Collision-induced fragmentation pathways including odd-electron ion formation from desorption electrospray ionisation generated protonated and deprotonated drugs derived from tandem accurate mass spectrometry

The rapid desorption electrospray ionisation (DESI) of some small molecules and their fragmentation using a triple-quadrupole and a hybrid quadrupole time-of-flight mass spectrometer (Q-ToF) have been investigated. Various scanning modes have been employed using the triple-quadrupole instrument to elucidate fragmentation pathways for the product ions observed in the collision-induced dissociation (CID) spectra. Together with accurate mass tandem mass spectrometry (MS/MS) measurements performed on the hybrid Q-ToF mass spectrometer, unequivocal product ion identification and fragmentation pathways were determined for deprotonated metoclopramide and protonated aspirin, caffeine and nicotine. Ion structures and fragmentation pathway mechanisms have been proposed and compared with previously published data. The necessity for elevated resolution for the differentiation of isobaric ions are discussed.     

Fragmentation of protonated dipeptides containing arginine. Effect of activation method

The fragmentation reactions of the protonated dipeptides Gly-Arg and Arg-Gly have been studied using collision-induced dissociation (CID) in a quadrupole ion trap, by in-source CID in a single-quadrupole mass spectrometer and by CID in the quadrupole cell of a QqTOF mass spectrometer. In agreement with earlier quadrupole ion trap studies (Farrugia, J. M.; O'Hair, R. A. J., Int. J. Mass Spectrom., 2003, 222, 229), the CID mass spectra obtained with the ion trap for the MH(+) ions and major fragment ions are very similar for the two isomers indicating rearrangement to a common structure before fragmentation. In contrast, in-source CID of the MH(+) ions and QqTOF CID of the MH(+), [MH - NH(3)](+) and [MH <23 HN = C(NH(2))(2)](+) ions provide distinctly different spectra for the isomeric dipeptides, indicating that rearrangement to a common structure has not occurred to a significant extent under these conditions even near the threshold for fragmentation in the QqTOF instrument. Clearly, under normal operating conditions significantly different fragmentation behavior is observed in the ion trap and beam-type experiments. This different behavior probably can be attributed to the shorter observation times and concomitant higher excitation energies in the in-source and QqTOF experiments compared to the long observation times and lower excitation energies relevant to the ion trap experiments. Based largely on elemental compositions derived from accurate mass measurements in QqTOF studies fragmentation schemes are proposed for the MH(+), [MH - NH(3)](+), and [MH - (HN = C(NH(2))(2))](+) ions.     

Comparison of Triple Quadrupole and Double Focusing Mass Spectrometers to Investigate Collision-Induced Dissociation and Metastable Ions of Glycoconjugates

Glycoconjugates, such as chromophore-labeled disaccharides and permethylated glycosphingolipids (GSL) were used for comparison of triple quadrupole and double focusing mass spectrometers in analysis of product ions. A profound effect of collision energy was observed in the product ion spectra of ceramide ions (fragment ions of permethylated GSL): more product ions were observed from a double focusing mass spectrometer. Besides collision energy, the structure of the analyte had a significant effect on the formation of product ions. Despite the fact that masses of protonated molecular ions (MH⁺) of permethylated GSL are significantly larger than their ceramide fragments, the low-energy and high-energy product ion spectra of MH⁺ are, in general, similar. In a double focusing mass spectrometer of reversed geometry, more metastable ions were observed in the first field free region (FFR) than in the second FFR. The metastable ions observed in the second FFR were similar to those observed in low-energy collision-induced dissociation (CID). Although a double focusing mass spectrometer is superior to triple quadrupole instrument for detection of product ions, the poor resolution in either the selection of precursor ion or in the product ion spectra can be a serious problem in analysis of a mixture with similar masses.     

Effect of the basic residue on the energetics, dynamics, and mechanisms of gas-phase fragmentation of protonated peptides

The effect of the basic residue on the energetics, dynamics, and mechanisms of backbone fragmentation of protonated peptides was investigated. Time-resolved and collision energy-resolved surface-induced dissociation (SID) of singly protonated peptides with the N-terminal arginine residue and their analogues, in which arginine is replaced with less basic lysine and histidine residues, was examined using a specially configured Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). SID experiments demonstrated different kinetics of formation of several primary product ions of peptides with and without arginine residue. The energetics and dynamics of these pathways were determined from Rice-Ramsperger-Kassel-Marcus (RRKM) modeling of the experimental data. Comparison between the kinetics and energetics of fragmentation of arginine-containing peptides and the corresponding methyl ester derivatives provides important information on the effect of dissociation pathways involving salt bridge (SB) intermediates on the observed fragmentation behavior. Because pathways involving SB intermediates are characterized by low threshold energies, they efficiently compete with classical oxazolone and imine/enol pathways of arginine-containing peptides on a long time scale of the FTICR instrument. In contrast, fragmentation of histidine- and lysine-containing peptides is largely determined by canonical pathways. Because SB pathways are characterized by negative activation entropies, fragmentation of arginine-containing peptides is kinetically hindered and observed at higher collision energies as compared to their lysine- and histidine-containing analogues.     

Implementation of dipolar direct current (DDC) collision-induced dissociation in storage and transmission modes on a quadrupole/time-of-flight tandem mass spectrometer

Means for effecting dipolar direct current collision-induced dissociation (DDC CID) on a quadrupole/time-of-flight in a mass spectrometer have been implemented for the broadband dissociation of a wide range of analyte ions. The DDC fragmentation method in electrodynamic storage and transmission devices provides a means for inducing fragmentation of ions over a large mass-to-charge range simultaneously. It can be effected within an ion storage step in a quadrupole collision cell that is operated as a linear ion trap or as ions are continuously transmitted through the collision cell. A DDC potential is applied across one pair of rods in the quadrupole collision cell of a QqTOF hybrid mass spectrometer to effect fragmentation. In this study, ions derived from a small drug molecule, a model peptide, a small protein, and an oligonucleotide were subjected to the DDC CID method in either an ion trapping or an ion transmission mode (or both). Several key experimental parameters that affect DDC CID results, such as time, voltage, low mass cutoff, and bath gas pressure, are illustrated with protonated leucine enkephalin. The DDC CID dissociation method gives a readily tunable, broadband tool for probing the primary structures of a wide range of analyte ions. The method provides an alternative to the narrow resonance conditions of conventional ion trap CID and it can access more extensive sequential fragmentation, depending upon conditions. The DDC CID approach constitutes a collision analog to infrared multiphoton dissociation (IRMPD).     

Cone voltage and collision cell collision-induced dissociation study of triphenylethylenes of pharmaceutical interest.

Fragmentations of three triphenylethylene compounds (toremifene and its two metabolites) with different functional side-chain groups (alcohol, acid and amine) were studied. The compounds were dissociated by collision-induced dissociation (CID) in the interface region of an electrospray ionization source (ESI(+)) and in the collision cell of a triple quadrupole mass spectrometer. Fragmentation pathways for these molecules are proposed, based on accurate mass measurements of in-source fragment ions and MS/MS experiments using product and precursor ion scanning. The side-chain functional groups were found to strongly affect the fragmentations of the molecular ions. The fragmentation pathways of the protonated molecule and sodium ion adduct were quite similar, but the subsequent stabilities of certain common fragments were surprisingly different.     

Specific photodissociation of peptides with multi-stage mass spectrometry

We report collision-induced dissociation (CID) and laser-induced dissociation (LID) performed at different wavelengths between 220 and 280 nm of the peptides leucine-enkephalin (protonated) and gramicidin A (sodiated). Hydrogen-atom losses and side-chain cleavages were observed in LID experiments. These losses depend on the laser wavelength and lead to the formation of radical ions. The fragmentations of these radicals, which are not observed in CID experiments, were investigated in multi-stage mass spectrometry experiments.     

ECD of Tyrosine Phosphorylation in a Triple Quadrupole Mass Spectrometer with a Radio-Frequency-Free Electromagnetostatic Cell

A radio frequency-free electromagnetostatic (EMS) cell devised for electron-capture dissociation (ECD) of ions has been retrofitted into the collision-induced dissociation (CID) section of a triple quadrupole mass spectrometer to enable recording of ECD product-ion mass spectra and simultaneous recording of ECD-CID product-ion mass spectra. This modified instrument can be used to produce easily interpretable ECD and ECD-CID product-ion mass spectra of tyrosine-phosphorylated peptides that cover over 50% of their respective amino-acid sequences and readily identify their respective sites of phosphorylation. ECD fragmentation of doubly protonated, tyrosine-phosphorylated peptides, which was difficult to observe with FT-ICR instruments, occurs efficiently in the EMS cell. Figure

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Sequence-specific fragmentation of deprotonated peptides containing H or alkyl side chains

The [M - H]- ions of a variety of di- to pentapeptides containing H or alkyl side chains have been prepared by electrospray ionization and low-energy collision-induced dissociation (CID) of the deprotonated species carried out in the interface region between the atmospheric pressure source and the quadrupole mass analyzer. Using the nomenclature applied to the fragmentation of protonated peptides, deprotonated dipeptides fragment to give a2 ions (CO2 loss) and y1 ions, where the y1 ion has two fewer hydrogens than the y"1 ions formed from protonated peptides. Deprotonated tri- and tetrapeptides fragment to give primarily y1, c1, and "b2 ions, where the "b2 ion has two fewer hydrogens than the b2 ion observed for protonated peptides. More minor yields of y2, c2, and a2 ions also are observed. The a ion formed by loss of CO2 from the [M - H]- ion shows loss of the N-terminal residue for tripeptides and sequential loss of two amino acid residues from the N-terminus for tetrapeptides. The formation of c(n) ions and the sequential loss of N-terminus residues from the [M - H - CO2]- ion serves to sequence the peptide from the N-terminus, whereas the formation of y(n) ions serves to sequence the peptide from the C-terminus. It is concluded that low-energy CID of deprotonated peptides provides as much (or more) sequence information as does CID of protonated peptides, at least for those peptides containing H or alkyl side chains. Mechanistic aspects of the fragmentation reactions observed are discussed.     

Electrospray Ionization Tandem Mass Spectrometry of Ammonium Cationized Polyethers

Quaternary ammonium salts (Quats) and amines are known to facilitate the MS analysis of high molar mass polyethers by forming low charge state adduct ions. The formation, stability, and behavior upon collision-induced dissociation (CID) of adduct ions of polyethers with a variety of Quats and amines were studied by electrospray ionization quadrupole time-of-flight, quadrupole ion trap, and linear ion trap tandem mass spectrometry (MS/MS). The linear ion trap instrument was part of an Orbitrap hybrid mass spectrometer that allowed accurate mass MS/MS measurements. The Quats and amines studied were of different degree of substitution, structure, and size. The stability of the adduct ions was related to the structure of the cation, especially the amine’s degree of substitution. CID of singly/doubly charged primary and tertiary ammonium cationized polymers resulted in the neutral loss of the amine followed by fragmentation of the protonated product ions. The latter reveals information about the monomer unit, polymer sequence, and endgroup structure. In addition, the detection of product ions retaining the ammonium ion was observed. The predominant process in the CID of singly charged quaternary ammonium cationized polymers was cation detachment, whereas their doubly charged adduct ions provided the same information as the primary and tertiary ammonium cationized adduct ions. This study shows the potential of specific amines as tools for the structural elucidation of high molar mass polyethers.     

Application of the statistical test of equivalent pathways (STEP) method to the triple quadrupole mass spectrometer

Recently, we demonstrated a new method, STEP (Statistical Test of Equivalent Pathways) analysis, which differentiates first-generation product ions (primary product ions) from second-generation product ions (secondary product ions) obtained in tandem mass spectrometric (MS/MS) experiments on a quadrupole ion trap mass spectrometer. The study presented here defines how to adapt the STEP method to a more routinely used mass analyzer, the triple quadrupole. New ion activation conditions were developed to adapt the STEP method to the triple quadrupole mass spectrometer using peptides and carbohydrates. The application of this method to the triple quadrupole is useful because it provides an efficient approach to differentiate primary and secondary ions on this instrument. Out of the total number of ions that were subjected to the STEP analysis, this method correctly identified 96% of ions as primary or secondary, indicating that this analysis is effective for carbohydrates and peptides undergoing collision-induced dissociation (CID) on a triple quadrupole mass spectrometer.     

Peptide photodissociation at 157 nm in a linear ion trap mass spectrometer

The photodissociation by 157 nm light of singly- and doubly-charged peptide ions containing C- or N-terminal arginine residues was studied in a linear ion trap mass spectrometer. Singly-charged peptides yielded primarily x- and a-type ions, depending on the location of the arginine residue, along with some related side-chain fragments. These results are consistent with our previous work using a tandem time-of-flight (TOF) instrument with a vacuum matrix-assisted laser desorption/ionization (MALDI) source. Thus, the different internal energies of precursor ions in the two experiments seem to have little effect on their photofragmentation. For doubly-charged peptides, the dominant fragments observed in both photodissociation and collisionally induced dissociation (CID) experiments are b- and y-type ions. Preliminary experiments demonstrating fragmentation of multiply-charged ubiquitin ions by 157 nm photodissociation are also presented.     

The Radical Ion Chemistry of S-Nitrosylated Peptides

The radical ion chemistry of a suite of S-nitrosopeptides has been investigated. Doubly and triply-protonated ions of peptides NYCGLPGEYWLGNDK, NYCGLPGEYWLGNDR, NYCGLPGERWLGNDR, NACGAPGEKWAGNDK, NYCGLPGEKYLGNDK, NYGLPGCEKWYGNDK and NYGLPGEKWYGCNDK were subjected to electron capture dissociation (ECD), and collision-induced dissociation (CID). The peptide sequences were selected such that the effect of the site of S-nitrosylation, the nature and position of the basic amino acid residues, and the nature of the other amino acid side chains, could be interrogated. The ECD mass spectra were dominated by a peak corresponding to loss of ^?NO from the charge-reduced precursor, which can be explained by a modified Utah-Washington mechanism. Some backbone fragmentation in which the nitrosyl modification was preserved was also observed in the ECD of some peptides. Molecular dynamics simulations of peptide ion structure suggest that the ECD behavior was dependent on the surface accessibility of the protonated residue. CID of the S-nitrosylated peptides resulted in homolysis of the S?CN bond to form a long-lived radical with loss of ^?NO. The radical peptide ions were isolated and subjected to ECD and CID. ECD of the radical peptide ions provided an interesting comparison to ECD of the unmodified peptides. The dominant process was electron capture without further dissociation (ECnoD). CID of the radical peptide ions resulted in cysteine, leucine, and asparagine side chain losses, and radical-induced backbone fragmentation at tryptophan, tyrosine, and asparagine residues, in addition to charge-directed backbone fragmentation.     

High mass accuracy in-source collision-induced dissociation tandem mass spectrometry and multi-step mass spectrometry as complementary tools for fragmentation studies of quaternary ammonium herbicides

Fragmentation studies using both an ion-trap mass analyzer and a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer were performed in order to establish the fragmentation pathways of organic molecules. A general strategy combining MSn data (n = 1-4) in an ion-trap analyzer with tandem mass spectrometry and in-source collision-induced dissociation tandem mass spectrometry (CID MS/MS) in a Q-TOF instrument was applied. The MSn data were used to propose a tentative fragmentation pathway following genealogical relationships. When several assignments were possible, MS/MS and in-source CID MS/MS (Q-TOF) allowed the elemental compositions of the fragments to be confirmed. Quaternary ammonium herbicides (quats) were used as test compounds and their fragmentation pathways were established. The elemental composition of the fragments was confirmed using the TOF analyzer with relative errors <0.0023 Da. Some fragments previously reported in the literature were reassigned taking advantage of the high mass resolution and accuracy of the Q-TOF instrument, which made it possible to solve losses where nitrogen was involved.     

Development of a tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source

A new tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source ‘ESI‐TOF/quadTOF’ was designed and constructed to achieve the desired aim of structural elucidation via high‐energy collision‐induced dissociation (CID), and the simultaneous detection of all fragment ions. The instrument consists of an orthogonal acceleration‐type ESI ion source, a linear TOF mass spectrometer, a collision cell, a quadratic‐field ion mirror and a microchannel plate detector. High‐energy CID spectra of doubly protonated angiotensin II and bradykinin were obtained. Several fragment ions such as a‐, d‐, v‐ and w‐type ions, characteristic of high‐energy CID, were clearly observed in these spectra. These high‐energy CID fragment ions enabled confirmation of the complete sequence, including leucine–isoleucine determinations. It was demonstrated that high‐energy CID of multiply protonated peptides could be achieved in the ESI‐TOF/quadTOF. Copyright © 2010 John Wiley & Sons, Ltd.     

Substituent effects on the gas-phase fragmentation reactions of sulfonium ion containing peptides

The multistage mass spectrometric (MS/MS and MS3) gas-phase fragmentation reactions of methionine side-chain sulfonium ion containing peptides formed by reaction with a series of para-substituted phenacyl bromide (XBr where X=CH2COC6H4R, and R=--COOH, --COOCH3, --H, --CH3 and --CH2CH3) alkylating reagents have been examined in a linear quadrupole ion trap mass spectrometer. MS/MS of the singly (M+) and multiply ([M++nH](n+1)+) charged precursor ions results in exclusive dissociation at the fixed charge containing side chain, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility). However, loss of the methylphenacyl sulfide side-chain fragment as a neutral versus charged (protonated) species was observed to be highly dependent on the proton mobility of the precursor ion, and the identity of the phenacyl group para-substituent. Molecular orbital calculations were performed at the B3LYP/6-31+G** level of theory to calculate the theoretical proton affinities of the neutral side-chain fragments. The log of the ratio of neutral versus protonated side-chain fragment losses from the derivatized side chain were found to exhibit a linear dependence on the proton affinity of the side-chain fragmentation product, as well as the proton affinities of the peptide product ions. Finally, MS3 dissociation of the nominally identical neutral and protonated loss product ions formed by MS/MS of the [M++H]2+ and [M++2H]3+ precursor ions, respectively, from the peptide GAILM(X)GAILK revealed significant differences in the abundances of the resultant product ions. These results suggest that the protonated peptide product ions formed by gas-phase fragmentation of sulfonium ion containing precursors in an ion trap mass spectrometer do not necessarily undergo intramolecular proton 'scrambling' prior to their further dissociation, in contrast to that previously demonstrated for peptide ions introduced by external ionization sources.     

New method to study the effects of peptide sequence on the dissociation energetics of peptide ions

A new method has been developed to study the dissociation patterns of singly protonated peptides by using a quadrupole ion trap mass spectrometer. The new approach involves using boundary-activated dissociation to characterize the ease of dissociation of peptide ions. Insight can be gained into the effect of specific peptide sequences on the dissociation energetics of protonated peptides. Increased knowledge of the effects of specific sequences on the dissociation patterns of peptide ions should improve the ability to interpret complex spectra from tandem mass spectrometry (MS/MS) experiments. This method has confirmed the previously observed increase in the energy needed for the dissociation of peptide ions containing basic residues. In addition, this technique has revealed the effect of the location of proline residues on the dissociation energetics of peptides with this amino acid.     

Comparison of collision-induced dissociation and electron-induced dissociation of <Emphasis Type="Italic">singly protonated</Emphasis> aromatic amino acids,cystine and related simple peptides using a hybrid linear ion trap–FT-ICR mass spectrometer

The gas-phase fragmentation reactions of singly protonated aromatic amino acids, their simple peptides as well as simple models for intermolecular disulfide bonds have been examined using a commercially available hybrid linear ion trap-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Low-energy collision-induced dissociation (CID) reactions within the linear ion trap are compared with electron-induced dissociation (EID) reactions within the FT-ICR cell. Dramatic differences are observed between low-energy CID (which occurs via vibrational excitation) and EID. For example, the aromatic amino acids mainly fragment via competitive losses of NH(3) and (H(2)O+CO) under CID conditions, while side-chain benzyl cations are major fragment ions under EID conditions. EID also appears to be superior in cleaving the S-S and S-C bonds of models of peptides containing an intermolecular disulfide bond. Systematic studies involving fragmentation as a function of electron energy reveal that the fragmentation efficiency for EID occurs at high electron energy (more than 10 eV) compared with the low-electron energy (less than 0.2 eV) typically observed for electron capture dissociation fragmentation. Finally, owing to similarities between the types of fragment ions observed under EID conditions and those previously reported in ultraviolet photodissociation experiments and the electron-ionization mass spectra, we propose that EID results in fragmentation via electronic excitation and vibrational excitation. EID may find applications in analyzing singly charged molecular ions formed by matrix-assisted laser desorption ionization.     

Energy-dependent collision-induced dissociation study of buprenorphine and its synthetic precursors

The collision-induced dissociation (CID) of protonated buprenorphine ([M+H](+) ) and four related compounds was studied by electrospray quadrupole/time-of-flight mass spectrometry (ESI-QTOF MS). The fragmentation pathways were investigated by using energy-dependent CID and pseudo-MS(3) (in-source CID combined with tandem mass spectrometry (MS/MS)) methods. The first steps of the fragmentation are the parallel losses of the substituents from the non-aromatic ring moieties. Depending on the applied collision energies, a large number of further fragment ions arising from the cross-ring cleavages of the core-ring structure were observed. Based on the experimental results, a generalized fragmentation scheme was developed for the five buprenorphine derivatives highlighting the differences for the alternatively substituted compounds. The collision-energy-dependent fragmentation profile of buprenorphine is visualized in a two-dimensional plot to aid its fingerprint identification.     

Protein identification via surface-induced dissociation in an FT-ICR mass spectrometer and a patchwork sequencing approach

Surface-induced dissociation (SID) and collision-induced dissociation (CID) are ion activation techniques based on energetic collisions with a surface or gas molecule, respectively. One noticeable difference between CID and SID is that SID does not require a collision gas for ion activation and is, therefore, directly compatible with the high vacuum requirement of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Eliminating the introduction of collision gas into the ICR cell for collisional activation dramatically shortens the acquisition time for MS/MS experiments, suggesting that SID could be utilized for high-throughput MS/MS studies in FT-ICR MS. We demonstrate for the first time the utility of SID combined with FT-ICR MS for protein identification. Tryptic digests of standard proteins were analyzed using a hybrid 6-tesla FT-ICR mass spectrometer with SID and CID capabilities. SID spectra of mass-selected singly and doubly charged peptides were obtained using a diamond-coated target mounted at the rear trapping plate of the ICR cell. The broad internal energy distribution deposited into the precursor ion following collision with the diamond surface allowed a variety of fragmentation channels to be accessed by SID. Composition and sequence qualifiers produced by SID of tryptic peptides were used to improve the statistical significance of database searches. Protein identification MASCOT scores obtained using SID were comparable or better than scores obtained using sustained off-resonance irradiation collision-induced dissociation (SORI-CID), the conventional ion activation technique in FT-ICR MS.