Structural characterization of glycerophospholipids by combinations of ozone- and collision-induced dissociation mass spectrometry: The next step towards "top-down" lipidomics

The complete structural elucidation of complex lipids, including glycerophospholipids, using only mass spectrometry represents a major challenge to contemporary analytical technologies. Here, we demonstrate that product ions arising from the collision-induced dissociation (CID) of the [M + Na] + adduct ions of phospholipids can be isolated and subjected to subsequent gas-phase ozonolysis-known as ozone-induced dissociation (OzID)-in a linear ion-trap mass spectrometer. The resulting CID/OzID experiment yields abundant product ions that are characteristic of the acyl substitution on the glycerol backbone (i.e., sn-position). This approach is shown to differentiate sn-positional isomers, such as the regioisomeric phosphatidylcholine pair of PC 16:0/18:1 and PC 18:1/16:0. Importantly, CID/OzID provides a sensitive diagnostic for the existence of an isomeric mixture in a given sample. This is of very high value for the analysis of tissue extracts since CID/OzID analyses can reveal changes in the relative abundance of isomeric constituents even within different tissues from the same animal. Finally, we demonstrate the ability to assign carbon-carbon double bond positions to individual acyl chains at specific backbone positions by adding subsequent CID and/or OzID steps to the workflow and that this can be achieved in a single step using a hybrid triple quadrupole-linear ion trap mass spectrometer. This unique approach represents the most complete and specific structural analysis of lipids by mass spectrometry demonstrated to date and is a significant step towards comprehensive top-down lipidomics. This journal is © The Royal Society of Chemistry 2014. Grant Number ARC/DP0986628, ARC/FT110100249, ARC/LP110200648

Ozone-induced dissociation : Elucidation of double bond position within mass-selected lipid ions

Ions formed from lipids during electrospray ionization of crude lipid extracts have been mass-selected within a quadrupole linear ion trap mass spectrometer and allowed to react with ozone vapor. Gas-phase ion-molecule reactions between unsaturated lipid ions and ozone are found to yield two primary product ions for each carbon-carbon double bond within the molecule. The mass-to-charge ratios of these chemically induced fragments are diagnostic of the position of unsaturation within the precursor ion. This novel analytical technique, dubbed ozone-induced dissociation (OzID), can be applied both in series and in parallel with conventional collision-induced dissociation (CID) to provide near-complete structural assignment of unknown lipids within complex mixtures without prior fractionation or derivatization. In this study, OzID is applied to a suite of complex lipid extracts from sources including human lens, bovine kidney, and commercial olive oil, thus demonstrating the technique to be applicable to a broad range of lipid classes including both neutral and acidic glycerophospholipids, sphingomyelins, and triacylglycerols. Gas-phase ozonolysis reactions are also observed with different types of precursor ions including \[M + H](+), \[M + Li](+), \[M + Na](+), and \[M H](-): in each case yielding fragmentation data that allow double bond position to be unambiguously assigned. Within the human lens lipid extract, three sphingomyelin regioisomers, namely SM(d18:0/15Z-24:1), SM(d18:0/17Z-24:1), and SM(d18:0/19Z-24:1), and a novel phosphatidylethanolamine alkyl ether, GPEtn(11Z-18:1e/9Z18:1), are identified using a combination of CID and OzID. These discoveries demonstrate that lipid identification based on CID alone belies the natural structural diversity in lipid biochemistry and illustrate the potential of OzID as a complementary approach within automated, high-throughput lipid analysis protocols.

Analysis of unsaturated lipids by ozone-induced dissociation

Recent developments in analytical technologies have driven significant advances in lipid science. The sensitivity and selectivity of modern mass spectrometers can now provide for the detection and even quantification of many hundreds of lipids in a single analysis. In parallel, increasing evidence from structural biology suggests that a detailed knowledge of lipid molecular structure including carbon-carbon double bond position, stereochemistry and acyl chain regiochemistry is required to fully appreciate the biochemical role(s) of individual lipids. Here we review the capabilities and limitations of tandem mass spectrometry to provide this level of structural specificity in the analysis of lipids present in complex biological extracts. In particular, we focus on the capabilities of a novel technology termed ozone-induced dissociation to identify the position (s) of double bonds in unsaturated lipids and discuss its possible role in efforts to develop workflows that provide for complete structure elucidation of lipids by mass spectrometry alone: so-called top-down lipidomics. This article is part of a Special Issue entitled: Lipodomics and Imaging Mass Spectrom. (C) 2011 Elsevier B.V. All rights reserved.

Ozone-Induced Dissociation on a Modified Tandem Linear Ion-Trap : Observations of Different Reactivity for Isomeric Lipids

Ozone-induced dissociation (OzID) exploits the gas-phase reaction between mass-selected lipid ions and ozone vapor to determine the position(s) of unsaturation In this contribution, we describe the modification of a tandem linear ion-trap mass spectrometer specifically for OzID analyses wherein ozone vapor is supplied to the collision cell This instrumental configuration provides spatial separation between mass-selection, the ozonolysis reaction, and mass-analysis steps in the OzID process and thus delivers significant enhancements in speed and sensitivity (ca 30-fold) These improvements allow spectra revealing the double-bond position(s) within unsaturated lipids to be acquired within 1 s significantly enhancing the utility of OzID in high-throughput lipidomic protocols The stable ozone concentration afforded by this modified instrument also allows direct comparison of relative reactivity of isomeric lipids and reveals reactivity trends related to (1) double-bond position, (2) substitution position on the glycerol backbone, and (3) stereochemistry For cis- and trans-isomers, differences were also observed in the branching ratio of product ions arising from the gas-phase ozonolysis reaction, suggesting that relative ion abundances could be exploited as markers for double-bond geometry Additional activation energy applied to mass-selected lipid ions during injection into the collision cell (with ozone present) was found to yield spectra containing both OzID and classical-CID fragment ions This combination CID-OzID acquisition on an ostensibly simple monounsaturated phosphatidylcholine within a cow brain lipid extract provided evidence for up to four structurally distinct phospholipids differing in both double-bond position and sn-substitution U Am Soc Mass Spectrom 2010, 21, 1989-1999) (C) 2010 American Society for Mass Spectrometry

Ozone-induced dissociation of conjugated lipids reveals significant reaction rate enhancements and characteristic odd-electron product ions

Ozone-induced dissociation (OzID) is an alternative ion activation method that relies on the gas phase ion-molecule reaction between a mass-selected target ion and ozone in an ion trap mass spectrometer. Herein, we evaluated the performance of OzID for both the structural elucidation and selective detection of conjugated carbon-carbon double bond motifs within lipids. The relative reactivity trends for \[M + X](+) ions (where X = Li, Na, K) formed via electrospray ionization (ESI) of conjugated versus nonconjugated fatty acid methyl esters (FAMEs) were examined using two different OzID-enabled linear ion-trap mass spectrometers. Compared with nonconjugated analogues, FAMEs derived from conjugated linoleic acids were found to react up to 200 times faster and to yield characteristic radical cations. The significantly enhanced reactivity of conjugated isomers means that OzID product ions can be observed without invoking a reaction delay in the experimental sequence (i.e., trapping of ions in the presence of ozone is not required). This possibility has been exploited to undertake neutral-loss scans on a triple quadrupole mass spectrometer targeting characteristic OzID transitions. Such analyses reveal the presence of conjugated double bonds in lipids extracted from selected foodstuffs. Finally, by benchmarking of the absolute ozone concentration inside the ion trap, second order rate constants for the gas phase reactions between unsaturated organic ions and ozone were obtained. These results demonstrate a significant influence of the adducting metal on reaction rate constants in the fashion Li > Na > K.

Differentiation of complex lipid isomers by radical-directed dissociation mass spectrometry

Contemporary lipidomics protocols are dependent on conventional tandem mass spectrometry for lipid identification. This approach is extremely powerful for determining lipid class and identifying the number of carbons and the degree of unsaturation of any acyl-chain substituents. Such analyses are however, blind to isomeric variants arising from different carbon carbon bonding motifs within these chains including double bond position, chain branching, and cyclic structures. This limitation arises from the fact that conventional, low energy collision-induced dissociation of even-electron lipid ions does not give rise to product ions from intrachain fragmentation of the fatty acyl moieties. To overcome this limitation, we have applied radical-directed dissociation (RDD) to the study of lipids for the first time. In this approach, bifunctional molecules that contain a photocaged radical initiator and a lipid-adducting group, such as 4-iodoaniline and 4-iodobenzoic acid, are used to form noncovalent complexes (i.e., adduct ions) with a lipid during electrospray ionization. Laser irradiation of these complexes at UV wavelengths (266 nm) cleaves the carbon iodine bond to liberate a highly reactive phenyl radical. Subsequent activation of the nascent radical ions results in RDD with significant intrachain fragmentation of acyl moieties. This approach provides diagnostic fragments that are associated with the double bond position and the positions of chain branching in glycerophospholipids, sphingomyelins and triacylglycerols and thus can be used to differentiate isomeric lipids differing only in such motifs. RDD is demonstrated for well-defined lipid standards and also reveals lipid structural diversity in olive oil and human very-low density lipoprotein.

The collision induced loss of carbon monoxide from deprotonated benzyl benzoate in the gas phase. An anionic 1,2-Wittig type rearrangement

The ion PhCO2--CHPh, upon collision activation, undergoes competitive losses of CO and CO2 of which the former process produces the base peak of the spectrum. Product ion and substituent effect (Hammett) studies indicate that PhCO2--CHPh cyclises to a deprotonated hydroxydiphenyloxirane which ring opens to PhCOCH(O-)Ph. This anion then undergoes an anionic 1,2-Wittig type rearrangement {through [PhCO- (PhCHO)]} to form Ph2CHO- and CO. The mechanism of the 1,2-rearrangement has been probed by an ab initio study [at MP4(SDTQ)/6-31++G(d,p) level] of the model system HCOCH2O- →; MeO- + CO The analogous system RCO2--CHPh (R = alkyl) similarly loses CO, and the migratory aptitudes of the alkyl R groups in this reaction are Bu′ > Me > Et ∼Pri). This trend correlates with the order of anion basicities (i.e. the order of ΔG○acid values of RH), supporting the operation of an anion migration process. The loss of CO2 from PhCO2--CHPh yields Ph2CH- as the anionic product: several mechanistic scenarios are possible, one of which involves an initial ipso nucleophilic substitution.

Advances in mass spectrometry for lipidomics

Recent expansion in research in the field of lipidomics has been driven by the development of new mass spectrometric tools and protocols for the identification and quantification of molecular lipids in complex matrices. Although there are similarities between the field of lipidomics and the allied field of mass spectrometry (e.g., proteomics), lipids present some unique advantages and challenges for mass spectrometric analysis. The application of electrospray ionization to crude lipid extracts without prior fractionation-the so-called shotgun approach-is one such example, as it has perhaps been more successfully applied in lipidomics than in any other discipline. Conversely, the diverse molecular structure of lipids means that collision-induced dissociation alone may be limited in providing unique descriptions of complex lipid structures, and the development of additional, complementary tools for ion activation and analysis is required to overcome these challenges. In this article, we discuss the state of the art in lipid mass spectrometry and highlight several areas in which current approaches are deficient and further innovation is required.

Does Addition of NO2 to Carbon-Centered Radicals Yield RONO or RNO2? An Investigation Using Distonic Radical Ions

Nitrogen dioxide is used as a "radical scavenger" to probe the position of carbon-centered radicals within complex radical ions in the gas phase. As with analogous neutral radical reactions, this addition results in formation of an \[M + NO2](+) adduct, but the structural identity of this species remains ambiguous. Specifically, the question remains: do such adducts have a nitro-(RNO2) or nitrosoxy-(RONO) moiety, or are both isomers present in the adduct population? In order to elucidate the products of such reactions, we have prepared and isolated three distonic phenyl radical cations and observed their reactions with nitrogen dioxide in the gas phase by ion-trap mass spectrometry. In each case, stabilized \[M + NO2](+) adduct ions are observed and isolated. The structure of these adducts is probed by collision-induced dissociation and ultraviolet photodissociation action spectroscopy and a comparison made to the analogous spectra of authentic nitro-and nitrosoxy-benzenes. We demonstrate unequivocally that for the phenyl radical cations studied here, all stabilized \[M + NO2](+) adducts are exclusively nitrobenzenes. Electronic structure calculations support these mass spectrometric observations and suggest that, under low-pressure conditions, the nitrosoxy-isomer is unlikely to be isolated from the reaction of an alkyl or aryl radical with NO2. The combined experimental and theoretical results lead to the prediction that stabilization of the nitrosoxy-isomer will only be possible for systems wherein the energy required for dissociation of the RO-NO bond (or other low energy fragmentation channels) rises close to, or above, the energy of the separated reactants.

Rapid differentiation of isomeric lipids by photodissociation mass spectrometry of fatty acid derivatives

RATIONALE Both traditional electron ionization and electrospray ionization tandem mass spectrometry have demonstrated limitations in the unambiguous identification of fatty acids. In the former case, high electron energies lead to extensive dissociation of the radical cations from which little specific structural information can be obtained. In the latter, conventional collision-induced dissociation (CID) of even-electron ions provides little intra-chain fragmentation and thus few structural diagnostics. New approaches that harness the desirable features of both methods, namely radical-driven dissociation with discrete energy deposition, are thus required. METHODS Herein we describe the derivatization of a structurally diverse suite of fatty acids as 4-iodobenzyl esters (FAIBE). Electrospray ionization of these derivatives in the presence of sodium acetate yields abundant [M+Na]+ ions that can be mass-selected and subjected to laser irradiation (=266nm) on a modified linear ion-trap mass spectrometer. RESULTS Photodissociation (PD) of the FAIBE derivatives yields abundant radical cations by loss of atomic iodine and in several cases selective dissociation of activated carboncarbon bonds (e.g., at allylic positions) are also observed. Subsequent CID of the [M+NaI]center dot+ radical cations yields radical-directed dissociation (RDD) mass spectra that reveal extensive carboncarbon bond dissociation without scrambling of molecular information. CONCLUSIONS Both PD and RDD spectra obtained from derivatized fatty acids provide a wealth of structural information including the position(s) of unsaturation, chain-branching and hydroxylation. The structural information obtained by this approach, in particular the ability to rapidly differentiate isomeric lipids, represents a useful addition to the lipidomics tool box. Copyright (c) 2013 John Wiley & Sons, Ltd.

Hydroxyl radical formation in the gas phase oxidation of distonic 2-methylphenyl radical cations

The reactions of distonic 4-(N, N, N-trimethylammonium)-2-methylphenyl and 5-(N, N, N-trimethylammonium)-2-methylphenyl radical cations (m/z 149) with O-2 are studied in the gas phase using ion-trap mass spectrometry. Photodissociation (PD) of halogenated precursors gives rise to the target distonic charge-tagged methylphenyl radical whereas collision-induced dissociation (CID) is found to produce unreactive radical ions. The PD generated distonic radicals, however, react rapidly with O-2 to form \[M + O2](center dot+) and \[M + O-2 - OH](center dot+) ions, detected at m/z 181 and m/z 164, respectively. Quantum chemical calculations using G3SX(MP3) and M06-2X theories are deployed to examine key decomposition pathways of the 5-(N, N, N-trimethylammonium)-2-methylphenylperoxyl radical and rationalise the observed product ions. The prevailing product mechanism involves a 1,5- H shift in the peroxyl radical forming a QOOH-type intermediate that subsequently eliminates (OH)-O-center dot to yield charge-tagged 2-quinone methide. Our study suggests that the analogous process should occur for the neutral methylphenyl + O-2 reaction, thus serving as a plausible source of (OH)-O-center dot radicals in combustion environments. Grants: ARC/DP0986738, ARC/DP130100862

Formation and Fragmentation of Unsaturated Fatty Acid M-2H+Na (-) Ions: Stabilized Carbanions for Charge-Directed Fragmentation

Fatty acids are long-chain carboxylic acids that readily produce \[M - H](-) ions upon negative ion electrospray ionization (ESI) and cationic complexes with alkali, alkaline earth, and transition metals in positive ion ESI. In contrast, only one anionic monomeric fatty acid-metal ion complex has been reported in the literature, namely \[M - 2H + (FeCl)-Cl-II](-). In this manuscript, we present two methods to form anionic unsaturated fatty acid-sodium ion complexes (i.e., \[M - 2H + Na](-)). We find that these ions may be generated efficiently by two distinct methods: (1) negative ion ESI of a methanolic solution containing the fatty acid and sodium fluoride forming an \[M - H + NaF](-) ion. Subsequent collision-induced dissociation (CID) results in the desired \[M - 2H + Na](-) ion via the neutral loss of HF. (2) Direct formation of the \[M - 2H + Na](-) ion by negative ion ESI of a methanolic solution containing the fatty acid and sodium hydroxide or bicarbonate. In addition to deprotonation of the carboxylic acid moiety, formation of \[M - 2H + Na](-) ions requires the removal of a proton from the fatty acid acyl chain. We propose that this deprotonation occurs at the bis-allylic position(s) of polyunsaturated fatty acids resulting in the formation of a resonance-stabilized carbanion. This proposal is supported by ab initio calculations, which reveal that removal of a proton from the bis-allylic position, followed by neutral loss of HX (where X = F- and -OH), is the lowest energy dissociation pathway.

Identification of abundant alkyl ether glycerophospholipids in the human lens by tandem mass spectrometry techniques

Previous studies have shown that the human lens contains glycerophospholipids with ether linkages. These lipids differ from conventional glycerophospholipids in that the sn-1 substituent is attached to the glycerol backbone via an 1-O-alkyl or an 1-O-alk-1'-enyl ether rather than an ester bond. The present investigation employed a combination of collision-induced dissociation (CID) and ozone-induced dissociation (OzID) to unambiguously distinguish such 1-O-alkyl and 1-O-alk-1'-enyl ethers. Using these methodologies the human lens was found to contain several abundant 1-O-alkyl glycerophos-phoethanolamines, including GPEtn(16:0e/9Z-18:1), GPEtn(11Z-18:1e/9Z-18:1), and GPEtn(18:0e/9Z-18:1), as well as a related series of unusual 1-O-alkyl glycerophosphoserines, including GPSer(16:0e/9Z-18:1), GPSer(11Z-18:1e/9Z-18:1), GPSer(18:0e/9Z-18:1) that to our knowledge have not previously been observed in human tissue. Isomeric 1-O-alk-1'-enyl ethers were absent or in low abundance. Examination of the double bond position within the phospholipids using OzID revealed that several positional isomers were present, including sites of unsaturation at the n-9, n-7, and even n-5 positions. Tandem CID/OzID experiments revealed a preference for double bonds in the n-7 position of 1-O-ether linked chains, while n-9 double bonds predominated in the ester-linked fatty acids [e.g., GPEtn(11Z-18:1e/9Z-18:1) and GPSer(11Z-18:1e/9Z-18:1)]. Different combinations of these double bond positional isomers within chains at the sn-1 and sn-2 positions point to a remarkable molecular diversity of ether-lipids within the human lens.

Ion-molecule reactions reveal facile radical migration in peptides

Ion-molecule reactions between molecular oxygen and peptide radicals in the gas phase demonstrate that radical migration occurs easily within large biomolecules without addition of collisional activation energy.