Characterization of alpha- and gamma-glutamyl dipeptides by negative ion collision-induced dissociation

The low-energy CID mass spectra of the [M-H](-) ions of a variety of dipeptides containing glutamic acid have been obtained using cone-voltage collisional activation. Dipeptides with the gamma-linkage, H-Glu(Xxx-OH)-OH, are readily distinguished from those with the alpha-linkage, H-Glu-Xxx-OH, by the much more prominent elimination of H-Xxx-OH from the [M-H](-) ions of the former isomers, resulting in formation of m/z 128, presumably deprotonated pyroglutamic acid. Dipeptides with the reverse linkage, H-Xxx-Glu-OH, show distinctive fragmentation reactions of the [M-H](-) ions including enhanced elimination of CO(2) and formation of deprotonated glutamic acid. Exchange of the labile hydrogens for deuterium has shown that there is considerable interchange of C-bonded hydrogens with labile (N- and O-bonded) hydrogens prior to most fragmentation reactions. All dipeptides show loss of H(2)O from [M-H](-). MS(3) studies show that the [M-H-H(2)O](-) ion derived from H-Glu-Gly-OH has the structure of deprotonated pyroglutamylglycine while the [M-H-H(2)O](-) ions derived from H-Glu(Gly-OH)-OH and H-Gly-Glu-OH show a different fragmentation behaviour indicating distinct structures for the fragment ions.     

Dissociative electron attachment to glycyl-glycine, glycyl-alanine and alanyl-alanine

The processes of negative ions formation of dipeptides glycyl-glycine, glycyl-alanine and alanyl-alanine in the conditions of resonant electron capture have been studied with a help of negative ions mass spectrometry. Using a thermochemical approach, the main channels of fragment negative ions formation were found and the structure of the ions were established. The isobaric ions have been identified by the experiments with high mass resolution. The cross sections of fragment ions formation were measured. The metastable fragmentation of [M-H](-) and [M-COOH](-) ions in the energy range 4.5-7.5 eV have been found.     

Elucidation of the double-bond position of long-chain unsaturated fatty acids by multiple-stage linear ion-trap mass spectrometry with electrospray ionization

Linear ion-trap (LIT) MS2 mass spectrometric approach toward locating the position of double bond(s) of unsaturated long-chain fatty acids and toward discerning among isomeric unsaturated fatty acids as dilithiated adduct ([M-H+2Li]+) ions are described in this report. Upon resonance excitation in a LIT instrument, charge-remote fragmentation that involves beta-cleavage with gamma-H shift (McLafferty rearrangement) is the predominant fragmentation pathway seen for the [M-H+2Li]+ ions of monoenoic long-chain fatty acids. The fragmentation process results in a dilithiated product ion of terminally unsaturated fatty acid, which undergoes consecutive McLafferty rearrangement to eliminate a propylene residue, and gives rise to another dilithiated adduct ion of terminally unsaturated fatty acid. In addition to the above-cited fragmentation process, the [M-H+2Li]+ ions of homoconjugated dienoic long-chain fatty acids also undergo alpha-cleavage(s) with shift of the allylic hydrogen situated between the homoconjugated double bonds to the unsaturated site. These fragmentation pathways lead to two types of CC bond cleavages that are allylic (alpha-cleavage) or vinylic, respectively, to the proximal CC double bond, resulting in two distinct sets of ion series, in which each ion series is separated by a CH2CHCH (40 Da) residue. These latter fragmentations are the predominant processes seen for the polyunsaturated long-chain fatty acids. The spectrum feature dependent on the position of unsaturated double bond(s) affords unambiguous assignment of the position of double bond(s) of long-chain unsaturated fatty acids.     

Formation and decay of the dehydrogenated parent anion upon electron attachment to dialanine

The dehydrogenated parent anion [M-H](-) is one of the most dominant anions formed in dissociative electron attachment to various small biomolecules like nucleobases and single amino acids. In the present study, we investigate the [M-H](-) channel for the dipeptide dialanine by utilizing an electron monochromator and a two-sector-field mass spectrometer. At electron energies below 2?eV, the measured high-resolution ion-efficiency curve has a different shape to that for the single amino acid alanine, which is explained by the altered threshold energies for formation of [M-H](-) determined in quantum chemical calculations. Moreover, the structure of the formed [M-H](-) anion is further studied by investigating the unimolecular and collision-induced decay of this anion. Trajectory calculations have been carried out to aid the interpretation of the experimentally observed fragmentation patterns.     

Resonant electron capture by some amino acids and their methyl esters

Resonant electron capture mass spectra of aliphatic and aromatic amino acids and their methyl esters show intense [M-H](-) negative ions in the low-energy range. Ion formation results from a predissociation mechanism mediated by the low-energy pi*oo resonant state. Methylation in general has little influence on the electronic structure according to quantum chemical calculations, but the corresponding ions from the methyl esters, [M-Me](-), could be ascertained to arise only at higher resonance energies. Aromatic amino acids are characterized by an additional low-energy fragmentation channel associated with the generation of negative ions with loss of the side chain. The complementary negative ions of the side chains are more efficiently produced at higher energies. The results have significant implications in biological systems as they suggest that amino acids can serve as radiation protectors since they have been found to efficiently thermalize electrons.     

Low energy (0-10 eV) electron driven reactions in the halogenated organic acids CCl3COOH, CClF2COOH, and CF3CHNH2COOH (trifluoroalanine)

Negative ion formation following resonant electron attachment to the three title molecules is studied by means of a beam experiment with mass spectrometric detection of the anions. All three molecules exhibit a pronounced resonance in the energy range around 1 eV which decomposes by the loss of a neutral hydrogen atom thereby generating the closed shell anion (M-H)(-) (or RCOO(-)), a reaction which is also a common feature in the non-substituted organic acids. The two chlorine containing molecules CCl(3)COOH and CClF(2)COOH exhibit an additional strong and narrow resonance at very low energy (close to 0 eV) which decomposes by the cleavage of the C-Cl bond with the excess charge finally localised on either of the two fragments Cl(-) and (M-Cl)(-). This reaction is by two to three orders of magnitude more effective than hydrogen loss. Apart from these direct bond cleavages (C-Cl, O-H) resonant attachment of subexcitation electrons trigger additional remarkably complex unimolecular decompositions leading, e.g., to the formation of the bihalide ions ClHCl(-) and ClHF(-) from CCl(3)COOH and CClF(2)COOH, respectively, or the loss of a neutral CF(2) unit from trifluoroalanine thereby generating the fluoroglycine radical anion. These reactions require substantial rearrangement in the transitory negative ion, i.e., the cleavage of different bonds and formation of new bonds. F(-) from both chlorodifluoroacetic acid and trifluoroalanine is formed at comparatively low intensity (more than three orders of magnitude less than Cl(-) from the chlorine containing molecules) and predominantly within a broad resonant feature around 7-8 eV characterised as core excited resonance.     

Mass spectrometric characterization of 4-oxopentanoic acid and gas-phase ion fragmentation mechanisms studied using a triple quadrupole and time-of-flight analyzer hybrid system and density functional theory

4-Oxopentanoic acid was characterized experimentally by electrospray ionization using a triple quadrupole and time-of-flight analyzer hybrid system. This compound was chosen as a model substance for small organic compounds bearing an acetyl and a carboxyl group. Collision-induced dissociation experiments at different activation energies were performed to elucidate possible fragmentation pathways. These pathways were also studied on the theoretical level using density functional theory (DFT) B3LYP/6-311++G(3df,3pd)//B3LYP/6-31+G(d)+ZPVE calculations. CO2 ejection from the [M-H](-) anion of 4-oxopentanoic acid was observed and the fragmentation pathway studied by DFT reveals a new concerted mechanism for CO2 elimination accompanied by an intramolecular proton transfer within a pentagonal transition state structure. Successive elimination of water and CO from the [M-H](-) anion of 4-oxopentanoic acid was also observed. A rearrangement in the primary deprotonated ketene anion produced after water elimination was found on the theoretical level and leads to CO elimination from the primary product anion [M-H-H2O](-). Energy diagrams along the reaction coordinates of the fragmentation pathways are presented and discussed in detail. Mulliken charge distributions of some important structures are presented.     

Phenol, chlorobenzene and chlorophenol isomers: resonant states and dissociative electron attachment

This paper reports a study of resonant dissociative electron attachment (DEA) to the phenol, chlorobenzene, p-, m-, and o-chlorophenol molecules. On the basis of spectroscopic and thermochemical approaches the resonant states of the molecular negative ions (NIs) and the structures of some dissociative decay products are assigned. In the electron energy range up to 3 eV, DEA processes are determined by the two 2[pi*]-shape resonances resulting mainly in formation of [M-H]- and/or Cl- ions. At higher electron energies the energy correlation between peaks in the negative ion effective yield curves and bands of UV spectra allowed identification of the core-excited resonances. The peculiarities of Cl- ion formation and the vibrational fine structure on the effective yield curves of the [M-H]- ions are discussed. The mass spectrometric procedures for measurement of relative cross sections for NI formation are described.     

Generation of alkoxide anions from a series of aliphatic diols and alcohols and their ion-molecule reactions with carbon dioxide in the gas phase

Alkoxide anions, [M-H](-) from a series of aliphatic diols and alcohols are generated in the source under negative ion electrospray ionisation conditions by cone-voltage fragmentation of the corresponding [M + F](-) ions. The collision-induced dissociation (CID) spectra of [M-H](-) ions consist of [M-H-2H](-) ions, in addition to the other characteristic fragment ions, and the relative abundance of [M-H-2H(-) ions among the series of diols varies as a function of chain length that could be explained based on their stabilities through intramolecular hydrogen bonding. The reactivity of alkoxide anions is studied through ion-molecule reactions with CO(2) in the collision cell of a triple quadrupole mass spectrometer. All the alkoxide anions reacted with CO(2) and formed corresponding carbonate anions, [M-H + CO(2)](-) ions. The reactivity of alkoxide anions within the series of diols also reflected the stability of their [M-H](-) ions.     

Differentiation of common diastereoisomeric ursane-type triterpenoids by high-performance liquid chromatography/tandem mass spectrometry

A rapid and stable method consisting of high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry was established for the identification and differentiation of common diastereoisomeric ursane-type triterpenoids at the C-3 position. Two characteristic fragment ions, [M-H-H(2) O-CO(2)](-) and [M-H-H(2)O-HCOOH](-) , exhibited significant stereochemical effects and were utilized to distinguish 3-OH epimers. Based on reference standards, the abundance of the fragment ion [M-H-H(2)O-HCOOH](-) in 3β-OH compounds in the MS(3) experiment was dramatically higher compared to [M-H-H(2) O-CO(2)](-); however, for 3α-OH compounds, the product ion [M-H-H(2) O-CO(2)](-) was noted to be higher than [M-H-H(2)O-HCOOH](-). Energy-resolved mass spectrometric experiments were carried out to support the differentiation of these diastereoisomeric triterpenoids at the C-3 position. Using this method, a total of nine ursane-type triterpenoids from a plant crude extract, including four pairs of epimers at the C-3 position, were identified and distinguished rapidly. Furthermore, offline Fourier transform ion cyclotron resonance tandem mass spectrometry was also performed to assign accurate elemental compositions.     

Resonant dissociative electron capture by the simplest amino acids and dipeptides

The formation of ions from amino acids (glycine and alanine) and dipeptides (glycylglycine, alanylalanine, and glycylalanine) under the resonant electron capture conditions was studied by negative ion resonant electron capture mass spectrometry. The isobaric ions were found, their effective yield curves were experimentally separated, and the elemental composition was determined. The thermochemical aspect of ion formation was considered, and probable dissociative channels of fragmentation ion formation and their structures were established on the basis of this aspect. Bond cleavage reactions only and H-shift processes were revealed. The rearrangements occur presumably through the stage of formation of intramolecular hydrogen bonds. The cross-sections of formation of ions [M − H]⁻ were measured in the energy range 1.1–1.3 eV. The metastable decay channels of ions [M − H]⁻ and [M − COOH]⁻ were found in the energy range 4.5–7.5 eV for dipeptides, which enabled establishing the genetic relationship between the parental and daughter ions and revealing hidden fragmentation pathways.     

An unexpected ion-molecule adduct in negative-ion collision-induced decomposition ion-trap mass spectra of halogenated benzoic acids

The ion observed at m/z 145 when product ion spectra of iodobenzoate anions are recorded using ion-trap mass spectrometers corresponds to the adduct ion [I(H(2)O)](-). The elements of water required for the formation of this adduct do not originate from the precursor ion but from traces of moisture present in the helium buffer gas. A collision-induced decomposition (CID) spectrum recorded from the [M-H](-) ion (m/z 251) derived from 3-iodo[2,4,5,6-(2)H(4)]benzoic acid also showed an ion at m/z 145. This observation confirmed that the m/z 145 is not a product ion resulting from a direct neutral loss from the carboxylate anion. (79)Bromobenzoate anions produce similar results showing an ion at m/z 97 for [(79)Br(H(2)O)](-). The ion-molecule reaction observed here is unique to ion-trap mass spectrometers since a corresponding ion was not observed under our experimental conditions in spectra recorded with in-space tandem mass spectrometers such as triple quadrupole or quadrupole time-of-flight instruments.     

A study of resonance electron capture ionization on a quadrupole tandem mass spectrometer

Procedures that allow the realization of resonance electron capture (REC) mode on a commercial triple-quadrupole mass spectrometer, after some simple modifications, are described. REC mass spectrometry (MS) and tandem mass spectrometry (MS/MS) experiments were performed and spectra for some compounds were recorded. In particular, the charge-remote fragmentation (CRF) spectra of [M - H](-) ions of docosanoic and docosenoic acids under low-energy collisionally activated dissociation (CAD) conditions were obtained, and showed that there were no significant differences for [M - H](-) ions produced at different resonances (i.e. for [M - H](-) ions with different structures). This observation was explained on the basis of results obtained from deuterium-labeled fatty acids, which showed that different CRF ions (but with the same m/z value in the absence of labels) could be produced by different mechanisms, and all of them were obviously realized under CAD conditions that made spectra practically indistinguishable. The other example, which compared the REC-MS/MS spectrum of [M - H](-) ions and EI-MS/MS spectrum of M(+.) ions of daidzein, demonstrated the potential of the REC-MS/MS technique for more complex structure elucidation.     

Structural determination of modified fatty acids by collisional activation of cationized molecules

The nature and location of a variety of modifications of fatty acids are determined by collisional activation (CA) of [M + 2Li ? H]⁺ ions. The sample molecules are cationized in situ on the probe tip, desorbed by fast atom bombardment and, upon CA, undergo charge-remote decompositions. This approach is a direct, totally instrumental method for structure elucidation. Advantages of CA of [M + 2Li ? H]⁺ ions are that fatty acids with substituents in close proximity to the carboxylate terminus and modified short-chain acids are readily determined: decompositions of carboxylate anions of these fatty acids result in collision-activated dissociation (CAD) spectra that give incomplete structural information. However, the CAD spectra of some [M ? H]^? ions, such as those from epoxy acids, are simpler to interpret than those of the [M + 2Li ? H]⁺ ions. Thus, CA of fatty acid [M + 2Li ? H]⁺ ions is a complementary approach to CA of [M ? H]^? ions for determining the fatty acid structures investigated here. The use of this approach for analyzing complex mixtures of modified fatty acids is also evaluated.     

The role of free electrons in MALDI: electron capture by molecules of alpha-cyano-4-hydroxycinnamic acid

Low-energy (0-12 eV) electron attachment to molecules of a typical matrix substance used for matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), namely alpha-cyano-4-hydroxicynnamic acid, has been investigated in the gas phase at different temperatures ranging from 140 degrees C to 260 degrees C by means of electron capture negative-ion mass spectrometry (ECNI MS). The yield of negative ions, formed by electron capture, was measured as a function of incident electron energy for four different temperatures. The long-lived parent molecular anion, [M]- (m/z 189), was observed in the negative-ion mass spectra of the substance under investigation. Its autodetachment lifetime was estimated to be approximately 600 micros. It was found that at 140 degrees C the main decay channel of the long-lived temporary molecular anion of alpha-cyano-4-hydroxicynnamic acid is a formation of the [M-COOH]-; fragment negative ion (m/z 144) with an intensity of 37.2% in percentage terms in respect of the total anion current. There are also [M-H]-, [M-CO2]- and [CN]- fragments in the spectra with intensities of about 7.7%, 21.6% and 3.1% at 140 degrees C. It was shown that the escape of the CO2 molecule from the parent molecular anion is a slow process. It takes [M]- about 10 micros to decay on carbon dioxide molecules and [M-CO2]- fragment anions. Increasing the temperature of the target molecule alters the negative-ion mass spectra of alpha-cyano-4-hydroxicynnamic acid significantly. A possible role for the findings in typical MALDI MS experiments is discussed.     

Amide bond cleavage in deprotonated tripeptides: a newly discovered pathway to "b2 ions

The fragmentation reactions of the [M-H](-) ions of the tripeptides H-Gly-Leu-Sar-OH, H-Leu-Gly-Pro-OH and H-Gly-Leu-Gly-OH have been investigated in detail using energy-resolved mass spectrometry, isotopic labelling and MS(3) experiments. It is shown that the major route to the "b(2) ions involves loss of a neutral amine from the a(3) ([M-H-CO(2)](-)) ion rather than being formed directly by fragmentation of the [M-H](-) ion. When there is no C-terminal amidic hydrogen (Sar, Pro), loss of a neutral amine is the dominant primary fragmentation reaction of the a(3) ion. However, when there is a C-terminal amidic hydrogen (Gly), elimination of the N-terminal amino acid residue is the major fragmentation reaction of the a(3) ion and formation of the "b(2) ion is greatly reduced in importance. It is proposed that the "b(2) ions are deprotonated oxazolones.     

Chiral NMR discrimination of secondary amines using (18-crown-6)-2,3,11,12-tetracarboxylic acid

[reaction: see text] Enantiomeric discrimination is observed in the (1)H NMR spectra of chiral secondary amines in the presence of (R)-(+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid. Secondary amines are protonated by one of the carboxylic acid groups of the crown ether to produce the corresponding ammonium and carboxylate ions. The secondary ammonium ion likely forms two hydrogen bonds to crown ether oxygen atoms and an ion pair with the carboxylate anion.     

Electron capture negative ion mass spectra of some typical matrix-assisted laser desorption/ionization matrices

A series of seven typical matrix-assisted laser desorption/ionization (MALDI) matrices has been investigated by means of electron capture negative ion mass spectrometry (ECNI-MS). It has been shown that the most effective matrices form deprotonated negative ions predominantly in the low-energy region. Relative dissociative cross sections have been measured for all molecules under investigation. The relative integrated abundance of [M - H](-) ion formation in the series changes by four orders of magnitude. It has been shown that 2,5-DHB (gentisic acid), one of the most effective MALDI matrices, has maximal relative intensity of [M - H](-) formation at the energy approximately equal 0.8 eV. This result is in accordance with a finding of Frankevich and Zenobi [Book of Abstracts, Workshop-school "Mass spectrometry in chemical physics, bio-physics and environmental sciences", Zvenigorod, Russia, April, 25-26, 2002, p. 40] that a probable origin of negative ions in MALDI is the process of low-energy (0.5-1 eV) dissociative electron capture by matrix molecules.     

A new charge-associated mechanism to account for the production of fragment ions in the high-energy CID spectra of fatty acids

A new mechanism, termed a charge-assisted process, is proposed as an additional mechanism to the charge-remote process to account for ions of the [M - CnH2n+2] series found in the positive and negative high energy CID spectra of fatty acids and related compounds when ionized as closed-shell ([M - H]- or [M + X]+) species. The new mechanism is based on that commonly invoked to account for similar ions in the electron-impact spectra of derivatized fatty acids whereby the positive charge on the derivative abstracts a hydrogen atom from various positions of the alkyl chain to leave a radical that initiates a radical-induced cleavage of the chain. It is proposed that in the high energy CID spectra of closed-shell ions, similar hydrogen migrations occur but unpairing of electrons is avoided by charge transfer to the alkyl chain. This charge then initiates a concerted cleavage of the chain to give an allylic carbonium (positive ion spectrum) or carbanion (negative ion spectrum). The mechanism avoids the need to involve radicals or loss of hydrogen atoms from even-electron (closed shell) ions and provides a driving force for the reaction, namely, the formation of ions with a stabilized charge. An extension of the mechanism is also proposed to account for the formation of odd-electron ions from these compounds. The charge-assisted mechanism does not rule out the occurrence of other mechanisms that have been accepted for many years but provides an alternative process that can account for some spectral features which were difficult to explain earlier.     

Electron attachment to a hydrated DNA duplex: the dinucleoside phosphate deoxyguanylyl-3',5'-deoxycytidine

The minimal essential section of DNA helices, the dinucleoside phosphate deoxyguanylyl-3',5'-deoxycytidine dimer octahydrate, [dGpdC](2), has been constructed, fully optimized, and analyzed by using quantum chemical methods at the B3LYP/6-31+G(d,p) level of theory. Study of the electrons attached to [dGpdC](2) reveals that DNA double strands are capable of capturing low-energy electrons and forming electronically stable radical anions. The relatively large vertical electron affinity (VEA) predicted for [dGpdC](2) (0.38 eV) indicates that the cytosine bases are good electron captors in DNA double strands. The structure, charge distribution, and molecular orbital analysis for the fully optimized radical anion [dGpdC](2)(·-) suggest that the extra electron tends to be redistributed to one of the cytosine base moieties, in an electronically stable structure (with adiabatic electron affinity (AEA) 1.14 eV and vertical detachment energy (VDE) 2.20 eV). The structural features of the optimized radical anion [dGpdC](2)(·-) also suggest the probability of interstrand proton transfer. The interstrand proton transfer leads to a distonic radical anion [d(G-H)pdC:d(C+H)pdG](·-), which contains one deprotonated guanine anion and one protonated cytosine radical. This distonic radical anion is predicted to be more stable than [dGpdC](2)(·-). Therefore, experimental evidence for electron attachment to the DNA double helices should be related to [d(G-H)pdC:d(C+H)pdG](·-) complexes, for which the VDE might be as high as 2.7 eV (in dry conditions) to 3.3 eV (in fully hydrated conditions). Effects of the polarizable medium have been found to be important for increasing the electron capture ability of the dGpdC dimer. The ultimate AEA value for cytosine in DNA duplexes is predicted to be 2.03 eV in aqueous solution.