Mass spectral fragmentation pathways in nitramines. A collision-induced dissociation study

A collision-induced dissociation (CID) study of five synthesized nitramines was carried out using a hybrid EBQQ mass spectrometer. CID spectra were obtained in two modes: B/E linked-scan mode and MS/MS mode using the EB sector combination as the first mass spectrometer and the QQ as collision cell and second mass spectrometer, respectively. Fragmentation pathways of the compounds were determined in the electron-impact mode. It was found that dominant fragmentation pathways included the loss of OH, NO₂ and HNO₂ in addition to the loss of CH₂NNO and CH₂NNO₂.     

Improving fragmentation of poorly fragmenting peptides and phosphopeptides during collision-induced dissociation by malondialdehyde modification of arginine residues

Despite significant technological and methodological advancements in peptide sequencing by mass spectrometry, analyzing peptides that exhibit only poor fragmentation upon collision-induced dissociation (CID) remains a challenge. A major cause for unfavorable fragmentation is insufficient proton 'mobility' due to charge localization at strongly basic sites, in particular, the guanidine group of arginine. We have recently demonstrated that the conversion of the guanidine group of the arginine side chain by malondialdehyde (MDA) is a convenient tool to reduce the basicity of arginine residues and can have beneficial effects for peptide fragmentation. In the present work, we have focused on peptides that typically yield incomplete sequence information in CID-MS/MS experiments. Energy-resolved tandem MS experiments were carried out on angiotensins and arginine-containing phosphopeptides to study in detail the influence of the modification step on the fragmentation process. MDA modification dramatically improved the fragmentation behavior of peptides that exhibited only one or two dominant cleavages in their unmodified form. Neutral loss of phosphoric acid from phosphopeptides carrying phosphoserine and threonine residues was significantly reduced in favor of a higher abundance of fragment ions. Complementary experiments were carried out on three different instrumental platforms (triple-quadrupole, 3D ion trap, quadrupole-linear ion trap hybrid) to ascertain that the observation is a general effect.     

Mass spectral fragmentation pathways in phthalate esters. A tandem mass spectrometric collision-induced dissociation study

A collision-induced dissociation study of a series of phthalate esters was carried out using a tandem BB mass spectrometer. Fragmentation pathways of the phthalates were determined in the electron impact mode. Two major daughter ions are formed, one by a McLafferty rearrangement and hydrogen transfer and the other by loss of an alkoxy radical Another major daughter ion, at m/z 149—which is the base peak in the electron impact mass spectra of most phthalate esters—is being formed through four alternate pathways.     

Charge-remote fragmentation characteristics of functionalized alkanes in high-energy collision-induced dissociation

In this study we report on high-energy, collision-induced dissociation processes leading to charge-remote fragmentations, using three alkyl cations, namely n-hexadecylpyridinium, n-hexadecyltriphenylphosphonium and n-hexadecyltriethylammonium, each with and without (2)H(2)-labelling at the C(9) position of the hexadecyl chain. The characteristic patterns corresponding to the formal elimination of alkane elements were observed, and the (2)H(2)-labelling at C(9) clearly affected only one charge-remote fragment ion of the homologous series. However, in addition to the expected fragment ion containing only one deuterium atom, a significant ion retaining two deuterium atoms was observed. MS/MS/MS experiments demonstrated clearly that the latter ion showed partial deuteration around the charge site, the level of deuteration depending on the structure of the original precursor cation. These results can be interpreted in terms of two novel, distinct mechanisms, one of which involves an excited state in an aromatic ring. Mixed-site fragmentation (MSF) ions were also observed from the phosphonium and ammonium ion precursors. We believe that the observation of the MSF process occurring at an sp(2)-hybridized center in the phosphonium series has not been reported previously. It thus becomes apparent that high-energy collisions leading to charge-remote reactions in fact lead to a broad range of pathways. Copyright 2000 John Wiley & Sons, Ltd.     

Scrambling of sequence information in collision-induced dissociation of peptides

Collision-induced dissociation (CID) of protonated YAGFL-NH2 leads to nondirect sequence fragment ions that cannot directly be derived from the primary peptide structure. Experimental and theoretical evidence indicate that primary fragmentation of the intact peptide leads to the linear YAGFLoxa b5 ion with a C-terminal oxazolone ring that is attacked by the N-terminal amino group to induce formation of a cyclic peptide b5 isomer. The latter can undergo various proton transfer reactions and opens up to form something other than the YAGFLoxa linear b5 isomer, leading to scrambling of sequence information in the CID of protonated YAGFL-NH2.     

Mass spectral fragmentation pathways in some dinitroaromatic compounds studied by collision-induced dissociation and tandem mass spectrometry

A collision-induced dissociation study of a series of dinitroaromatic compounds was carried out using a tandem BB mass spectrometer. Fragmentation pathways were determined in the electron impact mode. Loss of NO₂˙ from the molecular ion was observed In most of the investigated compounds. In some compounds loss of NO₂˙ occurred only after loss of OH˙. In other compounds it was not observed at all because of competitive processes, such as loss of NO˙, CO₂, CH₂O, C₂H₄ or H₂O. Loss of NO˙ was a major decomposition pathway, forming ‘dished peaks’ in some of the compounds having a nitro group ortho to a phenyl group, indicating a release of kinetic energy associated with the decomposition. Loss of OH˙ due to an ‘ortho effect’ occurred in compounds where a nitro group was ortho to a group containing a labile hydrogen, but was not observed when competitive processes such as loss of NO˙, NO₂˙ or H₂O occurred. ‘Nitro to nitrite’ isomerization was suggested to explain the decarboxylation process in 2,4- and 2,5-dinitrobenzoic acid and the loss of COH₂ in 2,4-dinitroanisole.     

Mass spectral fragmentation pathways in some glycoluril-type explosives. A study by collision-induced dissociation and isotope labeling

Electron impact (EI), chemical ionization and negative-ion chemical ionization (NCI) mass spectra of 1,4-dinitroglycoluril (DINGU), its ¹⁵N- and ²H-labeled analogues and the dimethyl-substituted derivatives were recorded. Tandem mass spectrometry with collision induced dissociation was used to study the fragmentation pathways of these compounds. It was found that the main EI fragmentation processes of DINGU are due to the cleavage of C? N bonds and some rearrangement reactions.     

Three competitive dissociation pathways of glyoxalate

Three possible lower barrier dissociation pathways of the deportonated glyoxylic acid in aqueous solution are predicated by means of B3LYP and CCSD（T） （single-point） methods. The calculated results suggest that the formation of formate ion is the energetically most favorable process, and that the decomposition reaction is occurring through nucleophilic attack of negatively charged carboxylic oxygen on the α-carbon. This is in good agreement with the proposed mechanisms of the mass spectrometry experiment.     

Investigation of the neutral loss of a full amino acid mass during collision-induced dissociation of the b(3)+ ion derived from a model peptide containing a 4-aminobutyric acid residue

In a previous study we found that a dominant fragmentation pathway observed for collision-induced dissociation (CID) of b(3)+ derived from peptides with sequence AXAG, where X is gamma-aminobutyric acid (gammaAbu) or epsilon-aminocaproic acid (Cap), involved the loss of 89 mass units (u). A neutral loss of 89 u corresponded to the free acid mass of an alanine (A) residue. This specific pathway was studied in greater detail here using a series of A(gammaAbu)AG peptides with strategic positioning of (15)N, (13)C and (2)H isotope labels. Based on the extensive labeling, several possible routes to the net elimination of 89 u are proposed. One is based on initial elimination of either aziridinone or imine and CO, followed by opening of an oxazolinone, tautomerization and elimination of H2O. Another involves formation of an aziridinone by cleavage of the N-terminal amide bond, and transfer of O and H atoms to this fragment via an H-bonded ion-molecule complex to complete the loss of 89 u. Both types of pathway include the transfer/migration of H atoms from the alpha-carbon position of gammaAbu or A residues.     

Collision-induced dissociation of protonated tetrapeptides containing beta-alanine, gamma-aminobutyric acid, epsilon-aminocaproic acid or 4-aminomethylbenzoic acid residues

The influence of the presence and position of a single beta-alanine, gamma-aminobutyric acid, epsilon-aminocaproic acid or 4-aminomethylbenzoic acid residue on the tendency to form b(n)+ -and y(n)+ -type product ions was determined using a group of protonated tetrapeptides with general sequence XAAG, AXAG and AAXG (where X refers to the position of amino acid substitution). The hypothesis tested was that the 'alternative' amino acids would influence product ion signal intensities by inhibiting or suppressing either the nucleophilic attack or key proton transfer steps by forcing the adoption of large cyclic intermediates or blocking cyclization altogether. We found that specific b ions are diminished or eliminated completely when betaA, gammaAbu, Cap or 4AMBz residues are positioned such that they should interfere with the intramolecular nucleophilic attack step. In addition, differences in the relative proton affinities of the alternative amino acids influence the competition between complementary b(n) and y(n) ions. For both the AXAG and the XAAG series of peptides, collision-induced dissociation (CID) generated prominent b ions despite potential inhibition or suppression of intramolecular proton migration by the betaA, gammaAbu, Cap or 4AMBz residues. The prominent appearance of b ions from the AXAG and XAAG peptide is noteworthy, and suggests either that proton migration occurs through larger, 'whole' peptide cyclic intermediates or that fragmentation proceeds through a population of [M+H]+ isomers that are initially protonated at amide O atoms.     

Comparison of infrared multiphoton dissociation and collision-induced dissociation of supercharged peptides in ion traps

The number and types of diagnostic ions obtained by infrared multiphoton dissociation (IRMPD) and collision-induced dissociation (CID) were evaluated for supercharged peptide ions created by electrospray ionization of solutions spiked with m-nitrobenzyl alcohol. IRMPD of supercharged peptide ions increased the sequence coverage compared with that obtained by CID for all charge states investigated. The number of diagnostic ions increased with the charge state for IRMPD; however, this trend was not consistent for CID because the supercharged ions did not always yield the greatest number of diagnostic ions. Significantly different fragmentation pathways were observed for the different charge states upon CID or IRMPD with the latter yielding far more immonium ions and often fewer uninformative ammonia, water, and phosphoric acid neutral losses. Pulsed-Q dissociation resulted in an increase in the number of internal product ions, a decrease in sequence-informative ions, and reduced overall ion abundances. The enhanced sequence coverage afforded by IRMPD of supercharged ions was demonstrated for a variety of model peptides, as well as for a tryptic digest of cytochrome c.     

On performing simultaneous electron transfer dissociation and collision-induced dissociation on multiply protonated peptides in a linear ion trap

We propose a tandem mass spectrometry method that combines electron-transfer dissociation (ETD) with simultaneous collision-induced dissociation (CID), termed ETD/CID. This technique can provide more complete sequence coverage of peptide ions, especially those at lower charge states. A selected precursor ion is isolated and subjected to ETD. At the same time, a residual precursor ion is subjected to activation via CID. The specific residual precursor ion selected for activation will depend upon the charge state and m/z of the ETD precursor ion. Residual precursor ions, which include unreacted precursor ions and charge-reduced precursor ions (either by electron-transfer or proton transfer), are often abundant remainders in ETD-only reactions. Preliminary results demonstrate that during an ETD/CID experiment, b, y, c, and z-type ions can be produced in a single experiment and displayed in a single mass spectrum. While some peptides, especially doubly protonated ones, do not fragment well by ETD, ETD/CID alleviates this problem by acting in at least one of three ways: (1) the number of ETD fragment ions are enhanced by CID of residual precursor ions, (2) both ETD and CID-derived fragments are produced, or (3) predominantly CID-derived fragments are produced with little or no improvement in ETD-derived fragment ions. Two interesting scenarios are presented that display the flexibility of the ETD/CID method. For example, smaller peptides that show little response to ETD are fragmented preferentially by CID during the ETD/CID experiment. Conversely, larger peptides with higher charge states are fragmented primarily via ETD. Hence, ETD/CID appears to rely upon the fundamental reactivity of the analyte cations to provide the best fragmentation without implementing any additional logic or MS/MS experiments. In addition to the ETD/CID experiments, we describe a novel dual source interface for providing front-end ETD capabilities on a linear ion trap mass spectrometer.     

On the dynamics of fragment isomerization in collision-induced dissociation of peptides

The structures of peptide collision-induced dissociation (CID) product ions are investigated using ion mobility/mass spectrometry techniques combined with theoretical methods. The cross-section results are consistent with a mixture of linear and cyclic structures for both b4 and a4 fragment ions. Direct evidence for cyclic structures is essential in rationalizing the appearance of fragments with scrambled (i.e., permutated) primary structures, as the cycle may not open up where it was initially formed. It is demonstrated here that cyclic and linear a4 structures can interconvert freely as a result of collisional activation, implying that isomerization takes place prior to dissociation.     

The promotion of d-type ions during the low energy collision-induced dissociation of some cysteic acid-containing peptides

Low energy collisionally activated dissociations (CAD) of doubly protonated peptides incorporating cysteic acid and arginine residues have been studied. Deuterium labeling experiments have established that loss of the elements of H₂SO₃ occurs with cleavage of one CH bond and transfer of the hydrogen to a neutral fragment. Prominent d-type ions were observed corresponding to cleavage at the cysteic acid residue. The analysis of structural analogs suggested that the unexpectedly low energy requirement for this process is attributable to a charge-proximal process promoted by intra-ionic interaction of the arginine and cysteic acid side chains. CAD (in the collision hexapole of a tandem quadrupole instrument) of electrospray source-formed fragment ions established that the d-type ions can form via b-type ions; there was no evidence of formation via (a _n + 1) or (b _n — H₂SO₃) ions. The equivalent d-ion was observed, albeit with lesser abundance, when the cysteic acid residue was replaced by aspartic acid, but not by glutamic acid.     

Detection and characterization of methionine oxidation in peptides by collision-induced dissociation and electron capture dissociation

Electron capture dissociation (ECD) and collision-induced dissociation (CID), the two complementary fragmentation techniques, are demonstrated to be effective in the detection and localization of the methionine sulfoxide [Met(O)] residues in peptides using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. The presence of Met(O) can be easily recognized in the low-energy CID spectrum showing the characteristic loss of methanesulfenic acid (CH(3)SOH, 64 Da) from the side chain of Met(O). The position of Met(O) can then be localized by ECD which is capable of providing extensive peptide backbone fragmentation without detaching the labile Met(O) side chain. We studied CID and ECD of several Met(O)-containing peptides that included the 44-residue human growth hormone-releasing factor (GRF) and the human atrial natriuretic peptide (ANP). The distinction and complementarity of the two fragmentation techniques were particularly remarkable in their effects on ANP, a disulfide bond-containing peptide. While the predominant fragmentation pathway in CID of ANP was the loss of CH(3)SOH (64 Da) from the molecular ion, ECD of ANP resulted in many sequence-informative products, including those from cleavages within the disulfide-bonded cyclic structure, to allow for the direct localization of Met(O) without the typical procedures for disulfide bond reduction followed by [bond]SH alkylation.     

Search of sequence databases with uninterpreted high-energy collision-induced dissociation spectra of peptides

We have broadened the utility of the SEQUEST computer algorithms to permit correlation of uninterpreted high-energy collision-induced dissociation spectra of peptides with all sequences in a database. SEQUEST now allows for the additional fragment ion types observed under high-energy conditions. We analyzed spectra from peptides isolated following trypsin digestion of 13 proteins. SEQUEST ranked the correct sequence first for 90% (18/20) of the spectra in searches of the OWL database, without constraint by enzyme cleavage specificity or species of origin. All false-positives were flagged by the scoring system. SEQUEST searches databases for sequences that correspond to the precursor ion mass ±0.5 u. Preliminary ranking of the top 500 candidates is done by calculation of fragment ion masses for each sequence, and comparison to the measured ion masses on the basis of ion series continuity, summed ion intensity, and immonium ion presence. Final ranking is done by construction of model spectra for the 500 candidates and constructing/performing of a cross-correlation analysis with the actual spectrum. Given the need to relate mounting genome sequence information with corresponding suites of proteins that comprise the cellular molecular machinery, tandem mass spectrometry appears destined to play the leading role in accelerating protein identification on the large scale required.     

Energy-dependent collision-induced dissociation of lithiated polytetrahydrofuran: Effect of the size on the fragmentation properties

The fragmentation properties of singly and doubly lithiated polytetrahydrofuran (PTHF) were studied using energy-dependent collision-induced dissociation. The product ion spectrum of [PTHF + Li]⁺ showed the formation of three different series corresponding to product ions with hydroxyl, aldehyde and vinyl end-groups. Interestingly, besides these series, two additional, non-lithiated product ions C₄H₉O⁺ and C₄H ₇ ⁺ were identified in the MS/MS spectra. The MS/MS of the doubly lithiated PTHF ([PTHF + 2Li]²⁺) with a number of repeat units ranging from 8 to 27 showed the formation of product ions similar to those of the singly lithiated series, however, doubly lithiated product ions and product ions formed by the loss of one Li⁺-ion from the precursor ion also appeared with significant abundances. Analysis of the breakdown curves for the singly and doubly charged PTHF indicated that the series A ions are formed most probably together with the series B ions, while members of the series C ions appeared at significantly higher collision energies. The fragmentation properties of [PTHF + Li]⁺ and [PTHF + 2Li]²⁺ were also interpreted using the survival yield method. It was found that the collision energy/voltage necessary to obtain 50% fragmentation (CV₅₀) was dependent linearly on the number of the repeat units, i.e., on the size, or the number of degrees of freedom (DOF).     

Dehydration versus deamination of N-terminal glutamine in collision-induced dissociation of protonated peptides

Some of the most prominent "neutral losses" in peptide ion fragmentation are the loss of ammonia and water from N-terminal glutamine. These processes are studied by electrospray ionization mass spectrometry in singly- and doubly-protonated peptide ions undergoing collision-induced dissociation in a triple quadrupole and in an ion trap instrument. For this study, four sets of peptides were synthesized: (1) QLLLPLLLK and similar peptides with K replaced by R, H, or L, and Q replaced by a number of amino acids, (2) QLnK (n = 0, 1, 3, 5, 7, 9, 11), (3) QLnR (n = 0, 1, 3, 5, 7, 9), and (4) QLn (n = 1, 2, 3, 4, 8). The results for QLLLPLLLK and QLLLPLLLR show that the singly protonated ions undergo loss of ammonia and to a smaller extent loss of water, whereas the doubly protonated ions undergo predominant loss of water. The fast fragmentation next to P (forming the y5 ion) occurs to a larger extent than the neutral losses from the singly protonated ions but much less than the water loss from the doubly protonated ions. The results from these and other peptides show that, in general, when N-terminal glutamine peptides have no "mobile protons", that is, the number of charges on the peptide is no greater than the number of basic amino acids (K, R, H), deamination is the predominant neutral loss fragmentation, but when mobile protons are present the predominant process is the loss of water. Both of these processes are faster than backbone fragmentation at the proline. These results are rationalized on the basis of resonance stabilization of the two types of five-membered ring products that would be formed in the neutral loss processes; the singly protonated ion yields the more stable neutral pyrrolidinone ring whereas the doubly protonated ion yields the protonated aminopyrroline ring (see Schemes). The generality of these trends is confirmed by analyzing an MS/MS spectra library of peptides derived from tryptic digests of yeast. In the absence of mobile protons, glutamine deamination is the most rapid neutral loss process. For peptides with mobile protons, dehydration from glutamine is far more rapid than from any other amino acid. Most strikingly, end terminal glutamine is by far the most labile source of neutral loss in excess-proton peptides, but not highly exceptional when mobile protons are not available. In addition, rates of deamination are faster in lysine versus arginine C-terminus peptides and 20 times faster in positively charged than negatively charged peptides, demonstrating that these formal neutral loss reactions are not "neutral reactions" but depend on charge state and stability.     

Characterization of the product ions from the collision-induced dissociation of argentinated peptides

Tandem mass spectrometry performed on a pool of 18 oligopeptides shows that the product ion spectra of argentinated peptides, the [bn + OH + Ag]+ ions and the [yn - H + Ag]+ ions bearing identical sequences are virtually identical. These observations suggest strongly that these ions have identical structures in the gas phase. The structures of argentinated glycine, glycylglycine, and glycylglycylglycine were calculated using density functional theory (DFT) at the B3LYP/DZVP level of theory; they were independently confirmed using HF/LANL2DZ. For argentinated glycylglycylglycine, the most stable structure is one in which Ag+ is tetracoordinate and attached to the amino nitrogen and the three carbonyl oxygen atoms. Mechanisms are proposed for the fragmentation of this structure to the [b2 + OH + Ag]+ and the [Y2 - H + Ag]+ ions that are consistent with all experimental observations and known calculated structures and energetics. The structures of the [b2 - H + Ag]+ and the [a2 - H + Ag]+ ions of glycylglycylglycine were also calculated using DFT. These results confirm earlier suggestions that the [b2 - H + Ag]+ ion is an argentinated oxazolone and the [a2 - H + Ag]+ an argentinated immonium ion.     

Five-membered ring formation in unimolecular reactions of peptides: a key structural element controlling low-energy collision-induced dissociation of peptides

Unimolecular fragmentation reactions of peptides in low-energy collision-induced dissociation are reviewed in the mechanistic context of five-membered ring formation. This structure of intermediates or of fragment ions is recognized as a key element that governs unimolecular peptide fragmentation within the structural framework determined by the peptide backbone and its side-chains. A collection of collision-induced dissociation reactions is presented covering (i) b-ion formation, (ii) the fragmentation of N-terminally acylated peptides, (iii) neutral loss of the C-terminal amino acid in alkali or silver cationized peptides, (iv) the fragmentation of isoAsp-containing peptides and (v) the fragmentation of negatively charged Asp- or Glu-containing peptides. It appears that for all possible nucleophile-electrophile interactions leading to a five-membered ring structure an associated unimolecular peptide fragmentation reaction can be observed.