Negative ion electrospray tandem mass spectrometric structural characterization of leukotriene B4 (LTB4) and LTB4-derived metabolites

The low energy collision induced dissociation (CID) of the carboxylate anions generated by electrospray ionization of leukotriene B₄ (LTB₄) and 16 of its metabolites was studied in a tandem quadrupole mass spectrometer. LTB₄ is a biologically active lipid mediator whose activity is terminated by metabolism into a wide variety of structural variants. The collision-induced dissociation spectra of the carboxylate anions revealed structurally informative ions whose formation was determined by the position of hydroxyl substituents and double bonds present in the LTB₄ metabolite. Major ions resulted from charge remote α-hydroxy fragmentation or charge directed α-hydroxy fragmentation. The conjugated triene moiety present in some metabolites was proposed to undergo cyclization to a 1,3-cyclohexadiene structure prior to charge remote or charge driven a-hydroxy fragmentation. The mechanisms responsible for all major ions observed in the CID spectra were studied using stable isotope labeled analogs of the LTB₄ metabolites. In general, the collision-induced decomposition of carboxylate anions produced unique spectra for all LTB₄ derived metabolites. The observed decomposition product ions from the carboxylate anion could be useful in developing assays for these molecules in biological fluids.     

Gas-Phase Fragmentation of [M + nH + OH]<Superscript>n•+</Superscript> Ions Formed from Peptides Containing Intra-Molecular Disulfide Bonds

In this study, we systematically investigated gas-phase fragmentation behavior of [M + nH + OH]^n•+ ions formed from peptides containing intra-molecular disulfide bond. Backbone fragmentation and radical initiated neutral losses were observed as the two competing processes upon low energy collision-induced dissociation (CID). Their relative contribution was found to be affected by the charge state (n) of [M + nH + OH]^n•+ ions and the means for activation, i.e., beam-type CID or ion trap CID. Radical initiated neutral losses were promoted in ion-trap CID and for lower charge states where mobile protons were limited. Beam-type CID and dissociation of higher charge states of [M + nH + OH]^n•+ ions generally gave abundant backbone fragmentation, which was highly desirable for characterizing peptides containing disulfide bonds. The amount of sequence information obtained from CID of [M + nH + OH]^n•+ ions was compared with that from CID of disulfide bond reduced peptides. For the 11 peptides studied herein, similar extent of sequence information was obtained from these two methods.     

Loss of isocyanic acid from the internal oxadiazole ring of protonated molecules of some 2,5-diaryl-1,3,4-oxadiazoles

The fragmentation pattern of some protonated 2,5-diaryl-1,3,4-oxadiazoles is discussed. An unusual decomposition consisting of elimination of the isocyanic acid molecule from the internal oxadiazole ring was found. This fragmentation pathway was deduced on the basis of B/E linked scan mass spectra of metastable ions with liquid secondary ion mass spectrometry as the ionization method and also of low-energy CID mass spectra where electrospray was used as the ionization technique. High resolution measurements were also performed.     

一些双电荷离子的气相碎裂反应

借助质量分析离子动能谱和串联质谱研究了由电子轰击产生的双电荷离子的单分子亚稳碎裂及碰撞诱导分解过程,讨论了两种实验方法导致的差别因素.此外,根据质量分析离子动能谱提供的双电荷离子电荷分离反应的动能释放值计算了两电荷中心间距的最小值,以判别按不同电荷分离方式碎裂的双电荷离子的过渡态结构.     

A comparison of the peptide fragmentation obtained from a reflector matrix-assisted laser desorption-ionization time-of-flight and a tandem four sector mass spectrometer

The types, extent, and overall distribution of peptide fragmentation produced by matrix-assisted laser desorption-ionization-postsource decay (MALDI-PSD) on a reflector time-of-flight mass spectrometer were compared with those obtained from high and low energy collision-induced dissociation (CID) on a four-sector mass spectrometer and from liquid secondary ion mass spectrometry (LSIMS) ion source fragmentation and LSIMS metastable ion (MI) decomposition on a two-sector mass spectrometer. The model peptides studied had sequences and compositions that yielded predominantly either N- or C-terminal fragmentation from CID. For des-Arg¹ and des-Arg⁹ bradykinin (i.e., H-PPGFSPFR-OH and H-RP-PGFSPF-OH, respectively), the types of fragment ions and the extent to which each type is formed in both MALDI-PSD and low energy CID spectra are remarkably similar. This observation suggests that both methods deposit comparable internal energies (IE) into [M + H]⁺ precursor ions. The distribution of N-terminal, C-terminal, immonium, and internal fragmentation from MALDI-PSD spectra of des-Arg¹ and des-Arg⁹ bradykinin did not change dramatically with respect to the terminal arginine position, contrary to those from LSIMS MI decomposition, high and low energy CID spectra. This observation in combination with the prominent immonium, internal, and minus 17 fragment ion types in PSD indicates that the imparted IE from MALDI and the 14 µs of flight time may promote steady-state decomposition kinetics. Fragmentation distributions of MALDI-PSD spectra are also similar to those in LSIMS spectra. This implies that the distribution of protonation sites in [M + H]⁺ is comparable for both techniques.     

High-energy collision-induced dissociation of small polycyclic aromatic hydrocarbons

High-energy collision-induced dissociation (CID) experiments on polycyclic aromatic hydrocarbons (PAHs) having 2-6 rings, naphthalene, anthracene, phenanthrene, fluoranthene, pyrene and coronene, were performed, and the relative abundances of their fragment ions were investigated as a function of collision energy. The results revealed that the PAHs except naphthalene showed a bimodal-type distribution of positive fragmentation ions, which is closely similar to the fragment-ion distribution reported for the CID of three-dimensional fullerene, C(60)(+) and C(70)(+). The three-ring isomers of anthracene and phenanthrene and the four-ring isomers of fluoranthene and pyrene can be distinguishable in their spectra under an electron ionization energy of 70 eV, but the high-energy CID spectra of the three- and four-ring isomers were almost identical. The fragmentation corresponding to fragment ions in the low-mass region of the bimodal CID spectra could be interpreted by the simple statistical model that fragment ions are formed by random evaporation from the molecular ions after a considerable structural rearrangement, 'phase transition', occurring at some high-energy state.     

Characterization of five [C4H7O]+ Ion structures. Fragmentation of the 2-pentanone molecular ion

The [C₄H₇0]⁺ ions [CH₂?CH? C(?OH)CH₃]⁺ (1), [CH₃CH?CH? C(?OH)H]⁺ (2), [CH₂?C(CH₃)C(?OH)H]⁺ (3), [Ch₃CH₂CH₂C?O]⁺ (4) and [(CH₃)₂CHC?O]⁺ (5) have been characterized by their collision-induced dissociation (CID) mass spectra and charge stripping mass spectra. The ions 1–3 were prepared by gas phase protonation of the relevant carbonyl compounds while 4 and 5 were prepared by dissociative electron impact ionization of the appropriate carbonyl compounds. Only 2 and 3 give similar spectra and are difficult to distinguish from each other; the remaining ions can be readily characterized by either their CID mass spectra or their charge stripping mass spectra. The 2-pentanone molecular ion fragments by loss of the C(1) methyl and the C(5) methyl in the ratio 60:40 for metastable ions; at higher internal energies loss of the C(1) methyl becomes more favoured. Metastable ion characteristics, CID mass spectra and charge stripping mass spectra all show that loss of the C(1) methyl leads to formation of the acyl ion 4, while loss of the C(5) methyl leads to formation of protonated vinyl methyl ketone (1). These results are in agreement with the previously proposed potential energy diagram for the [C₅H₁₀O]⁺˙ system.     

Comparison of CID,ETD and metastable atom‐activated dissociation (MAD) of doubly and triply charged phosphorylated tau peptides

The fragmentation behavior of the 2+ and 3+ charge states of eleven different phosphorylated tau peptides was studied using collision‐induced dissociation (CID), electron transfer dissociation (ETD) and metastable atom‐activated dissociation (MAD). The synthetic peptides studied contain up to two known phosphorylation sites on serine or threonine residues, at least two basic residues, and between four and eight potential sites of phosphorylation. CID produced mainly b‐/y‐type ions with abundant neutral losses of the phosphorylation modification. ETD produced c‐/z‐type ions in highest abundance but also showed numerous y‐type ions at a frequency about 50% that of the z‐type ions. The major peaks observed in the ETD spectra correspond to the charge‐reduced product ions and small neutral losses from the charge‐reduced peaks. ETD of the 2+ charge state of each peptide generally produced fewer backbone cleavages than the 3+ charge state, consistent with previous reports. Regardless of charge state, MAD achieved more extensive backbone cleavage than CID or ETD, while retaining the modification(s) in most cases. In all but one case, unambiguous modification site determination was achieved with MAD. MAD produced 15–20% better sequence coverage than CID and ETD for both the 2+ and 3+ charge states and very different fragmentation products indicating that the mechanism of fragmentation in MAD is unique and complementary to CID and ETD. Copyright © 2012 John Wiley & Sons, Ltd.     

Collision gas effects in the collision-induced decomposition of protonated and cationized molecules of carbohydrate antibiotics

The collision-induced decomposition (CID) mass spectra of the protonated and cationized molecules of a number of carbohydrate antibiotics of RMM ranging from 700 to 1500 were studied by means of a four-sector mass spectrometer with a floated collision cell. Helium and argon were used as collision gases. This work illustrates that cationized rather than protonated carbohydrate antibiotics give an increased yield of high-mass ions of diagnostic value. Further, when helium is replaced by argon as collision gas, differences in the CID spectra of MH⁺ ions become apparent only for molecules of RMM > 1400 whereas for [M + Na]⁺ ions differences are observed for molecules of RMM as low as 1000. These results have been attributed to the deposition of more internal energy in the precursor ion when argon is used, resulting in increased fragmentation.     

Ion activation methods for tandem mass spectrometry

This tutorial presents the most common ion activation techniques employed in tandem mass spectrometry. In-source fragmentation and metastable ion decompositions, as well as the general theory of unimolecular dissociations of ions, are initially discussed. This is followed by tandem mass spectrometry, which implies that the activation of ions is distinct from the ionization step, and that the precursor and product ions are both characterized independently by their mass/charge ratios. In collision-induced dissociation (CID), activation of the selected ions occurs by collision(s) with neutral gas molecules in a collision cell. This experiment can be done at high (keV) collision energies, using tandem sector and time-of-flight instruments, or at low (eV range) energies, in tandem quadrupole and ion trapping instruments. It can be performed using either single or multiple collisions with a selected gas and each of these factors influences the distribution of internal energy that the activated ion will possess. While CID remains the most common ion activation technique employed in analytical laboratories today, several new methods have become increasingly useful for specific applications. More recent techniques are examined and their differences, advantages and disadvantages are described in comparison with CID. Collisional activation upon impact of precursor ions on solid surfaces, surface-induced dissociation (SID), is gaining importance as an alternative to gas targets and has been implemented in several different types of mass spectrometers. Furthermore, unique fragmentation mechanisms of multiply-charged species can be studied by electron-capture dissociation (ECD). The ECD technique has been recognized as an efficient means to study non-covalent interactions and to gain sequence information in proteomics applications. Trapping instruments, such as quadrupole ion traps and Fourier transform ion cyclotron resonance instruments, are particularly useful for the photoactivation of ions, specifically for fragmentation of precursor ions by infrared multiphoton dissociation (IRMPD). IRMPD is a non-selective activation method and usually yields rich fragmentation spectra. Lastly, blackbody infrared radiative dissociation is presented with a focus on determining activation energies and other important parameters for the characterization of fragmentation pathways. The individual methods are presented so as to facilitate the understanding of each mechanism of activation and their particular advantages and representative applications.     

Energetics of retro-Diels–Alder fragmentation in limonene as characterized by surface-induced dissociation,and energy- and angle-resolved mass spectrometry

Ionized limonene and related isomeric compounds have been examined by collisional activation at both gaseous and solid targets. The gas-phase collision-induced dissociation (CID) experiments were performed as a function of collision energy and scattering angle and the surface-induced dissociation (SID) experiments as a function of collision energy, in order to vary systematically the internal energy deposited in the molecular ion. The virtual absence of retro-Diels–Alder (RDA) fragmentation upon conventional CID, as compared to its importance in the electron impact (EI) mass spectrum, the subject of a study by Boyd and coworkers, was confirmed. However, as the ion internal energy was increased by raising the collision energy or the scattering angle, RDA fragmentation was observed and it became a dominant mode of fragmentation for SID at collision energies in the range of 25–50 eV. The energy deposited into the colliding ion in the SID technique is compared with that deposited upon CID in the eV and keV energy ranges and upon EI. The order obtained is: SID > EI > low-energy, multiple-collision CID > high-energy, single-collision CID > low-energy, single-collision CID. The distribution of energies in SID is narrower than in the other techniques. High internal energies are accessible by increasing the scattering angle in CID; however, this is accompanied by an increase in the width of the internal energy distribution, and it is therefore not possible to channel fragmentation predominantly into RDA by this method. It is concluded that RDA fragmentation of limonene is a high-energy process and that this is the explanation for its behavior. Isomerization, occurring through 1,3-hydrogen migrations of the molecular ions of limonene, isolimonene, terpinolene and α-terpinene, was investigated and long-lived molecular ions of the first three compounds were found to maintain distinct structures.     

Comparison of the activation time effects and the internal energy distributions for the CID,PQD and HCD excitation modes

Reproducibility among different types of excitation modes is a major bottleneck in the field of tandem mass spectrometry library development in metabolomics. In this study, we specifically evaluated the influence of collision voltage and activation time parameters on tandem mass spectrometry spectra for various excitation modes [collision‐induced dissociation (CID), pulsed Q dissociation (PQD) and higher‐energy collision dissociation (HCD)] of Orbitrap‐based instruments. For this purpose, internal energy deposition was probed using an approach based on Rice–Rampserger–Kassel–Marcus modeling with three thermometer compounds of different degree of freedom (69, 228 and 420) and a thermal model. This model treats consecutively the activation and decomposition steps, and the survival precursor ion populations are characterized by truncated Maxwell–Boltzmann internal energy distributions. This study demonstrates that the activation time has a significant impact on MS/MS spectra using the CID and PQD modes. The proposed model seems suitable to describe the multiple collision regime in the PQD and HCD modes. Linear relationships between mean internal energy and collision voltage are shown for the latter modes and the three thermometer molecules. These results suggest that a calibration based on the collision voltage should provide reproducible for PQD, HCD to be compared with CID in tandem in space instruments. However, an important signal loss is observed in PQD excitation mode whatever the mass of the studied compounds, which may affect not only parent ions but also fragment ions depending on the fragmentation parameters. A calibration approach for the CID mode based on the variation of activation time parameter is more appropriate than one based on collision voltage. In fact, the activation time parameter in CID induces a modification of the collisional regime and thus helps control the orientation of the fragmentation pathways (competitive or consecutive dissociations). Copyright © 2014 John Wiley & Sons, Ltd.     

De novo sequencing of proteolytic peptides by a combination of C-terminal derivatization and nano-electrospray/ collision-induced dissociation mass spectrometry

A series of synthetic peptides (3-15 residues), C-terminally derivatized with 4-aminonaphthalenesulfonic acid (ansa), have been analyzed on a hybrid magnetic sector-orthogonal acceleration time-of-flight tandem mass spectrometer, fitted with a nano-electrospray (nano-ES) interface. Deprotonated molecules generated by negative-ion ES were subjected to collision-induced dissociation (CID) using either methane or xenon as the collision gas, at a collision energy of 400 eV (laboratory frame of reference). As a consequence of charge localization on the sulfonate group, only C-terminal fragment ions were formed, presumably by charge-remote fragmentation mechanisms. Interpretable CID spectra were obtained from fmol amounts of the small peptides (up to 6 residues), whereas low pmol amounts were required for the larger peptides. CID spectra were also recorded of derivatized, previously noncharacterised peptides obtained by proteolysis of cytosolic hamster liver aldehyde dehydrogenase. Interpretation of these CID spectra was based on rules established for the fragmentation of the synthetic peptides. This study shows that derivatization with ansa may be useful in the de novo sequencing of peptides.     

Ionization and fragmentation of monochloro-isomers of 3-hydroxy-2-phenyl-4(1H)-quinolinone

Electron ionization (EI), methane chemical ionization (CI), and collision-induced dissociation (CID) mass spectra of complete series of positional monochloro-isomers of 3-hydroxy-2-phenyl-4(1H)-quinolinone are evaluated and discussed. It is shown that in the CI experiments, in addition to the protonated precursor molecules, odd-electron molecular ions are formed and this affects the appearance of the CID spectra. The influence of different direct probes and other experimental parameters such as the pressure of the reagent gas, isolation width, or collision energy was studied. EI, CI and CID spectra of the positional isomers show essentially the same fragmentation pathways but comparisons of the relative signal intensities of various product ions reveal some positional effects. Different isomers are also distinguished. The compounds can be divided into two groups using diagnostic ions (chloro substitution of the quinolinone moiety or the phenyl ring) or identified using a created spectral database. It was demonstrated that the reproducibility of the CID spectra is fully satisfactory for isomer identification, and that the created database can be applied for comparison of spectra measured over an extended time period (1 month) or spectra obtained during the direct analysis of a reaction mixture extract. Explanation of the fragmentation of the isomers is supported by exploratory density functional theory (DFT) calculations, e.g. rationalization of the relatively higher importance of the M(+.)-H(.)-Cl(.)-CO fragmentation pathway during EI than during CID, and vice versa for the pathway M(+.)-Cl(.)-CO. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Fast-atom bombardment mass spectrometry and low energy collision-induced tandem mass spectrometry of tauroconjugated bile acid anions

Fragmentation of negative ions produced by fast-atom bombardment (FAB) from 14 tauroconjugated bile acids and some of their deuterated analogs has been studied by mass spectrometry and by collision-induced dissociation (CED) tandem mass spectrometry at low energy. Low energy collision-induced dissociation of the deprotonated molecules [M - H]^? of these tauroconjugated bile acids leads to both charge-driven and charge-remote fragmentations (CRF). The former yields neutral loss from the side chain with charge migration during the fragmentation process. These fragments dominate the CID spectra, but are absent from the FAB spectra. Their relative abundances are dependent on the number and the positions of the hydroxyl groups in the steroid nucleus and thus permit distinction among some positional isomers. The CRF fragments correspond to cleavages in the side chain up to fragmentations across the steroid rings with charge retention on the sulfonate group. These CRF fragments, which also are useful for structural identification, are less intense in CID than in FAB spectra. It appears that these charge-remote fragments are favored by unsaturation in the steroid rings, either as keto groups or as endocyclic double bonds. Tandem mass spectrometry combined with the use of deuterated analogs demonstrates that the structures of the survivor pseudomolecular ions and of the CRF fragments are not rearranged.     

Effects of stepwise excitation on fragmentation of gas phase ions: An especially striking case

Dramatically different fragmentation patterns are obtained for 4-ethyl-2,6,7-trioxa-1-phosphabicyclo-[2.2.2]octane-1-oxide upon electron ionization (EI) and for the corresponding molecular ion on collision-induced dissociation (CID). Two reasons for this behaviour have been discovered. (i) Irreversible multistep isomerization of the molecular ions occurs prior to collisional activation in mass spectrometry/mass spectrometry (MS/MS). Isomerization reactions have been characterized by isotopic labelling and by examining structures of relevant unlabelled and labelled fragment ions by MS/MS. The extent of isomerization can be controlled by varying the amount of internal energy of the molecular ions. This has been done by changing the number of thermalizing collisions which the ions undergo with neutral molecules in the ion source. (ii) When multiple collisions are used to dissociate the molecular ions, the initially stable fragmentation products undergo extensive further decomposition. As a result, abundant phosphorus-containing fragment ions are obtained for the bicyclic phosphate in high-pressure CID, whereas electron ionization leads to predominant hydrocarbon ions. A minor change in the structure of this molecule has major effects on the fragmentation behaviour: high- and low-energy collisional activation spectra of the molecular ion of the corresponding phosphite are identical with the 12 e V EI mass spectrum of the neutral.     

Internal energy deposition in chemical ionization/tandem mass spectrometry

The efficiency of the collision-induced dissociation (CID) process as a function of the internal energy deposited into the ion during the ionization event was evaluated. (M + H)+ ions of pyrrole, pyrrolidine, pyridine and piperidine (five and six-membered ring heterocyclics) were generated by chemical ionization (CI). The internal energy of the ions was varied by using different reagent gases. Both high-energy (keV) and low-energy (eV) CID were performed on these ions. The experiments showed that the (M + H)+ ions of the five-membered ring compounds, pyrrole and pyrrolidine, have higher fragmentation efficiencies than the six-membered ring compounds, pyridine and piperidine. Fragmentation efficiencies in high-energy CID clearly correlate with the internal energy deposited by the ionization technique. Experiments showed that the low-energy CID process is more sensitive than high-energy CID to changes in internal energy.     

Peptide sequence scrambling through cyclization of b5 ions

The CID mass spectra of the MH(+) ions and the b(5) ions derived therefrom have been determined for the hexapeptides YAAAAA, AYAAAA, AAYAAA, AAAYAA, and AAAAYA. The CID mass spectra for the b(5) ions derived from the five isomers are essentially identical and show abundant ion signals for nonsequence b ions. This result is consistent with cyclization of the b(5) ions to a cyclic pentapeptide before fragmentation; this cyclic peptide can open at various positions, leading to losses of amino acid residues that are not characteristic of the original amino acid sequence. These nonsequence b ions are also observed in the fragmentation of the MH(+) ions and increase substantially in importance with increasing collision energy. A comparison of the fragmentation of AAAYAA and Ac-AAAYAA indicates that N-acetylation eliminates the cyclization of b(5) ions and, thus, eliminates the nonsequence ions in the CID mass spectra of both b(5) and MH(+) ions.     

Dissociation of multiply charged negative ions for hirudin (54–65), fibrinopeptide B, and insulin A (oxidized)

Collision-induced dissociation (CID) was performed on multiply deprotonated ions from three commercial peptides: hirudin (54-65), fibrinopeptide B, and oxidized insulin chain A. Ions were produced by electrospray ionization in a Fourier transform ion cyclotron resonance mass spectrometer. Each of these peptides contains multiple acidic residues, which makes them very difficult to ionize in the positive mode. However, the peptides deprotonate readily making negative ion studies a viable alternative. The CID spectra indicated that the likely deprotonation sites are acidic residues (aspartic, glutamic, and cysteic acids) and the C-terminus. The spectra are rife with c, y, and internal ions, although some a, b, x, and z ions form. Many of the fragment ions were formed from cleavage adjacent to acidic residues, both N- and C-terminal to the acidic site. In addition, neutral loss (e.g., NH3, CH3, H2O, and CO2) was prevalent from both the parent ions and from fragment ions. These neutral eliminations were often indicative of specific amino acid residues. The fragmentation patterns from several charge states of the parent ions, when combined, provide significant primary sequence information. These results suggest that negative mode CID of multiply deprotonated ions provides useful structural information and can be worthwhile for highly acidic peptides that do not form positive ions in abundance.     

Resonance excitation and dynamic collision-induced dissociation in quadrupole ion traps using higher-order excitation frequencies

Fragmentation of the pentapeptide leucine enkephalin (YGGFL) is accomplished via higher-order resonances combined with simultaneous analysis of low-mass product ions. Two methods of achieving excitation are explored: (1) 0.5 ms resonant excitation at the omega and at Omega-omega secular frequencies of ion motion (where Omega is the radio-frequency (rf) drive frequency) in a manner similar to both pulsed q collision-induced dissociation (PQD) and high amplitude short time excitation (HASTE), and (2) 0.5 ms pulse of the omega or at Omega-omega excitation frequencies when the secular frequency of the ions is quickly swept across resonance conditions (pulsed q dynamic CID, PqDCID). In both methods of excitation, the rf amplitude on the ring electrode is rapidly decreased after excitation, therefore enabling analysis of low-mass product ions. Maximum fragmentation efficiencies of approximately 20% can be obtained with pulsed CID with both regular and high-order frequency excitation, while pulsed DCID offers maximum efficiencies of approximately 12%. All the excitation methods studied offer increased internal energy depositions when compared to conventional CID, as measured by the a4/b4 product ion ratios of leucine enkephalin. These ratios were as high as 13:1 for pulsed CID and 8:1 for PqDCID. Successful mass analysis of the low-mass ions is observed with both pulsed CID and PqDCID. The combined benefit of high internal energy deposition and wider dynamic mass range offers the possibility of increased sequence coverage and the identification of unique internal fragments or high-energy product ions which may provide complementary information to biological applications of conventional CID. This is the first report on deliberate fragmentation of precursor ions at a higher-order component of the ion secular frequency combined with a successful mass analysis of the low-mass ions through pulsed CID and PqDCID.