Determination of the fatty acyl profiles of phosphatidylethanolamines by tandem mass spectrometry of sodium adducts

Phosphatidylethanolamines (PEs) are one of the major constituents of cellular membranes, and, along with other phospholipid classes, have an essential role in the physiology of cells. Profiling of phospholipids in biological samples is currently done using mass spectrometry (MS). In this work we describe the MS fragmentation of sodium adducts of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and 2-linoleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine (PLPE). This study was performed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) using three different instruments and also by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). All MS/MS spectra show product ions related to the polar head fragmentation and product ions related to the loss of acyl chains. In ESI-MS/MS spectra, the product ions [M+Na-R1COOH-43]+ and [M+Na-R2COOH-43]+ show different relative abundance, as well as [M+Na-R1COOH]+ and [M+Na-R2COOH]+ product ions, allowing identification of both fatty acyl residues of PEs, and their specific location. MALDI-MS/MS shows the same product ions reported before and other ions generated by charge-remote fragmentation of the C3-C4 bond (gamma-cleavage) of fatty acyl residues combined with loss of 163 Da. These fragment ions, [M+Na-(R2-C2H3)-163]+ and [M+Na-(R1-C2H3)-163]+, show different relative abundances, and the product ion formed by the gamma-cleavage of sn-2 is the most abundant. Overall, differences noted that are important for identification and location of fatty acyl residues in the glycerol backbone are: relative abundance between the product ions [M+Na-R1COOH-43]+ > [M+Na-R2COOH-43]+ in ESI-MS/MS spectra; and relative abundance between the product ions [M+Na-(R2-C2H3)-163]+ > [M+Na-(R1-C2H3)-163]+ in MALDI-MS/MS spectra.     

The behavior of stereoisomeric ions in the gas phase: The Case of cyclohexane-hexacarboxylic methyl esters

Not only strong differences in relative abundances of product ions, but also different fragmentation pathways are observed in the electron impact mass spectrosopy of six stereoisomeric cyclohexanehexacarboxylic methyl esters. This unusual behaviour has been studied in detail with the aid of different ionization methyods, B/E and B²/E linked scans, exact mass measurements, deuterium labelling experiments and collisionally activated decomposition spectrometry. A close analogy between the isomerization observed under acidic conditions in condensed phase and that observed under chemical ionization (CH₄) conditions is underlined.     

Studies on phosphatidylglycerol with triple quadrupole tandem mass spectrometry with electrospray ionization: Fragmentation processes and structural characterization

Negative-ion low-energy collisionally activated dissociation (CAD) tandem mass spectrometry of electrospray-produced ions permits structural characterization of phosphatidylglycerol (PG). The major ions that identify the structures arise from neutral loss of free fatty acid substituents ([M − H − R _x CO₂H]⁻) and neutral loss of the fatty acids as ketenes ([M − H − R′ _x CH = C = O]⁻), followed by consecutive loss of the glycerol head group. The abundances of the ions arising from neutral loss of the sn-2 substutient as a free fatty acid ([M − H − R₂CO₂H]⁻) or as a ketene ([M − H − R′₂CH = C = O]⁻) are greater than those of the product ions from the analogous losses at sn-1. Nucleophilic attack of the anionic phosphate site on the C-1 or the C-2 of the glycerol to which the carboxylates attached expels the sn-1 (R₁CO₂⁻) or the sn-2 (R₂CO₂⁻) carboxylate anion, resulting in a greater abundance of R₂COO⁻ than R₁COO⁻. These features permit assignments of fatty acid substituents and their position in the glycerol backbone. The results are also consistent with our earlier findings that pathways leading to those losses at sn-2 are sterically more favorable than those at sn-1. Fragment ions at m/z 227, 209 and 171 reflect the glycerol polar head group and identify the various PG molecules. Both charge-remote fragmentation (CRF) and charge-drive fragmentation (CDF) processes are the major pathways for the formation of [M − H − R _x COOH]⁻ ions. The CRF process involves participation of the hydrogen atoms on the glycerol backbone, whereas the CDF process involves participation of the exchangeable hydrogen atoms of the glycerol head group. The proposed fragmentation pathways are supported by CAD tandem mass spectrometry of the analogous precursor ions arising from the H-D exchange experiment, and further confirmed by source CAD in combination with tandem mass spectrometry.     

Differentiation of Regioisomeric Aromatic Ketocarboxylic Acids by Positive Mode Atmospheric Pressure Chemical Ionization Collision-Activated Dissociation Tandem Mass Spectrometry in a Linear Quadrupole Ion Trap Mass Spectrometer

Positive-mode atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS ⁿ ) was tested for the differentiation of regioisomeric aromatic ketocarboxylic acids. Each analyte forms exclusively an abundant protonated molecule upon ionization via positive-mode APCI in a commercial linear quadrupole ion trap (LQIT) mass spectrometer. Energy-resolved collision-activated dissociation (CAD) experiments carried out on the protonated analytes revealed fragmentation patterns that varied based on the location of the functional groups. Unambiguous differentiation between the regioisomers was achieved in each case by observing different fragmentation patterns, different relative abundances of ion-molecule reaction products, or different relative abundances of fragment ions formed at different collision energies. The mechanisms of some of the reactions were examined by H/D exchange reactions and molecular orbital calculations.     

Mass spectral cycloreversion reactions of 2-chloro-3,6-diaryl-3,4-dihydro-1,3,2-oxazaphosphorin-2-oxides

The mass spectral fragmentation modes of various 2-chloro-3,6-diaryl-3,4-dihydro-1,3,2-oxazaphosphorin-2-oxides—a novel ring system—reveal cycloreversion by two pathways. Retro Diels-Alder reaction by a stepwise mechanism is prominent in this system. The relative abundances of the enone and dienophile ions depend on the nature of the substituent attached to the double bond in this ring. Another retro Diels-Alder fragmentation process, involving the loss of PO₂Cl from the molecular ion, is preceded by a 1,3-allylic rearrangement and is the major fragmentation mode in the metastable time scale. Further fragmentation of the [M? PO₂Cl]⁺ imine ion seems to occur from cyclic dihydroquinoline intermediates by substitution elimination steps.     

Fragmentation Study of Limonoids from Turraae pubescens by Electrospray Ionization Quadrupole Time-of-flight Mass Spectrometry

Nine representative limonoids isolated from Turraae pubescens were investigated by electrospray ionization quadrupole time-of-flight tandem mass spectrometry in positive ion mode. Although the structures of these compounds are similar, the corresponding fragmentation patterns and mass spectrometry and tandem mass spectrometry (MS/MS) spectra are clearly different. For Turrapubin A–C, product ions can be detected in both low and high mass ranges. A McLafferty-type rearrangement is the only way for the cleavage of C9–C10. For 11-epi-toonacilin, Turrapubin E, Turraflorin A, 11-epi-23-hydroxytoonacilide, Turrapubin H and 11-epi-21-hydroxytoonacilide, the cleavage of C9–C10 goes through two different ways, including McLafferty-type rearrangement and homolytic cleavage. The relative abundances of product ions from McLafferty-type rearrangement for 11-epi-toonacilin, 11-epi-23-hydroxytoonacilide, and Turrapubin H are high, while those for Turrapubin E, Turraflorin A, and 11-epi-21-hydroxytoonacilide are low. A pair of epimers was distinguished unambiguously by MS/MS spectra. It was found that the substituent group at C-1, hydroxy group, O atom linked to C-14 and C-15, and the oxygenated furan ring were the important factors leading to the differences of their MS/MS spectra.     

Identification of isobaric product ions in electrospray ionization mass spectra of fentanyl using multistage mass spectrometry and deuterium labeling

Isobaric product ions cannot be differentiated by exact mass determinations, although in some cases deuterium labeling can provide useful structural information for identifying isobaric ions. Proposed fragmentation pathways of fentanyl were investigated by electrospray ionization ion trap mass spectrometry coupled with deuterium labeling experiments and spectra of regiospecific deuterium labeled analogs. The major product ion of fentanyl under tandem mass spectrometry (MS/MS) conditions (m/z 188) was accounted for by a neutral loss of N‐phenylpropanamide. 1‐(2‐Phenylethyl)‐1,2,3,6‐tetrahydropyridine (1) was proposed as the structure of the product ion. However, further fragmentation (MS³) of the fentanyl m/z 188 ion gave product ions that were different from the product ion in the MS/MS fragmentation of synthesized 1, suggesting that the m/z 188 product ion from fentanyl includes an isobaric structure different from the structure of 1. MS/MS fragmentation of fentanyl in deuterium oxide moved one of the isobars to 1 Da higher mass, and left the other isobar unchanged in mass. Multistage mass spectral data from deuterium‐labeled proposed isobaric structures provided support for two fragmentation pathways. The results illustrate the utility of multistage mass spectrometry and deuterium labeling in structural assignment of isobaric product ions. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of multiple fragmentation pathways initiated by collision‐induced dissociation of multifunctional anions formed by deprotonation of 2‐nitrobenzenesulfonylglycine

The correlation of anion structure with the fragmentation behavior of deprotonated nitrobenzenesulfonylamino acids was investigated using tandem mass spectrometry, isotopic labeling and computational methods. Four distinct fragmentation pathways resulting from the collision‐induced dissociation (CID) of deprotonated 2‐nitrobenzenesulfonylglycine (NsGly) were characterized. The unusual loss of the aryl nitro substituent as HONO was the lowest energy process. Subsequent successive losses of CO, HCN and SO₂ indicated that an ortho cyclization reaction had accompanied loss of HONO. Other pathways involving rearrangement of the ionized sulfonamide group, dual bond cleavage and intramolecular nucleophilic displacement were proposed to account for the formation of phenoxide, arylsulfinate and arylsulfonamide product ions at higher collision energies. The four distinct fragmentation pathways were consistent with precursor–product relationships established by CID experiments, isotopic labeling results and the formation of analogous product ions from 2,4‐dinitrobenzenesulfonylglycine and the Ns derivatives of alanine and 2‐aminoisobutyric acid. The computations confirmed a low barrier for ortho cyclization with loss of HONO and feasible energetics for each reaction step in the four pathways. Computations also indicated that three of the fragmentation pathways started from NsGly ionized at the carboxyl group. Overall, the pathways identified for the fragmentation of the NsGly anion differed from processes reported for anions containing a single functional group, demonstrating the importance of functional group interactions in the fragmentation pathways of multifunctional anions. Copyright © 2014 John Wiley & Sons, Ltd.     

The effect of cation size (H+, Li+, Na+, and K+) on McLafferty-type rearrangement of even-electron ions in mass spectrometry

Protonation and alkali-metal cation adduction are the most important ionization processes in soft-ionization mass spectrometry.Studies on the fragmentation mechanism of protonated and alkali-metal-cationized compounds in tandem mass spectrometry are essential and helpful for structural analysis.In some cases,it was often observed that a compound attached by different alkali-metal cations(or proton)exhibits similar fragmentation patterns but the relative abundances of product ions are different.This difference was considered to derive from the different electrostatic interactions of alkali-metal cations(or the bonded effect of proton)with the analyte.The alkali-metal cation with a smaller ionic radius shows stronger electrostatic interaction with the molecule because of its higher charge density.In addition,the bonded effect of the proton is stronger than the electrostatic interaction of the alkali-metal cation.In the present study,which used McLafferty-type rearrangements of even-electron ions([M+Cat]+,Cat=H,Li,Na,K)as model reactions,the effect of cation size in mass spectrometric fragmentation reactions is highlighted.These considerations were also successfully applied to interpret the similar but distinct fragmentation behavior of proton and alkali-metal cation adducts of a synthetic compound(2-(acetamido(phenyl)methyl)-3-oxobutanoate)and a drug(entecavir).     

Fragmentation behavior of disaccharides during desorption–ionization

The fragmentation behavior of native and singly derivatized protonated disaccharides were examined by fast ion bombardment Fourier transform mass spectrometry to determine the origin of various fragment ions produced during desorption–ionization. The lack of interfering matrix ions in this technique allowed the investigation of model disaccharides. Three major product ions were observed, corresponding in mass to a loss of H₂O, a loss of a monosaccharide unit and a combination of both losses. The oxygen of the lost H₂O molecule originates from an oxygen attached to the anomeric carbon. The other two product ions come from two different monosaccharide units and are due to competing processes. The relative ion intensities of these two fragment ions are governed by the basicities of the fragments. The basicity of the reducing end increases when the hemiacetal is converted into the methyl glycoside. This increase in basicity is due to the increased size of the alkyl group and the ability of the alkyl group to prevent the opening of the reducing glycosidic ring.     

Electrospray ionization and triple quadrupole tandem mass spectrometry study of some biologically relevant homo- and heterodimeric cadmium thiolate conjugates

A series of 19 compounds of general formula R₁S-Cd-SR₂, R₁, and R₂, being some biologically relevant thiol amino acids and peptides, were prepared by direct reaction of cadmium(II) ions and thiols in water at millimolar concentration. The obtained products were characterized by electrospray ionization and triple quadrupole tandem mass spectrometry. The source spectra of stoichiometric 1:2 Cd-thiol systems containing either an individual thiol or equimolar mixtures of two different thiols featured several Cd-containing signals, although at much lesser intensity than in the previously reported experiments with mercury(II) (J. Am. Soc. Mass Spectrom. 2004, 15, 288–300). Also, the relative intensity of the homo- and heterodimeric thiolates were significantly different from the theoretically expected 1:2:1 ratio, thus pointing at some degree of discrimination between the different thiols. In particular, homo-cysteine showed much less reactivity than cysteine, and penicillamine and cysteine methyl ester much less than the free amino acid. The fragment spectra show structure-specific ions for the different ligands bound to the metal ion and allow a stand-alone determination of the connectivity also of isomeric pairs. The fragmentation pathways are similar to those observed for the corresponding mercury(II) analogues, with the addition of further intense and specific fragments, one formally carrying a Cd-bound OH ligand and one connected as a five-membered oxazolone carrying a cadmium-bis-thiolate side chain, both formed with a high intensity. Energy-resolved fragmentation data show that metal-free ions can be generated from cysteine but not from glutathione conjugates and point to the possibility of unveiling differences in the biochemical behavior of the conjugates of different heavy metals through the detailed study of their mass spectrometric fragmentation.     

Triple quadrupole tandem mass spectrometry of sesquiterpene lactones: a study of goyazensolide and its congeners

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to investigate the fragmentation pattern of ten sesquiterpene lactones of the goyazensolide type under low-energy collision-induced dissociation (CID) using a triple quadrupole mass spectrometer. The analysis revealed that loss of CO(2)[M + H - 44](+) is the predominant process for compounds that exhibit a hydroxyl at C-8. In contrast, compounds with different acyloxy groups at C-8 fragment by means of elimination of the corresponding carboxylic acids [M + H - (R(2)CO(2)H)](+) and consecutive losses of CO and H(2)O. Our results also demonstrate the influence of both the stereochemistry of the acyloxy group at C-8 on the relative abundances of product ions and the hydroxyl at C-15, which creates an additional pathway, resulting in highly diagnostic product ions. This work clearly demonstrates the utility of tandem quadrupole low-resolution mass spectrometry for studies on the rationalization of the fragmentation of a series of compounds with a highly conserved core structure, but differing in substituent groups.     

Mass spectrometric methods for distinguishing structural isomers of glutathione conjugates of estrone and estradiol

Collisionally activated decompositions (CAD) of [M+H]⁺ ions from two sets (estrone and estradiol) of three isomeric glutathione (GSH) conjugates were studied by using five tandem mass spectrometric methods: (1) low energy (LE) CAD in an ion trap, (2) LE CAD in a triple quadrupole, (3) electrospray ionization (ESI)-source CAD in a tandem four sector, (4) high energy (HE) CAD of both ESI-produced and fast-atom bombardment (FAB)-produced ions in a tandem four-sector mass spectrometer, and (5) metastable-ion decompositions of FAB-produced ions. Four types of fragment ions are produced. The first type, formed from cleavage of the peptide backbone, gives rise to modified b₂, modified y₂, y₂, and b₁ ions. These fragments are observed with all the methods and show that the catechol estrogen attachment is at the cysteine moiety of the GSH. Internal fragment ions are the second type, and they also support that the modification is at cysteine. The third type involves fragmentation of the C–S bond to give an ion containing the steroid bonded to the sulfur. The fourth type of fragment ion is similar to the third but involves oxidation of the steroid ring and reduction of the GSH moiety; it is the most isomer specific of the four. The isomer-specific ions are of relatively low abundance in the product-ion spectra taken on the triple quadrupole and ion trap, but their abundances can be improved by increasing the collision energy. ESI source-CAD and the HE-CAD spectra of the isomers are the most distinctive because abundant product ions of all four types are seen in a single spectrum.     

Explanation through density functional theory of the unanticipated loss of CO2 and differences in mass fragmentation profiles of ritonavir and its rCYP3A4‐mediated metabolites

In the present study, the metabolism of ritonavir was explored in the presence of rCYP3A4 using a well‐established strategy involving liquid chromatography–mass spectrometry (LC–MS) tools. A total of six metabolites were formed, of which two were new, not reported earlier as CYP3A4‐mediated metabolites. During LC–MS studies, ritonavir was found to fragment through six principal pathways, many of which involved neutral loss of CO₂, as indicated through 44‐Da difference between masses of the precursors and the product ions. This was unusual as the drug and the precursors were devoid of a terminal carboxylic acid group. Apart from the neutral loss of CO₂, marked differences were also observed among the fragmentation pathways of the drug and its metabolites having intact N‐methyl moiety as compared to those lacking N‐methyl moiety. These unusual fragmentation behaviours were successfully explained through energy distribution profiles by application of the density functional theory. Copyright © 2014 John Wiley & Sons, Ltd.     

Matrix-assisted laser desorption/ionization tandem mass spectrometry and post-source decay fragmentation study of phenylhydrazones of <Emphasis Type="Italic">N</Emphasis>-linked oligosaccharides from ovalbumin

N-linked oligosaccharides were released from hen ovalbumin by PNGase F and derivatized with phenylhydrazine. They were then examined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Phenylhydrazones of N-glycans under MALDI-tandem mass spectrometry (MS/MS) and post-source decay (PSD) conditions produced relatively similar fragmentation patterns; however, more cross-ring cleavages and fragment ions corresponding to low abundance isomeric structures were detected by MS/MS and not in PSD. Most fragment ions corresponded to glycosidic cleavages with preferential loss of residues from the chitobiose core and the 3-antenna. Sialylated phenylhydrazone-N-glycans, characterized here for the first time in ovalbumin by tandem mass spectrometry, underwent losses of sialic acid residues followed the same fragmentation pathways observed with neutral derivatized glycans. The relative abundances of some fragment ions indicated the linkage position of sialic acid and provided information on the number of residues attached to the 6-antenna. Also, new structures of ovalbumin glycans were observed as part of this study and are reported here.     

Electron impact and chemical ionization mass spectra of some tetra-acetylated anomeric glycosides

Electron impact and chemical ionization (CH₄, iso-C₄H₁₀ and NH₃) mass spectra of some tetra-acetylated anomeric glycosides have been examined with a view to the characterization of anomeric pairs. Minor differences observed in the relative intensities of common ions in the anomeric pairs in the electron impact mass spectra are found to be enhanced in the methane and isobutane chemical ionization mass spectra. In the absence of thermal decomposition, the β-anomers show greater ion abundances of common glycosyl ions than the α-anomers. Ammonia chemical ionization mass spectra show complementary behaviour indicating strong adduct ions and practically no fragmentation.     

The influence of sialylation on glycan negative ion dissociation and energetics

For the analysis of native glycans using tandem mass spectrometry (MS), it is desirable to choose conditions whereby abundances of cross-ring cleavages indicative of branch positions are maximized. Recently, negative ion tandem mass spectrometry has been shown to produce significantly higher abundances of such ions in glycans compared to the positive ion mode. Much of this prior work has concerned fragmentation patterns in asialo glycans. The present work compares the abundances of critical cross-ring cleavage ions using negative mode tandem mass spectrometry for milk oligosaccharides and N-linked glycans. For comparison, product ion formation was studied for deprotonated and nitrated ions formed from asialo glycans and deprotonated ions from sialylated glycans. Breakdown profiles demonstrate clearly that more energy was required to fragment sialylated compounds to the same extent as either their asialo or nitrate adducted counterparts. The extraction of a proton from a ring hydroxyl group during the ionization process may be viewed, qualitatively, as imparting significantly more energy to the ion than would that from a molecule bearing an acidic group, so that acidic glycans are more stable in the gas phase, as the negative charge resides on the carboxyl group. These results have strong practical implications because a major portion of glycans released from mammalian proteins will be sialylated.     

Apparent inhibition by arginine of macrocyclic <Emphasis Type="Italic">b</Emphasis> ion formation from singly charged protonated peptides

There is now strong evidence for the existence of macrocyclic isomers of b_n⁺ ions, the formation and subsequent opening of which can lead to loss of sequence information from protonated peptides in multiple-stage tandem mass spectrometry experiments. In this study, the fragmentation patterns of protonated YARFLG and permuted isomers of the model peptide were investigated by collision-induced dissociation. Of interest was the potential influence of the arginine residue, and its position in the peptide sequence, on formation of the presumed macrocyclic b₅ ion isomer and potential loss of sequence information. We find that regardless of the sequence position (either internal or at the N- or C-terminus), only direct sequence ions or ions directly related to fragmentation of the arginine side chain are observed.     

Competing fragmentation processes in tandem mass spectra of heparin-like glycosaminoglycans

Heparin-like glycosaminoglycans (HLGAGs) are highly sulfated, linear carbohydrates attached to proteoglycan core proteins and expressed on cell surfaces and in basement membranes. These carbohydrates bind several families of growth factors and growth factor receptors and act as coreceptors for these molecules. Tandem mass spectrometry has the potential to increase our understanding of the biological significance of HLGAG expression by providing a facile means for sequencing these molecules without the need for time-consuming total purification. The challenge for tandem mass spectrometric analysis of HLGAGs is to produce abundant ions derived via glycosidic bond cleavages while minimizing the abundances of ions produced from elimination of the fragile sulfate groups. This work describes the competing fragmentation pathways that result from dissociation of high negative charge state ions generated from HLGAGs. Glycosidic bond cleavage ion formation competes with losses of equivalents of H2SO4, resulting in complex ion patterns. For the most highly sulfated structure examined, an octasulfated tetramer, an unusual loss of charge from the precursor ion was observed, accompanied by low abundance ions originating from subsequent backbone cleavages. These results demonstrate that fragmentation processes competing with glycosidic bond cleavages are more favored for highly sulfated HLGAG ions. In conclusion, reduction of charge-charge repulsions, such as is achieved by pairing the HLGAG ions with metal cations, is necessary in order to minimize the abundances of ions derived via fragmentation processes that compete with glycosidic bond cleavages.     

Differentiation of diastereomeric conduramine derivatives under electron ionization and chemical ionization mass spectral conditions

Diastereomeric conduramine derivatives, i.e., (1R,2S,3R/S,6S)-6-(N-carbomethoxyamino) 1,2-O-isopropylidenecyclohex-4-ene-1,2,3-triol (1 and 2) and their O-acetyl derivatives (3 and 4), were studied using gas chromatography (GC) with electron ionization (EI) and chemical ionization (CI). The EI mass spectra of diastereomeric pairs show consistent differences in the relative abundances of characteristic ions. The EI fragmentation patterns are based on precursor/product ion spectra, high-resolution mass spectrometry (HRMS) and deuterium labelling. The CI spectra show differences from the EI spectra, and the isobutane/CI spectra are much simpler than the methane/CI spectra. The differences shown in the CI spectra are similar to those shown in the product ion spectra of [M+H](+) ions generated under electrospray ionization (ESI) conditions. Theoretical calculations are performed to understand the observed differences. The differences in the relative stabilities of molecular ions, or protonated molecules at different sites, can explain the observed differences in the spectra.