,4,8,11‐tetramethyl‐1,4,8,11‐tetraazacyclotetradecane and X is CH₃CN ( 1 ‐NCCH₃), CF₃COO^? ( 1 ‐OOCCF₃), or N₃^? ( 1 ‐N₃), and ferrocene derivatives as electron donors. As the binding strength of the axial ligands increases, the one‐electron reduction potentials of 1 ‐X (E_red, V vs. saturated calomel electrode (SCE)) are more negatively shifted by the binding of the more electron‐donating axial ligands in the order of 1 ‐NCCH₃ (0.39) > 1 ‐OOCCF₃ (0.13) > 1 ‐N₃ (?0.05 V). Rate constants of electron transfer from ferrocene derivatives to 1 ‐X were analyzed in light of the Marcus theory of electron transfer to determine reorganization energies (λ) of electron transfer. The λ values decrease in the order of 1 ‐NCCH₃ (2.37) > 1 ‐OOCCF₃ (2.12) > 1 ‐N₃ (1.97 eV). Thus, the electron‐transfer reduction becomes less favorable thermodynamically but more favorable kinetically with increasing donor ability of the axial ligands. The net effect of the axial ligands is the deceleration of the electron‐transfer rate in the order of 1 ‐NCCH₃ > 1 ‐OOCCF₃ > 1 ‐N₃. In sharp contrast to this, the rates of the proton‐coupled electron‐transfer reactions of 1 ‐X are markedly accelerated in the presence of an acid in the opposite order: 1 ‐NCCH₃ < 1 ‐OOCCF₃ < 1 ‐N₃. Such contrasting effects of the axial ligands on the electron‐transfer and proton‐coupled electron‐transfer reactions of nonheme oxoiron(IV) complexes are discussed in light of the counterintuitive reactivity patterns observed in the oxo transfer and hydrogen‐atom abstraction reactions by nonheme oxoiron(IV) complexes (Sastri et al. Proc. Natl. Acad. Sci. U.S.A. 2007 , 104, 19 181–19 186).     

Nuclear‐Spin Polarization in Electron‐Transfer Reactions of Amines

Chemically induced dynamic nuclear polarization (CIDNP) observed during electron transfer (ET) reactions of tertiary amines such as DABCO ( 1 ) or Et₃N ( 2 ) with a wide range of electron acceptors support the involvement of amine radical‐cations (e.g., 1^{. +} or 2^{. +} ) as key intermediates. Radical ions such as 2^{. +} may be deprotonated, generating neutral aminoalkyl radicals (e.g., 2^. ). When generated by reaction with an electron acceptor of energetically low triplet state such as naphthalene (¹Naph*), the resulting pair 2^{. +} /Naph^.? reacts mostly by reverse electron transfer (RET) from triplet pairs populating the naphthalene triplet state.     

Comparison of collision‐ versus electron‐induced dissociation of sodium chloride cluster cations

The collision‐induced dissociation (CID) and electron‐induced dissociation (EID) spectra of the [(NaCl)_m(Na)_n]ⁿ⁺ clusters of sodium chloride have been examined in a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer. For singly charged cluster ions (n = 1), mass spectra for CID and EID of the precursor exhibit clear differences, which become more pronounced for the larger cluster ions. Whereas CID yields fewer product ions, EID produces all possible [(NaCl)_xNa]⁺ product ions. In the case of doubly charged cluster ions, EID again leads to a larger variety of product ions. In addition, doubly charged product ions have been observed due to loss of neutral NaCl unit(s). For example, EID of [(NaCl)₁₁(Na)₂]²⁺ leads to formation of [(NaCl)₁₀(Na)₂]²⁺, which appears to be the smallest doubly charged cluster of sodium chloride observed experimentally to date. The most abundant product ions in EID spectra are predominantly magic number cluster ions. Finally, [(NaCl)_m(Na)₂]^{+ .} radical cations, formed via capture of low‐energy electrons, fragment via the loss of [(NaCl)_n(Na)] ^. radical neutrals. Copyright © 2008 John Wiley & Sons, Ltd.     

Anharmonic Effect of N‐Propyl Peroxy Dissociation

The anharmonic and harmonic dissociation rate constants of alkylperoxy (RO₂) in different pathways, as well as those for the reactions of the n‐propyl peroxy radical, were calculated using the Rice–Ramsperger–Kassel–Marcus (RRKM) theory. When the temperature/total energy increased, the rate constants of the different pathways varied independently, causing changes in the dominating/leading products. Anharmonic rate constants were larger than harmonic rate constants, and their difference increased with increasing temperature/energy. Therefore, the anharmonic effect cannot be neglected. The rate‐determining steps of CH₃CH₂CH₂OO dissociation are discussed. Then the anharmonic effect was found clearly for CH₃CH₂CH₂OO dissociation, especially for the hydroperoxyalkyl radical (QOOH) dissociation. At low temperature, the rate constants were similar to those found from experiment, which indicated the RRKM theory was suitable for calculating the dissociation rates of RO₂ species.     

Dissociation of disulfide‐intact somatostatin ions: the roles of ion type and dissociation method

The dissociation chemistry of somatostatin‐14 was examined using various tandem mass spectrometry techniques including low‐energy beam‐type and ion trap collision‐induced dissociation (CID) of protonated and deprotonated forms of the peptide, CID of peptide‐gold complexes, and electron transfer dissociation (ETD) of cations. Most of the sequence of somatostatin‐14 is present within a loop defined by the disulfide linkage between Cys‐3 and Cys‐14. The generation of readily interpretable sequence‐related ions from within the loop requires the cleavage of at least one of the bonds of the disulfide linkage and the cleavage of one polypeptide backbone bond. CID of the protonated forms of somatostatin did not appear to give rise to an appreciable degree of dissociation of the disulfide linkage. Sequential fragmentation via multiple alternative pathways tended to generate very complex spectra. CID of the anions proceeded through CH₂? S cleavages extensively but relatively few structurally diagnostic ions were generated. The incorporation of Au(I) into the molecule via ion/ion reactions followed by CID gave rise to many structurally relevant dissociation products, particularly for the [M+Au+H]²⁺ species. The products were generated by a combination of S? S bond cleavage and amide bond cleavage. ETD of the [M+3H]³⁺ ion generated rich sequence information, as did CID of the electron transfer products that did not fragment directly upon electron transfer. The electron transfer results suggest that both the S? S bond and an N? C_α bond can be cleaved following a single electron transfer reaction. Copyright © 2009 John Wiley & Sons, Ltd.     

Studies on Intra‐Supramolecular and Intermolecular Electron‐Transfer Processes between Zinc Naphthalocyanine and Imidazole‐Appended Fullerene

Spectroscopic, computational, redox, and photochemical behavior of a self‐assembled donor‐acceptor dyad formed by axial coordination of zinc naphthalocyanine, ZnNc, and fulleropyrrolidine bearing an imidazole coordinating ligand (2‐(4′‐imidazolylphenyl)fulleropyrrolidine, C₆₀Im) was investigated in noncoordinating solvents, toluene and o‐dichlorobenzene, and the results were compared to the intermolecular electron transfer processes in a coordinating solvent, benzonitrile. The optical absorption and ab initio B3 LYP/3–21G(*) computational studies revealed self‐assembled supramolecular 1:1 dyad formation between the ZnNc and C₆₀Im entities. In the optimized structure, the HOMO was found to be entirely located on the ZnNc entity while the LUMO was found to be entirely on the fullerene entity. Cyclic voltammetry studies of the dyad exhibited a total of seven one‐electron redox processes in o‐dichlorobenzene, with 0.1 M tetrabutylammonium perchlorate. The excited‐state electron‐transfer processes were monitored by both optical‐emission and transient‐absorption techniques. Direct evidence for the radical‐ion‐pair (C₆₀Im^.?:ZnNc ^{. +} ) formation was obtained from picosecond transient‐absorption spectral studies, which indicated charge separation from the singlet‐excited ZnNc to the C₆₀Im moiety. The calculated rates of charge separation and charge recombination were 1.4×10¹⁰ s^?1 and 5.3×10⁷ s^?1 in toluene and 8.9×10⁹ s^?1 and 9.2×10⁷ s^?1 in o‐dichlorobenzene, respectively. In benzonitrile, intermolecular electron transfer from the excited triplet state of ZnNc to C₆₀Im occurs and the second‐order rate constant (k_q^triplet) for this quenching process was 5.3×10⁸ M ^?1 s^?1.     

Gas‐phase fragmentation reactions of protonated benzofuran‐ and dihydrobenzofuran‐type neolignans investigated by accurate‐mass electrospray ionization tandem mass spectrometry

We have investigated gas‐phase fragmentation reactions of protonated benzofuran neolignans (BNs) and dihydrobenzofuran neolignans (DBNs) by accurate‐mass electrospray ionization tandem and multiple‐stage (MSⁿ) mass spectrometry combined with thermochemical data estimated by Computational Chemistry. Most of the protonated compounds fragment into product ions B ([M + H–MeOH]⁺), C ([ B –MeOH]⁺), D ([ C –CO]⁺), and E ([ D –CO]⁺) upon collision‐induced dissociation (CID). However, we identified a series of diagnostic ions and associated them with specific structural features. In the case of compounds displaying an acetoxy group at C‐4, product ion C produces diagnostic ions K ([ C –C₂H₂O]⁺), L ([ K –CO]⁺), and P ([ L –CO]⁺). Formation of product ions H ([ D –H₂O]⁺) and M ([ H –CO]⁺) is associated with the hydroxyl group at C‐3 and C‐3′, whereas product ions N ([ D –MeOH]⁺) and O ([ N –MeOH]⁺) indicate a methoxyl group at the same positions. Finally, product ions F ([ A –C₂H₂O]⁺), Q ([ A –C₃H₆O₂]⁺), I ([ A –C₆H₆O]⁺), and J ([ I –MeOH]⁺) for DBNs and product ion G ([ B –C₂H₂O]⁺) for BNs diagnose a saturated bond between C‐7′ and C‐8′. We used these structure‐fragmentation relationships in combination with deuterium exchange experiments, MSⁿ data, and Computational Chemistry to elucidate the gas‐phase fragmentation pathways of these compounds. These results could help to elucidate DBN and BN metabolites in in vivo and in vitro studies on the basis of electrospray ionization ESI‐CID‐MS/MS data only.