Decompositions of odd- and even-electron anions derived from deoxy-polyadenylates

Radical anions have been formed via electron transfer from multiply charged 5′-d(AAA)-3′ and 5′-d(AAAA)-3′ anions to CCl₃ ⁺. These ions have been isolated in a quadrupole ion trap operated with helium bath gas at a pressure of 1 mtorr and subjected to resonance excitation (i. e., conventional ion trap collisional activation). Collisional activation of the even-electron species of the same charge state formed directly via electrospray was also performed by using essentially identical conditions. The collisional activation data can be compared directly without ambiguity arising from differences in parent ion internal energies and/or dissociation time frames. Both the odd- and even-electron anions yield extensive sequence-informative fragmentation but show significant differences in the extent of nucleobase loss and in the relative contributions from the various sequence diagnostic dissociation channels. The results of this study indicate that radical anions derived from multiply deprotonated oligo-deoxynucleotides that survive the electron transfer process are stable with respect to fragmentation in the ion trap environment under normal storage conditions and that the unimolecular dissociation behavior of these ions differs from the even-electron anions of the same charge state. These findings suggest, therefore, that odd- and even-electron anions might be used to provide complementary sequence information in cases in which neither ion type provides the full sequence.     

Ion Trap Collision-Induced Dissociation of Human Hemoglobin α-Chain Cations

Multiply protonated human hemoglobin alpha-chain species, ranging from [M + 4H]4+ to [M + 20H]20+, have been subjected to ion trap collisional activation. Cleavages at 88 of the 140 peptide bonds were indicated, summed over all charge states, although most product ion signals were concentrated in a significantly smaller number of channels. Consistent with previous whole protein ion dissociation studies conducted under similar conditions, the structural information inherent to a given precursor ion was highly sensitive to charge state. A strongly dominant cleavage at D75/M76, also noted previously in beam-type collisional activation studies, was observed for the [M + 8H]8+ to [M + 11H]11+ precursor ions. At lower charge states, C-terminal aspartic acid cleavages were also prominent but the most abundant products did not arise from the D75/M76 channel. The [M + 12H]12+-[M + 16H]16+ precursor ions generally yielded the greatest variety of amide bond cleavages. With the exception of the [M + 4H]4+ ion, all charge states showed cleavage at the L113/P114 bond. This cleavage proved to be the most prominent dissociation for charge states [M + 14H]14+ and higher. The diversity of dissociation channels observed within the charge state range studied potentially provides the opportunity to localize residues associated with variants via a top-down tandem mass spectrometry approach.     

Does Electron Capture Dissociation Cleave Protein Disulfide Bonds?

Peptide and protein characterization by mass spectrometry (MS) relies on their dissociation in the gas phase into specific fragments whose mass values can be aligned as ‘mass ladders’ to provide sequence information and to localize possible posttranslational modifications. The most common dissociation method involves slow heating of even-electron (M+n H)ⁿ⁺ ions from electrospray ionization by energetic collisions with inert gas, and cleavage of amide backbone bonds. More recently, dissociation methods based on electron capture or transfer were found to provide far more extensive sequence coverage through unselective cleavage of backbone N–Cα bonds. As another important feature of electron capture dissociation (ECD) and electron transfer dissociation (ETD), their unique unimolecular radical ion chemistry generally preserves labile posttranslational modifications such as glycosylation and phosphorylation. Moreover, it was postulated that disulfide bond cleavage is preferred over backbone cleavage, and that capture of a single electron can break both a backbone and a disulfide bond, or even two disulfide bonds between two peptide chains. However, the proposal of preferential disulfide bond cleavage in ECD or ETD has recently been debated. The experimental data presented here reveal that the mechanism of protein disulfide bond cleavage is much more intricate than previously anticipated.     

Fragmentation mechanisms of oxofatty acids via high-energy collisional activation

Upon high-energy collisional activation, oxofatty-acid ions undergo fragmentations to produce a unique pattern of product ions by which the position of the ketone is revealed. The reactions occurring in the vicinity of the ketone, which are the subject of this article, produce a spectral pattern that is not symmetrical. Although cleavages of α, β, and γ C-C bonds occur on the side proximal to the charge site, giving α, β, and γ ions, respectively, there is only a γ′ ion formed on the side distal to the charge site. The resulting lack of symmetry seemingly contradicts the concept that the reactions are independent of the charge (i.e., that they are charge remote). To eliminate any interaction between the charge and the reaction site, oxofatty acids were linked to glycyrrhetic acid, a steroid with a rigid polycyclic system. The fragmentation pattern remains the same, indicating that the effect does not depend on charge but rather on the ketone. Isotopic labeling and MS/MS/MS studies confirm that the fragmentations of C-C bonds in the vicinity of the ketone are complex, charge-remote processes. Formation of [M-H-H₂O]^? and [M-H-CO₂]^? anions and the ion that is formed by homolytic cleavage of the β bond at the side distal to the charge, however, are charge directed.     

Identification of esters by collisional charge inversion of their enolate ions

The collisional charge inversion and neutralization-reionization (^?NR) mass spectra of the enolate ions of m/z 115 derived from the four butyl acetates, the two propyl propionates, ethyl butyrate, ethyl isobutyrate, methyl valerate, methyl 2-methylbutyrate and methyl 3-methylbutyrate were recorded. The major primary fragmentation reactions of the unstable carbenium ion formed by charge inversion involve elimination of an alkoxy radical to form a ketene or alkylketene molecular ion and formation of an alkyl ion consisting of the R¹ group of RCOOR¹. A minor fragmentation reaction involves elimination of an alkyl radical by cleavage of a C? C bond α to the ether oxygen. The alkylketene ions fragment by β-cleavage eliminating an alkyl radical to form an olefinic acylium ion. In most cases the charge inversion mass spectra of the enolate ions allow identification of the ester.     

Distinction among isomeric unsaturated fatty acids as lithiated adducts by electrospray ionization mass spectrometry using low energy collisionally activated dissociation on a triple stage quadrupole instrument

Features of tandem mass spectra of dilithiated adduct ions of unsaturated fatty acids obtained by electrospray ionization mass spectrometry with low-energy collisionally activated dissociation (CAD) on a triple stage quadrupole instrument are described. These spectra distinguish among isomeric unsaturated fatty acids and permit assignment of double-bond location. Informative fragment ions reflect cleavage of bonds remote from the charge site on the dilithiated carboxylate moiety. The spectra contain radical cations reflecting cleavage of bonds between the first and second and between the second and third carbon atoms in the fatty acid chain. These ions are followed by a closed-shell ion series with members separated by 14 m/z units that reflect cleavage of bonds between the third and fourth and then between subsequent adjacent pairs of carbon atoms. This ion series terminates at the member reflecting cleavage of the carbon-carbon single bond vinylic to the first carbon-carbon double bond. Ions reflecting cleavages of bonds distal to the double bond are rarely observed for monounsaturated fatty acids and are not abundant when they occur. For polyunsaturated fatty acids that contain double bonds separated by a single methylene group, ions reflecting cleavage of carbon-carbon single bonds between double bonds are abundant, but ions reflecting cleavages distal to the final double bond are not. Cleavages between double bonds observed in these spectra can be rationalized by a scheme involving a six-membered transition state and subsequent rearrangement of a bis-allylic hydrogen atom to yield a terminally unsaturated charge-carrying fragment and elimination of a neutral alkene. The location of the beta-hydroxy-alkene moiety in ricinoleic acid can be demonstrated by similar methods. These observations offer the opportunity for laboratories that have tandem quadrupole instruments but do not have instruments with high energy CAD capabilities to assign double bond location in unsaturated free fatty acids by mass spectrometric methods without derivatization.     

Collisional activation of ions formed by [Li]+ ion attachment

Since no unimolecular fragmentation is observed with [M+Li]⁺ ions under normal operating conditions the collisional activation method was used to study the fragmentation behaviour of these ions. It was found that the liberation of the [Li]⁺ ion is a dominant process only with smaller molecules. In addition, direct bond cleavages and new types of rearrangement reactions lead to fragment ions in which the lithium is normally retained. The decomposition behaviour of [M+Li]⁺ ions represents an intermediate case between that of [M]+ ions and excited neutral molecules and is quite different from that of [M+H]⁺ ions.     

Electron impact studies. CXXIV—Negative ion mass spectra of organic functional groups. Thioacetanilides

The thioacetanilide negative molecular ion (produced by secondary electron capture) is stable, but it fragments after collisional activation to yield [C₆H₅NH]^? by cleavage α to the C?S grouping. The negative molecular ions of (substituted) o-nitrothioacetanilides undergo a series of extremely complex rearrangement reactions. For example, the molecular anion derived from o-nitro-N-methylthioacetanilide yields both acetate and thioacetate anions as major fragment ions.     

Charge reversal of the conjugate base of formamide

Charge reversal collisional activation mass spectremetry of negative ions has been used in conjunction with positive ion collisional activation to investigate several isomeric [H₂, C, N, O]⁺ ions. Generation of m/z 44 ions from formamide, acetamide, JV-methylformamide, acetaldoxime and by charge reversal of the [M–1]^? ion formed from formamide yields several different isomeric structures. Charge reversal of the conjugate base of formamide appears to yield a mixture of singlet and triplet H₂NC?O⁺ ions; experiments with deuterium-labeled compounds have been used to support this. Ab initio molecular orbital calculations indicate that the triplet ion is a stable structure, existing in a potential minimum 390.6 kJ mol^?1 above the ground state singlet.     

Collision Induced Dissociation Products of Disulfide-Bonded Peptides: Ions Result from the Cleavage of More Than One Bond

Disulfide bonds are a post-translational modification (PTM) that can be scrambled or shuffled to non-native bonds during recombinant expression, sample handling, or sample purification. Currently, mapping of disulfide bonds is not easy because of various sample requirements and data analysis difficulties. One step towards facilitating this difficult work is developing a better understanding of how disulfide-bonded peptides fragment during collision induced dissociation (CID). Most automated analysis algorithms function based on the assumption that the preponderance of product ions observed during the dissociation of disulfide-bonded peptides result from the cleavage of just one peptide bond, and in this report we tested that assumption by extensively analyzing the product ions generated when several disulfide-bonded peptides are subjected to CID on a quadrupole time of flight (QTOF) instrument. We found that one of the most common types of product ions generated resulted from two peptide bond cleavages, or a double cleavage. We found that for several of the disulfide-bonded peptides analyzed, the number of double cleavage product ions outnumbered those of single cleavages. The influence of charge state and precursor ion size was investigated, to determine if those parameters dictated the amount of double cleavage product ions formed. It was found in this sample set that no strong correlation existed between the charge state or peptide size and the portion of product ions assigned as double cleavages. These data show that these ions could account for many of the product ions detected in CID data of disulfide bonded peptides. We also showed the utility of double cleavage product ions on a peptide with multiple cysteines present. Double cleavage products were able to fully characterize the bonding pattern of each cysteine where typical single b/y cleavage products could not.     

Ion trap collision-induced dissociation of locked nucleic acids

Gas-phase dissociation of model locked nucleic acid (LNA) oligonucleotides and functional LNA-DNA chimeras have been investigated as a function of precursor ion charge state using ion trap collision-induced dissociation (CID). For the model LNA 5 and 8 mer, containing all four LNA monomers in the sequence, cleavage of all backbone bonds, generating a/w-, b/x-, c/y-, and d/z-ions, was observed with no significant preference at lower charge states. Base loss ions, except loss of thymine, from the cleavage of N-glycosidic bonds were also present. In general, complete sequence coverage was achieved in all charge states. For the two LNA-DNA chimeras, however, dramatic differences in the relative contributions of the competing dissociation channels were observed among different precursor ion charge states. At lower charge states, sequence information limited to the a-Base/w-fragment ions from cleavage of the 3′C-O bond of DNA nucleotides, except thymidine (dT), was acquired from CID of both the LNA gapmer and mixmer ions. On the other hand, extensive fragmentation from various dissociation channels was observed from post-ion/ion ion trap CID of the higher charge state ions of both LNA-DNA chimeras. This report demonstrates that tandem mass spectrometry is effective in the sequence characterization of LNA oligonucleotides and LNA-DNA chimeric therapeutics.     

Influence de l’énergie interne sur la réactivité de l’ion Fe(CO)2+ avec le diméthyléther, étudiée dans un spectromètre de masse FT–ICR

The influence of the internal energy on the reactivity of iron carbonyl cations with dimethylether CH₃OCH₃ (DME) has been studied using a triple cell Fourier Transform Ion Cyclotron Resonance mass spectrometer. The experimental set-up as well as the data analysis are briefly presented before being detailed on the example of the reactivity of Fe(CO)₂⁺. The strong energy dependence upon the reactivity of the ion is shown: when working with thermalised ions, the only channels observed are the two successive substitutions of a CO ligand by one DME molecule, whereas other channels are opened up for excited ions (the cleavage of the C–O bond may be homolytic or due to a rearrangement). A reaction mechanism of the C–O bond activation is then proposed.     

Charge reversal of three alkylamide ions: Free nitrenium ions

The methylnitrenium, ethylnitrenium and dimethylnitrenium ions are prepared by charge reversal collisional activation (CR CA) of the corresponding negative ions; their collisional activation mass spectra are shown to support the assigned structures. MINDO/3 energies are used to evaluate relative energies of [CH₄N]⁺ and [C₂H₆N]⁺ isomers, and to determine whether unstable forms rearrange spontaneously to stable ones. As in other examples, charge reversal here generates cations that do not exist in an energy well, but their transient existence is established because their fragmentation is more rapid than their rearrangement to a more stable form.     

On the Origin of the Methyl Radical Loss from Deprotonated Ferulic and Isoferulic Acids: Electronic Excitation of a Transient Structure

Formation of radical fragments from even-electron ions is an exception to the “even-electron rule”. In this work, ferulic acid (FA) and isoferulic acid (IFA) were used as the model compounds to probe the fragmentation mechanisms and the isomeric effects on homolytic cleavage. Elimination of methyl radical and CO₂ are the two competing reactions observed in the CID-MS of [FA – H]^? and [IFA – H]^?, of which losing methyl radical violates the “even-electron rule”. The relative intensity of their product ions is significantly different, and thereby the two isomeric compounds can be differentiated by tandem MS. Theoretical calculations indicate that both the singlet-triplet gap and the excitation energy decrease in the transient structures, as the breaking C–O bond is lengthened. The methyl radical elimination has been rationalized as the intramolecular electronic excitation of a transient structure with an elongating C–O bond. The potential energy diagrams, completed by the addition of the energy barrier of the radical elimination, have provided a reasonable explanation of the different CID-MS behaviors of [FA – H]^? and [IFA – H]^?.

Rearrangement process occurring in the fragmentation of adefovir derivatives

The fragmentation of the antiviral drug adefovir dipivoxil and its two active metabolites, adefovir and monopivoxil adefovir, was investigated using both ion trap and triple-quadrupole mass spectrometers. Fragment ions due to loss of 30 Da were observed and attributed to an unanticipated rearrangement process by loss of formaldehyde. The proposed mechanism is supported with the aid of three newly synthesized adefovir derivatives and with accurate mass measurement. Other fragmentations by loss of a pivaloyl group, loss of water, C-P bond cleavage and C-O bond cleavage were also observed for adefovir derivatives. It was concluded that the compounds containing a >POO-CHR-OCO- group generally displayed a rearrangement reaction by loss of RCHO in collision-induced dissociation, and the process generally required an activation energy lower than for a direct bond cleavage.     

The Chemistry of C6H6O radical cations: A study of rearrangement reactions of halogen substituted ethyl phenyl ethers

New experimental data on the rearrangement reaction of various phenoxyethyl halides to give [C₆H₆O]^+˙ are presented and compared with previous studies so that a coherent picture of this process can be developed. By examining the metastable kinetic energy release for low energy decomposing molecular ions of the phenoxyethyl halides, it has been concluded that formation of [C₆H₆O] occurs by competitive 1,2 and 1,3 hydrogen shifts from the alkyl carbons to oxygen followed by a rate determining C? O bond cleavage. This is substantiated by the absence of a primary hydrogen isotope effect. For more highly activated molecular ions, a new mechanism comes into play as evidenced by the appearance of a small hydrogen isotope effect. It is postulated that this third mechanism involves transfer of the alkyl hydrogen to the ortho position of the ring by a rate determining 1,5 shift, followed by a 1,3 hydrogen shift from the ortho methylene group to oxygen and rapid C? O bond cleavage. This 1,3 hydrogen shift to oxygen appears to be ‘catalysed’ by the halogen atoms yielding phenol ions. No indications have been found for the formation of tautomeric 2,4-cyclohexadienone ions. Furthermore, highly activated molecular ions produce [C₆H₆O]^+˙ which can undergo metastable decomposition to lose carbon monoxide. Kinetic energy release measurements for the latter reaction show that the majority of these [C₆H₆O]^+˙ions have been formed as phenol ions as well. These arguments are supported by energetic measurements and by comparisons with previous ion cyclotron resonance and collisional activation studies.     

Structural determination of oxofatty acids by charge-remote fragmentations

A strategy is described to locate the carbonyl position in oxofatty acids by utilizing charge-remote fragmentations of various molecular ions that are desorbed by fast atom bombardment (FAB). Oxofatty acids were cationized with alkali metal ions (Li⁺, Na⁺, K⁺, Rb⁺, and Cs⁺) to form [M+2Met?H]⁺ or alkaline earth metal ions (Mg²⁺, Ca²⁺, Sr²⁺ or Ba²⁺) to form [M+Met?H]⁺ in the gas phase. The cationized acids undergo charge-remote fragmentations upon high-energy activation, giving a product-ion pattern that has a gap corresponding to the oxo position and bordered by two high-intensity peaks. One of the peaks corresponds to an ion that is formed by the cleavage of the C-C bond β to the oxo position and proximal to the charge (β ion), whereas the other is formed from the cleavage of the C-C bond γ to the oxo position and distal to the charge (γ′ ion). The oxo position is easily determined by identifying the gap and the β and γ′ ions. Furthermore, there are two competing patterns of fragments in a CAD spectrum of an oxofatty acid or ester [M+Li]⁺ ion. These arise because Li⁺ attaches to either the oxo or the carboxylic end, as was confirmed by ab initio molecular orbital calculations. The results demonstrate that control of the fragmentation can be guided by an understanding of metal-ion affinities. Collisional activation of the anionic carboxylates gives results that are similar to those for positive ions, showing that the process is not related to the charge status. Collisional activation of [M+H]⁺ ions does not give structural information because the charge migrates, leading to charge-mediated fragmentations.     

Dissociation dynamics of halotoluene ions,production of tolyl,benzyl and tropylium ([C7H7]+)ions

The dissociation rates and energetics of the loss of halogen atoms from energy-selected halotoluene ions were investigated by photoelectron photoion coincidence (PEPICO) and collisional activation (CA) mass spectrometric experiments. Dissociation onsets, determined from the dissociation rates measured as a function of the internal energy of the parent ion, revealed the formation of three [C₇H₇]⁺ isomers, which were identified, on the basis of the CA data, as the tolyl, benzyl and tropylium ions. All of the ions investigated produced a mixture of isomeric ions. Only iodotoluene ions produced any tolyl product ions by a direct bond cleavage. The bromo- and chlorotoiuene ions produced mixtures of benzyl and tropyl ions. The observed two-component decay rates of the iodotoluene ions revealed the participation of a lower energy [C₇H₇I]^{+ ˙} isomer in the dissociation process. The identity of this isomer is not known but it probably does not have the cycloheptatriene ion structure because considerable kinetic energy was released in this dissociation.     

Dissociation and rearrangements of the molecular ions of sulfides,sulfoxides, and sulfones generated by a strong electric field

The field mass spectra of 18 sulfides, sulfoxides, and sulfones were investigated, and the principles of charge localization in the dissociation and rearrangements of the molecular ions of these compounds were established. A new type of fragmentation leading to the elimination of S^+., SO^+., and SO₂. was observed. A new mechanism, according to which cleavage of two C-S bonds and the formation of one new C-C bond due to radical recombination cocur in the cyclic transition complex, is proposed. Prior migration of alkyl radicals from sulfur to oxygen with subsequent cleavage of the S-O and C-S bonds via the above-mentioned mechanism may also occur in the molecular ions of sulfoxides and sulfones.Translated from Teoreticheskaya i Éksperimental'naya Khimiya, Vol. 23, No. 2, pp. 172–181, March–April, 1987.     

Mechanisms and energetics of free radical initiated disulfide bond cleavage in model peptides and insulin by mass spectrometry

We investigate the mechanism of disulfide bond cleavage in gaseous peptide and protein ions initiated by a covalently-attached regiospecific acetyl radical using mass spectrometry (MS). Highly selective S–S bond cleavages with some minor C–S bond cleavages are observed by a single step of collisional activation. We show that even multiple disulfide bonds in intact bovine insulin are fragmented in the MS2 stage, releasing the A- and B-chains with a high yield, which has been challenging to achieve by other ion activation methods. Yet, regardless of the previous reaction mechanism studies, it has remained unclear why (1) disulfide bond cleavage is preferred to peptide backbone fragmentation, and why (2) the S–S bond that requires the higher activation energy conjectured in previously suggested mechanisms is more prone to be cleaved than the C–S bond by hydrogen-deficient radicals. To probe the mechanism of these processes, model peptides possessing deuterated β-carbon(s) at the disulfide bond are employed. It is suggested that the favored pathway of S–S bond cleavage is triggered by direct acetyl radical attack at sulfur with concomitant cleavage of the S–S bond (S_H2). The activation energy for this process is substantially lower by ∼9–10 kcal mol^–1 than those of peptide backbone cleavage processes determined by density functional quantum chemical calculations. Minor reaction pathways are initiated by hydrogen abstraction from the α-carbon or the β-carbon of a disulfide, followed by β-cleavages yielding C–S or S–S bond scissions. The current mechanistic findings should be generally applicable to other radical-driven disulfide bond cleavages with different radical species such as the benzyl and methyl pyridyl radicals.