Effect of Cysteic Acid Position on the Negative Ion Fragmentation of Proteolytic Derived Peptides

A study on the effect of cysteic acid position on the types of fragment ions formed by collision-induced dissociation (CID) of [M – H]⁻ ions is presented. Of particular note is the observation of d-type fragment ions for peptides that contain an N-terminal cysteic acid (fixed negative charge) and cleavable amino acid side chains possessing a β-γ carbon–carbon bond. For example, the CID mass spectrum of oxidized cys-kemptide (C_oxLRRASLG) [M – H + O₃]⁻ ions contains abundant series of d-type fragment ions, and similar results are observed for oxidized cysteine-containing ribonuclease A proteolytic peptides. The d _i fragment ions are assumed to arise by a charge-remote and/or charge-assisted fragmentation mechanism, which both occur at high collision energies and involve consecutive reactions (i.e., the formation of a _i ions followed by the elimination of the side chain to form d _i 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.     

Observation of an unusually facile fragmentation pathway of gas-phase peptide ions: a study on the gas-phase fragmentation mechanism and energetics of tryptic peptides modified with 4-sulfophenyl isothiocyanate (SPITC) and 4-chlorosulfophenyl isocyanate (SPC) and their 18-crown-6 complexes

Various peptide modifications have been explored recently to facilitate the acquisition of sequence information. N-terminal sulfonation is an interesting modification because it allows unambiguous de novo sequencing of peptides, especially in conjunction with MALDI-PSD-TOF analysis; such modified peptide ions undergo fragmentation at energies lower than those required conventionally for unmodified peptide ions. In this study, we systematically investigated the fragmentation mechanisms of N-terminal sulfonated peptide ions prepared using two different N-terminal sulfonation reagents: 4-sulfophenyl isothiocyanate (SPITC) and 4-chlorosulfophenyl isocyanate (SPC). Collision-induced dissociation (CID) of the SPC-modified peptide ions produced a set of y-series ions that were more evenly distributed relative to those observed for the SPITC-modified peptides; y(n-1) ion peaks were consistently and significantly larger than the signals of the other y-ions. We experimentally investigated the differences between the dissociation energies of the SPITC- and SPC-modified peptide ions by comparing the MS/MS spectra of the complexes formed between the crown ether 18-crown-6 (CE) and the modified peptides. Upon CID, the complexes formed between 18-crown-6 ether and the protonated amino groups of C-terminal lysine residues underwent either peptide backbone fragmentation or complex dissociation. Although the crown ether complexes of the unmodified ([M + CE + 2H]2+) and SPC-modified ([M* + CE + 2H]2+) peptides underwent predominantly noncovalent complex dissociation upon CID, the low-energy dissociations of the crown ether complexes of the SPITC-modified peptides ([M' + CE + 2H]2+) unexpectedly resulted in peptide backbone fragmentations, along with a degree of complex dissociation. We performed quantum mechanical calculations to address the energetics of fragmentations observed for the modified peptides.     

A novel tandem quadrupole mass spectrometer allowing gaseous collisional activation and surface induced dissociation.

A novel tandem quadrupole mass spectrometer is described that enables gaseous collision-induced dissociation (CID) and surface-induced dissociation (SID) experiments. The instrument consists of a commercially available triple quadrupole mass spectrometer connected to an SID region and an additional, orthogonal quadrupole mass analyser. The performance of the instrument was evaluated using leucine-enkephalin, allowing a comparison between CID and SID, and with previous reports of other SID instruments. The reproducibility of SID data was assessed by replicate determinations of the collision energy required for 50% dissociation of leucine-enkephalin; excellent precision was observed (standard deviation of 0.6 eV) though, unexpectedly, the reproducibility of the equivalent figure for CID was superior. Several peptides were analysed using SID in conjunction with liquid secondary-ion mass spectrometry or electrospray; a comparison of the fragmentation of singly protonated peptide ions and the further dissociation of y-type fragments was consistent with the equivalence of the latter fragments to protonated peptides. Few product ions attributable to high-energy cleavages of amino acid side-chains were observed. The SID properties were investigated of a series of peptides differing only in the derivatization of a cysteine residue; similar decomposition efficiencies were observed for all except the cysteic acid analogue, which demonstrated significantly more facile fragmentation.     

Gas phase chemistry of the 2-tert-butyl-3-phenylphosphirenylium cation: novel onium ions by nucleophilic attack at phosphorus and de novo P-spiro bicyclic phosphonium ions via [4 + 2+] cycloaddition with dienes

The 2-tert-butyl-3-phenylphosphirenylium ion 13 is formed in abundance in the gas phase from 1-chloro-1H-phosphirene 6 upon 70 eV electron ionization. Collision-induced dissociation (CID) and ion-molecule reactions followed by CID of the product ions were performed via pentaquadrupole mass spectrometry to probe the structure and reactivity of 13 towards representative nucleophiles and dienes. Under CID conditions, 13 produces a variety of fragment ions mainly via dissociation processes that are preceded by isomerizations. In ion-molecule reactions, 13 reacts readily with ethers, sulfides, pyridine and aniline to form hitherto unknown oxonium, sulfonium and azonium ions via nucleophilic attack at phosphorus. With butadiene, isoprene, 1-acetoxybutadiene, and with Danishefsky's diene (1-methoxy-3-silyloxybuta-1,3-diene), 13 undergoes [4 + 2+] cycloaddition at phosphorus to generate novel P-spiro bicyclic phosphonium ions. With butadiene and isoprene, a second [4 + 2] cycloaddition occurs which generates P-spiro tricyclic phosphonium ions. Whereas 13 also reacts readily with 1-acetoxybutadiene via[4 + 2+] cycloaddition, most of the nascent P-spiro cycloadducts are unstable and dissociate by the loss of either a neutral ketene or acetic acid molecule. B3LYP/6-31G(d,p) calculations were performed to gain insight into the structures of the product ions. The present study constitutes the first successful attempt to unravel the chemistry of 13, a unique 2 pi-Hückel phosphirenylium ion for which no direct solution chemical reactivity data are as yet available. The present findings also create a parallel with the solution reactivity of 1-halo-1H-phosphirenes and 1-triflato-1H-phosphirenes as precursors to phosphirenylium ions.     

Hydrogen-shift isomerism: mass spectrometry of isomeric benzenesulfonate and 2-, 3- and 4-dehydrobenzenesulfonic acid anions in the gas phase

The isomeric 3- and 4-dehydrobenzenesulfonic acid anions b and c were prepared by collision induced dissociation (CID) of the [M - H](-) ions of isomeric sulfobenzoic acids obtained by negative electrospray ionization (ESI). The CID spectra (MS(3)) of anions b and c are different from each other, and both are different from that of the isomeric benzenesulfonate anion a, obtained from benzenesulfonic acid. The stability of ions b and c shows that 1,2-proton transfer does not take place in this system under the conditions of the CID experiment. Density functional (DFT) calculations at B3LYP/6-31+G(2d,p) level of theory show that benzenesulfonate anion a is the most stable isomer, and the energies of isomers b and c are higher by more than 65 kcal mol(-1). The calculated energies of the transition states involved in the 1,2-hydrogen migration leading to the interconversion of the isomeric anions are very high (>120 kcal mol(-1)relative to ion a, barrier energies >55 kcal mol(-1)), much higher than those of transition structures leading to fragmentation. This situation does not allow isomerization of ions b and c to a, under the conditions of the CID experiments. The isomeric 2-dehydrobenzenesulfonic acid anion isomerizes to the benzenesulfonate anion a by a facile proton transfer from the SO(3)H group to the adjacent position 2. The results of this work indicate that the gas phase deprotonation of meta- and para-sulfobenzoic acids is a kinetically controlled process.     

Infrared spectroscopy and theoretical studies on gas-phase protonated leu-enkephalin and its fragments: direct experimental evidence for the mobile proton

The gas-phase structures of the protonated pentapeptide Leu-enkephalin and its main collision-induced dissociation (CID) product ions, b4 and a4, are investigated by means of infrared multiple-photon dissociation (IR-MPD) spectroscopy and detailed molecular mechanics and density functional theory (DFT) calculations. Our combined experimental and theoretical approach allows accurate structural probing of the site of protonation and the rearrangement reactions that have taken place in CID. It is shown that the singly protonated Leu-enkephalin precursor is protonated on the N-terminus. The b4 fragment ion forms two types of structures: linear isomers with a C-terminal oxazolone ring, as well as cyclic peptide structures. For the former structure, two sites of proton attachment are observed, on the N-terminus and on the oxazolone ring nitrogen, as shown in a previous communication (Polfer, N. C.; Oomens, J.; Suhai, S.; Paizs, B. J. Am. Chem. Soc. 2005, 127, 17154-17155). Upon leaving the ions for longer radiative cooling delays in the ion cyclotron resonance (ICR) cell prior to IR spectroscopic investigation, one observes a gradual decrease in the relative population of oxazolone-protonated b4 and a corresponding increase in N-terminal-protonated b4. This experimentally demonstrates that the mobile proton is transferred between two sites in a gas-phase peptide ion and allows one to rationalize how the proton moves around the molecule in the dissociation process. The a4 fragment, which is predominantly formed via b4, is also confirmed to adopt two types of structures: linear imine-type structures, and cyclic structures; the former isomers are exclusively protonated on the N-terminus in sharp contrast to b4, where a mixture of protonation sites was found. The presence of cyclic b4 and a4 fragment ions is the first direct experimental proof that fully cyclic structures are formed in CID. These results suggest that their presence is significant, thus lending strong support to the recently discovered peptide fragmentation pathways (Harrison, A. G.; Young, A. B.; Bleiholder, B.; Suhai, S.; Paizs, B. J. Am. Chem. Soc. 2006, 128, 10364-10365) that result in scrambling of the amino acid sequence upon CID.     

Structural analysis of leukotriene C<Subscript>4</Subscript> isomers using collisional activation and 157 nm photodissociation

The fragmentation of 5-hydroxy-6-glutathionyl-7,9,11,14-eicosatetraenoic acid [leukotriene C4 or LTC4 (5, 6)] and its isomeric counterpart LTC4 (14, 15) were studied by low and high-energy collisional induced dissociation (CID) and 157 nm photofragmentation. For singly charged protonated LTC4 precursors, photodissociation significantly enhances the signal intensities of informative fragment ions that are very important to distinguish the two LTC4 isomers and generates a few additional fragment ions that are not usually observed in CID experiments. The ion trap enables MSn experiments on the fragment ions generated by photodissociation. Photofragmentation is found to be suitable for the structural identification and isomeric differentiation of cysteinyl leukotrienes and is more informative than low or high-energy CID. We describe for the first time the structural characterization of the LTC4 (14, 15) isomer by mass spectrometry using CID and 157 nm light activation methods.     

Histidine, lysine, and arginine radical cations: isomer control via the choice of auxiliary ligand (L) in the dissociation of [CuII(L)amino acid]*2+ complexes

Histidine, lysine, and arginine radical cations have been generated through collision-induced dissociation (CID) of complexes [CuII(auxiliary ligand)namino acid]*2+, using tri-, bi-, as well as monodentate auxiliary ligands. On the basis of the observed CID products, the existence of two isomeric amino-acid populations is postulated. The Type 1 radical cations of histidine and lysine, stable on the mass spectrometer time scale, were found to lose water, followed by the loss of carbon monoxide under more energetic CID conditions. The arginine Type 1 radical cation behaved differently, losing dehydroalanine. The Type 2 radical cations were metastable and easily fragmented by the loss of carbon dioxide, effectively preventing direct observation. Type 1 radical cations are proposed to result from neutral (canonical) amino-acid coordination, whereas Type 2 radical cations are from zwitterionic amino-acid coordination to copper in the complex. The ratio of Type 1/Type 2 ions was found to be dependent on the auxiliary ligand, providing a method of controlling which radical cation would be formed primarily. Density functional calculations at B3LYP/6-311++G(d,p) have been used to determine the relative energies of five His*+ isomers. Barriers against interconversion between the isomers and against fragmentation have been calculated, giving insight as to why the Type 1 ions are stable, while only fragmentation products of the Type 2 ions are observable under CID conditions.     

Multi-stage tandem mass spectrometry of metal cationized leucine enkephalin and leucine enkephalin amide

We have examined the multi-stage collision induced dissociation (CID) of metal cationized leucine enkephalin, leucine enkephalin amide, and the N-acetylated versions of the peptides using ion trap mass spectrometry. In accord with earlier studies, the most prominent species observed during the multi-stage CID of alkali metal cationized leucine enkephalin are the [b(n) + 17 + Cat]+ ions. At higher CID stages (i.e. >MS(4)), however, dissociation of the [b2 + 17 + Cat]+ ion, a cationized dipeptide, results in the production of [a(n) -1 + Cat]+ species. The multi-stage CID of Ag+ cationized leucine enkephalin can be initiated with either the [b(n) -1 + Ag]+ or [b(n) + 17 + Ag]+ ions produced at the MS/MS stage. For the former, sequential CID stages cause, in general, the loss of CO, and then the loss of the imine of the C-terminal amino acid, to reveal the amino acid sequence. Similar to the alkali cationized species, CID of [b2 -1 + Ag]+ produces prominent [a(n) -1 + Ag]+ ions. The multi-stage CID of argentinated peptides is reminiscent of fragmentation observed for protonated peptides, in that a series of (b(n)) and (a(n)) type ions are generated in sequential CID stages. The Ag+ cation is similar to the alkali metals, however, in that the [b(n) + 17 + Ag]+ product is produced at the MS/MS and MS3 stages, and that sequential CID stages cause the elimination of amino acid residues primarily from the C-terminus. We found that N-acetylation of the peptide significantly influenced the fragmentation pathways observed, in particular by promoting the formation of more easily interpreted (in the context of unambiguous sequence determination) dissociation spectra from the [b2 + 17 + Li]+, [b2 + 17 + Na]+ and [b2 -1 + Ag]+ precursor ions. Our results suggest, therefore, that N-acetylation may improve the efficacy of multi-stage CID experiments for C-terminal peptide sequencing in the gas phase. For leucine enkephalin amide, only the multi-stage CID of the argentinated peptide allowed the complete amino acid sequence to be determined from the C-terminal side.     

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.     

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.     

Gas-phase synthesis and reactivity of binuclear gold hydride cations, (R3PAu)2H+ (R = Me and Ph)

Electrospray ionization of a mixture of the two gold phosphine chlorides, R3PAuCl (R = Ph and Me), silver nitrate and the amino acid N,N-dimethylglycine (DMG) yields a range of gold containing cluster ions including: (R3P)Au(PR'3)+; (R3PAu)(R'3PAu)Cl+ and (R3PAu)(R'3PAu)(DMG-H)+ (where R = R' = Ph; R = R' = Me; R = Me and R' = Ph). Collision induced dissociation (CID) of the (R3PAu)(R'3PAu)(DMG-H)+ precursor ions yielded the hitherto unknown gold hydride dimers (R3PAu)(R'3PAu)H+. The gas-phase chemistry of these dimers was studied using ion-molecule reactions, collision induced dissociation, electronic excitation dissociation (EED) and DFT calculations on the (H3PAu)2H+ model system. A novel phosphine ligand migration was found to occur prior to fragmentation under CID conditions and this was supported by DFT calculations, which revealed a transition state with a bridging phosphine ligand.     

Study of protein modification by 4-hydroxy-2-nonenal and other short chain aldehydes analyzed by electrospray ionization tandem mass spectrometry

A convenient way to study lipid oxidation products-modified proteins by means of suitable model systems has been investigated. As a model peptide, the oxidized B chain of insulin has been chemically modified by either 4-hydroxy-2-nonenal (HNE) or hexanal and the extent, sites, and structure of modifications were assessed by electrospray mass spectrometry. A reduction step, using either NaCNBH(3) or NaBH(4), was also studied to stabilize the alkylated compounds. From the data gathered, it appeared that NaCNBH(3), when added at the beginning of incubation, dramatically influenced the HNE-induced modifications in terms of the addition mechanism (Schiff base formation instead of Michael addition) but also of the amino acid residues modified (N-terminal amino acid instead of histidine residues). However, by reducing the HNE-adducted species at the end of the reaction with NaBH(4), the fragment ions obtained in the product ion scan experiments become more stable and thus, easier to interpret in terms of origin and mechanism involved. With regard to hexanal induced modifications, we have observed that hexanal addition under reductive conditions led to an extensive modification of the peptide backbone. Moreover, as confirmed by "in-source" collision followed by collision induced dissociation (CID) experiments on selected precursor ions (pseudo-MS(3) experiments), N,N-di-alkylations were first observed on the N-terminal residue and further on Lys(29) residue. On the other hand, compared to the native peptide, no significant changes in MS/MS fragmentation patterns (b and y ions series) were observed whatever the basic site modified by the aldehyde-addition.     

Study of structural characteristic features of phenanthriondolizidine alkaloids by fast atom bombardment with tandem mass spectrometry

The mass spectrometric behavior of phenanthroindolizidine alkaloids has been investigated in detail by positive ion fast atom bombardment tandem mass spectrometry (FAB-MS/MS) combined with collision-induced dissociation (CID). The fragmentation has been correlated with the skeleton structure and positions of substituents. The product ions arising from retro-Diels-Alder cleavages differ clearly for compounds with different skeletons. The substituents at C-14 were observed to markedly influence the fragmentation pathway of [M + H](+) ions. A diversity of dissociation behavior initiated by the variation of substituents on the aromatic ring was also observed in the CID spectra. Product ions resulting from loss of CH(4) were obtained for compounds in which both C-6 and C-7 are occupied by methoxy groups. FAB-MS/MS spectra can reflect the connection between the fragmentation behavior and structural features of these phenanthroindolizidine alkaloids rapidly and effectively, and should prove to be a powerful method to determine the structures of related compounds.     

Solution-phase deuterium/hydrogen exchange at a specific residue using nozzle-skimmer and electron capture dissociation mass spectrometry

Information about protein conformation can be obtained with hydrogen/deuterium exchange (HDX) mass spectrometry. The isotopic solution-phase exchange of specific amide hydrogen atoms can be followed using low-vacuum nozzle-skimmer collision-induced dissociation (CID). In this study, the nozzle-skimmer technique was complemented by electron capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). The solution-phase exchange at a specific residue is monitored by comparing isotopic distributions of two consecutive b- or c-type ions. While nozzle-skimmer fragmentation takes place in the low-vacuum region of the mass spectrometer, ECD occurs at ultra-high vacuum within the mass analyzer cell of the FTICR mass spectrometer. The dissociations take place at 10(-4) and 10(-9) mbar, respectively. Low-vacuum nozzle-skimmer fragmentation can result in intramolecular exchange between product ions and solvent molecules in the gas phase. Consequently, the solution-phase information about protein or peptide conformation is lost. It was not possible to monitor isotopic solution-phase exchange at the eighth residue in substance P, (Phe)8, with nozzle-skimmer CID. By using the in-cell ECD fragmentation method, the solution-phase exchange at the (Phe)8 residue was preserved during mass spectrometric analysis. This result shows the complementary aspects of applying fragmentation at low and at high vacuum, when studying isotopic exchange in solution at specific residues using FTICRMS.     

Effect of metal cationization on the low-energy collision-induced dissociation of loganin, epi-loganin and ketologanin studied by electrospray ionization tandem mass spectrometry

The effect of alkali metal and silver cationization on the collision-induced dissociation (CID) of loganin (1), epi-loganin (2) and ketologanin (3) is discussed. Their protonated molecular ions fragment mainly by glycosidic cleavages. The epimeric pairs (1 and 2) show differences in the abundances of the resulting fragment ions. Lithium cationization induces new dissociation pathways such as the retro-Diels-Alder (RDA) fragmentation followed by rearrangement. Unlike the dissociation of protonated molecular ions, the dissociation of lithiated molecules also provides lithiated sugar fragments. The CID of dilithiated molecules is substantially different from that of the monolithiated precursors. RDA reaction appears to be favoured by the presence of the additional lithium atom in the molecule. In addition, other ring cleavages are also induced. The abundances of the various fragment ions are different in the CID spectra of the epimeric pairs. Extensive D labelling and (6)Li labelling experiments confirmed many of the ion structures proposed. The CID spectra of the sodiated ions are generally weaker, although similar to those of the corresponding lithiated species. Higher alkali metal ion (K(+), Rb(+) and Cs(+)) adducts generated only the corresponding metal ions as products of CID. Similar fragmentations were also observed in the CID of the [M + Ag](+) ions of these compounds, the epimeric pairs showing characteristic differences in their CID behaviour. Copyright 2000 John Wiley & Sons, Ltd.     

"Fast excitation" CID in a quadrupole ion trap mass spectrometer

Collision-induced dissociation (CID) in a quadrupole ion trap mass spectrometer is usually performed by applying a small amplitude excitation voltage at the same secular frequency as the ion of interest. Here we disclose studies examining the use of large amplitude voltage excitations (applied for short periods of time) to cause fragmentation of the ions of interest. This process has been examined using leucine enkephalin as the model compound and the motion of the ions within the ion trap simulated using ITSIM. The resulting fragmentation information obtained is identical with that observed by conventional resonance excitation CID. "Fast excitation" CID deposits (as determined by the intensity ratio of the a(4)/b(4) ion of leucine enkephalin) approximately the same amount of internal energy into an ion as conventional resonance excitation CID where the excitation signal is applied for much longer periods of time. The major difference between the two excitation techniques is the higher rate of excitation (gain in kinetic energy) between successive collisions with helium atoms with "fast excitation" CID as opposed to the conventional resonance excitation CID. With conventional resonance excitation CID ions fragment while the excitation voltage is still being applied whereas for "fast excitation" CID a higher proportion of the ions fragment in the ion cooling time following the excitation pulse. The fragmentation of the (M + 17H)(17+) of horse heart myoglobin is also shown to illustrate the application of "fast excitation" CID to proteins.     

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.     

Mass spectral analysis of N-oxides of Chemical Weapons Convention related aminoethanols under electrospray ionization conditions

N,N'-Dialkylaminoethanols are the hydrolyzed products or precursors of chemical warfare agents such as V-agents and nitrogen mustards, and they are prone to undergo oxidation in environmental matrices or during decontamination processes. Consequently, screening of the oxidized products of aminoethanols in aqueous samples is an important task in the verification of chemical weapons convention-related chemicals. Here we report the successful characterization of the N-oxides of N,N'-dialkylaminoethanols, alkyl diethanolamines, and triethanolamine using positive ion electrospray ionization mass spectrometry. The collision-induced dissociation (CID) spectra of the [M+H](+) and [M+Na](+) ions show diagnostic product ions that enable the unambiguous identification of the studied N-oxides, including those of isomeric compounds. The proposed fragmentation pathways are supported by high-resolution mass spectrometry data and product/precursor ion spectra. The CID spectra of [M+H](+) ions included [MH-CH(4)O(2)](+) as the key product ion, in addition to a distinctive alkene loss that allowed us to recognize the alkyl group attached to the nitrogen. The [M+Na](+) ions show characteristic product ions due to the loss of groups (R) attached to nitrogen either as a radical (R) or as a molecule [R+H or (R-H)] after hydrogen migration.