Chemistry and Physics of Lipids 156 (2008) 1–12

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Chemistry and Physics of Lipids

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Review

Mass spectrometry analysis of oxidized phospholipids

M. Rosário M. Domingues ∗, Ana Reis, Pedro DominguesMass Spectrometry Centre, Department of Chemistry, University of Aveiro, Campus Santiago, 3810-193 Aveiro, Portugal

a r t i c l e i n f o

Article history:Received 7 April 2008Received in revised form 24 June 2008Accepted 2 July 2008Available online 11 July 2008

Keywords:PhospholipidsOxidation

a b s t r a c t

The evidence that oxidized phospholipids play a role in signaling, apoptotic events and in age-relateddiseases is responsible for the increasing interest for the study of this subject. Phospholipid changesinduced by oxidative reactions yield a huge number of structurally different oxidation products whichdifficult their isolation and characterization. Mass spectrometry (MS), and tandem mass spectrometry(MS/MS) using the soft ionization methods (electrospray and matrix-assisted laser desorption ionization)is one of the finest approaches for the study of oxidized phospholipids. Product ions in tandem mass spectraof oxidized phospholipids, allow identifying changes in the fatty acyl chain and specific features such aspresence of new functional groups in the molecule and their location along the fatty acyl chain. This review

Mass spectrometryTandem mass spectrometryEM

describes the work published on the use of mass spectrometry in identifying oxidized phospholipids fromthe different classes.

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lectrosprayALDI

© 2008 Elsevier Ireland Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.1. Biological activity of oxidized glycerophospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2. The nature of oxidized glycerophospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.3. Mass spectrometry analysis of oxidized phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2. Characterization of oxidized phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.1. Glycerophosphocholines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.1.1. Characteristic fragmentation of PC under MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.1.2. Identification of short chain oxidation products of PC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.1.3. Identification of long-chain oxidation products of PC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.1.4. Identification of PC adducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2.2. Glycerophosphoethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72.3. Glycerophosphoserine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2.4. Cardiolipin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.5. Other phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: CL, cardiolipin; ESI, electrospray ionization; LC, liquid chromatographmyeloperoxidase; MS, mass spectrometry; MS/MS, tandem mass spectrometry; PC, phosPI, glycerophosphatidylinositol; PS, glycerophosphoserine; SM, Sphingomyelins.∗ Corresponding author. Tel.: +351 234 370 698; fax: +351 234 370 084.

E-mail address: mrd@ua.pt (M.R.M. Domingues).

009-3084/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.chemphyslip.2008.07.003

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

y; LOX, lipoxygenase; MALDI, matrix-assisted laser desorption ionization; MPO,phatidylcholines or glycerophosphocholines; PE, glycerophosphoethanolamine;

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M.R.M. Domingues et al. / Chemistr

. Introduction

.1. Biological activity of oxidized glycerophospholipids

Phospholipids are the major lipid constituents of cell mem-ranes and lipoproteins and play different roles in biologicalystems such as fuels, signaling agents or surfactants. Oxidativetress causes changes in a diversity of native phospholipids found iniving beings. These changes result in a vast number of structurallyifferent oxidation products, which may have different biologicalctivities (reviewed in Fruhwirth et al., 2007; Spickett and Dever,005). The biological role of these oxidized species depends notnly on the location, but also on the nature of the changes. Oxidizedhospholipids are the result of a series of radical catalyzed chem-

cal reactions and processes physiopathological roles in diseaseevelopment as in age-related and chronic diseases, atheroscle-osis, inflammation and immune response (Fruhwirth et al., 2007;eitinger, 2003, 2005; Subbanagounder et al., 2002a; Spickett andever, 2005; Niki et al., 2005). For example, oxidized phospho-

ipids induce platelet aggregation through activating the receptoror platelet-activating factor (PAF) (Subbanagounder et al., 1999;ndroulakis et al., 2005; Gopfert et al., 2005). Oxidized phospho-

ipids induce monocyte adhesion to endothelial cells, accumulaten atherosclerotic lesions, and play a role in inflammation and sig-aling inflammatory response (Cole et al., 2003; Pegorier et al.,006; Subbanagounder et al., 2002b; Zhang and Salomon, 2005).lso, bioactive phospholipid oxidation products may present anti-

nflammatory properties, playing an important role in modulationf the inflammatory process (Bochkov and Leitinger, 2003; Nonast al., 2006; Erridge and Spickett, 2007). In addition, the presence ofxidized phospholipids in biological membranes induces changesn physical properties such as fluidity (Borst et al., 2000) and acylacking (Megli et al., 2005; Megli and Sabatini, 2004; Sabatini etl., 2006). This can have an impact on the integrity of the mem-rane, causing apoptotic events (Fruhwirth et al., 2007; Megli andusso, 2008). The biological activity of oxidized glycerophospho-

ipids has been reviewed elsewhere (Leitinger, 2005; Spickett andever, 2005; Spiteller, 2006; Fruhwirth et al., 2007; Bochkov, 2007)nd will not be further discussed in this review.

.2. The nature of oxidized glycerophospholipids

Phospholipids are biomolecules that contain one or more phos-hate groups and have amphipathic properties. There are twoajor classes of phospholipids, one with a glycerol backbone

glycerophospholipids) and the other with a sphingosine groupsphingolipids). Glycerophospholipids are a class of phospholipidsith a phosphatidyl group linked to a glycerol molecule, which

s substituted (through ester or ether linkages) by two fattycids (sn-1 and sn-2 acyl chains). Depending on the alkyl chainsttached to the phosphatidyl group, they can be classified as glyc-rophosphatidylcholines (PC), glycerophosphatidylethanolaminesPE), glycerophosphatidylserines (PS) and glycerophosphatidyli-ositols (PI) (Yorek, 1993). The sphingolipids contain sphingosine,long-chain amino alcohol etherified with a fatty acid, which are

lassified in sphingomyelins and glycosphingolipids (cerebrosides,ulfatides, globosides and gangliosides). Sphingomyelins (SM) arehe only sphingolipids classified as phospholipids and have a phos-hatidyl group linked to the sphingosine molecule. CardiolipinsCL) are complex glycerophospholipids, having two diacylphos-

hatidylglycerol molecules linked by a third glycerol unit, and areharacteristic of mitochondria membrane. Phospholipids from theholine class (PC and SM) are the most abundant in mammalsbetween 40 and 80%, dry weight of the cell membrane), followedy PE and all the remaining classes (Yorek, 1993). Among these, the

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Physics of Lipids 156 (2008) 1–12

iacyl cholines prevail in most tissue membranes, while the cholinend ethanolamine plasmalogen (alkenyl chains) predominate in therain (Yorek, 1993).

The unsaturated fatty acid chains present in phospholipids arehe main targets of oxidation (as reviewed by Spiteller, 2006; Niki etl., 2005). In biological systems, these changes may occur throughadical and nonradical reactions involving enzymatic (LOX, MPO) oronenzymatic systems (•OOH, •OH, Fe2+, Cu+, radiation) (Fruhwirtht al., 2007). Depending on the predominating oxidative process,he phospholipid oxidation products formed can be different, ashown in previous studies (Niki et al., 2005; Spiteller, 2006). Oxi-ation reactions involving phospholipids produces a wide varietyf compounds that can be classified according to the nature ofhe modifications: (i) long-chain products, which are products thatreserve the phospholipid skeleton; (ii) short chain or truncatedroducts, formed by cleavage of the fatty acyl chains (unsaturated

atty acid); and (iii) adducts, formed by reaction between oxida-ion products and/or molecules containing nucleophilic groups.hospholipid adducts include the products usually formed byross-linking reactions between phospholipid oxidation productsith carbonyl groups and amino groups present in neighboring

iomolecules, such as peptides, proteins and ethanolamine phos-holipids (PE) (Hoff et al., 2003; Reis et al., 2006; Bacot et al.,003; Zamora and Hidalgo, 2003; Sayre et al., 2006). In Scheme 1,e show, as an example of all these structures, changes that are

bserved on lineloyl–palmitoyl-containing phospholipid.

.3. Mass spectrometry analysis of oxidized phospholipids

Mass spectrometry is increasingly being employed to identifyhe structure of biomolecules, especially of proteins (proteomic)nd lipids (lipidomic) and metabolism assessment (metabolomic),rom in vivo samples. This is mostly as the result of the discoveryf soft ionization techniques, such as electrospray ionization (ESI)Yamashita and Fenn, 1984) and matrix-assisted laser desorptiononization (MALDI (Tanaka et al., 1988; Karas and Hillenkamp, 1988)ver 20 years ago. Also, the design of new more robust instrumentsnd user-friendly software that allow multiple data processing andnalysis has enabled mass spectrometry as an accessible techniquen the research of biomolecules in general. Briefly, organic masspectrometry is an analytical technique which allows measuringhe molecular weight and relative abundances of an analyte. Inddition, generally by using tandem mass spectrometry, structuralnformation of molecules can also be obtained. The most com-

on ionization methods used in the analysis of phospholipids arelectrospray ionization (ESI) and matrix-assisted laser desorptiononization (MALDI). These are soft ionization methods, producinglmost no fragmentation, which allow the ionization of non-volatilend thermolabile samples. Usually, this is an advantage, especiallyn the analysis of mixtures, but only information about the molecu-ar weigh of the compounds can be obtained. However, it is possibleo induce the fragmentation of the sample ions, commonly by usingollision induced dissociation (CID). In this technique called tan-em mass spectrometry, MS/MS, fragmentation of a selected ion byollision with an inert gas is induced and the product ions are ana-yzed. Ion trap mass spectrometers and FT-ICR mass spectrometersllow performing sequential selection and fragmentation of ions,hus enabling multistage tandem mass spectrometry (MSn). Highensitivity analysis, usually in the fmol range, of complex sampleixtures and the capacity of coupling with separation techniques,

amely liquid chromatography (LC) are other advantages of thisechnique. It is beyond the scope of this paper to give a compre-ensive outline of the mass spectrometry fundaments.

Encompassing this emergent trend, mass spectrometry isecoming increasingly important in phospholipid oxidation

M.R.M. Domingues et al. / Chemistry and Physics of Lipids 156 (2008) 1–12 3

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Scheme 1. Oxidative mo

esearch. One of the main reasons for this, other than the increasingpread of mass spectrometers, is that interpreting mass spectra ofxidized phospholipids is usually straightforward. Mass spectra ofxidized phospholipids can be divided into three different regions:ong-chain products region, which show a higher m/z value than theative phospholipid, and include the products resulting from inser-ion of oxygen and hypohalous acid (HOX, X = Cl or Br) moleculesuch as hydroxy-, epoxy-, keto-, hydroperoxy-, polyhydroxy- andalohydrin derivatives; Short chain products region, with a lower/z value than the native phospholipid and result from cleavage

f the unsaturated fatty acyl chain, includes carbonyl productsuch as aldehydes, (keto)hydroxy-aldehydes, and carboxylic acidsketo)hydroxy-carboxylic acids esterified to the glycerol group andther products that are the result of saponification reactions ofhe fatty acyl chains, leading to the lyso-glycerophospholipids andalohydrin lyso-glycerophospholipid; phospholipid adducts regionhow products formed by reaction between oxidation productsnd/or molecules containing nucleophilic functional groups.

Early work published on the use of mass spectrometry for thenalysis of oxidized phospholipids dates from the end of last cen-ury. These studies were done using as ionization methods fasttom bombardment (FAB) (Kayganich-Harrison and Murphy, 1994;acino et al., 1996) or thermospray (TS) (Zhang et al., 1995). Sincehe introduction of electrospray as an ionization method, thisas become the method of choice for identifying oxidized phos-holipids. Only few studies describe the use of matrix-assisted

aser desorption (MALDI) as the ionization method, most likelyecause of the presence of matrix clusters ions, in the low massange of MALDI–MS spectra, between 100 and 800 Da. These ionsinder phospholipid ions and difficults the analysis and screen-

ng of oxidized phospholipids (Schiller et al., 2004). Traditionally,

1smfb

tions in phospholipids.

uantification of phospholipids and oxidized phospholipids iserformed using GC–MS or HPLC, usually after derivatization.lthough quantification of oxidized phospholipids has been done

n underivatized samples using mass spectrometry (Podrez et al.,002b), this subject is beyond the objective of this review. Detec-ion and identification of oxidative modifications in phospholipidssing mass spectrometry has been used mainly for the study of PChospholipids, and only few papers report the oxidation in PE, PSnd CL. So far, oxidized sphingomyelins and inositol phospholipidsave not been studied using mass spectrometry.

. Characterization of oxidized phospholipids

.1. Glycerophosphocholines

Glycerophosphocholines (PC) are the most studied class of oxi-ized phospholipids by mass spectrometry (Postle et al., 2007;pickett and Dever, 2005). Most of the published work that focusedn the investigation of oxidized PC use ESI as ionization method andC standards oxidized through either enzymatic or nonenzymaticystems. The PC standards used in these studies usually containne saturated fatty acid chain (palmitoyl or stearoyl) in sn-1 andsecond unsaturated fatty acid (oleoyl, linoleoyl, arachidonoyl orocosahexanoyl) located in sn-2. In fact, more than 90% of the unsat-rated fatty acid content in phospholipids of most mammalianissues have the composition 18:1, 18:2, 20:4 and 22:6 (Yorek,

993). Free radical oxidation of the unsaturated fatty acid chainstarts with the abstraction of a hydrogen atom from a bis-allylicethylene position in polyunsaturated fatty acid, or in less extend,

rom an allylic methylene group. The carbon–hydrogen bond ofis-allylic positions is weak (75 kcal/mol) when compared with

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M.R.M. Domingues et al. / Chemistr

ono-allylic carbon–hydrogen linkage (88 kcal/mol) (Wagner et al.,994) thus turning the bis-allylic position the preferential target ofeactive oxygen species. As a result, oxidation of a fatty acyl with aingle double bond, such as the case of oleic acid, is more difficulthen compared with polyunsaturated linoleic or arachidonic acid

xidation. Because of the lack of methylene groups, the saturatedatty acids are resistant to free radical oxidation, limiting changeso the sn-2 residue of the phospholipid. Several studies have used

ass spectrometry to identify oxidized phosphocholines in biolog-cal fluids (Zhang et al., 1995; Nakamura et al., 1997; Nakamura etl., 1998; Harrison et al., 2000; Marathe et al., 2000; Hoff et al.,003; Jiang et al., 2004; Adachi et al., 2006; Milne et al., 2005) andissues (Hoff et al., 2003; Adachi et al., 2005). In most of the pub-ished work, the m/z value observed in the mass spectra was thenly information used to infer on the nature of the oxidized speciesnd were consequently only described as short or long oxidationroducts. However, more information is obtainable by analysis ofroduct ion mass spectra (MS/MS).

.1.1. Characteristic fragmentation of PC under MS/MSIonization of glycerophosphocholines by ESI or MALDI, pro-

uces protonated molecules, [MH]+, or cationized molecules, suchs [MNa]+. The fragmentation pathway observed in tandem masspectra (MS/MS) of PC ions depends on the selected precursor ion.he MS/MS mass spectra of the [MH]+ ions are simple and of easynterpretation, while the MS/MS spectra of the [MNa]+ ions showigher number of product ions (Jensen et al., 1986; Domingues etl., 1998; Domingues et al., 2001; Pulfer and Murphy, 2003), but notecessarily more informative.

The product ion spectra of [MH]+ ions show an abun-ant product ion at m/z 184 (base peak) which is the polaread [H2PO4(CH2)2N(CH3)3]+. Other product ions with low rel-tive abundance (<5%) include those formed from loss ofPO4(CH2)2N(CH3)3 (183 Da) and loss of fatty acyl chains placed at

n-1 (R1COOH and R1 C O) and sn-2 (R2COOH and R2 C O) (Hsund Turk, 2003). The product ion spectra of [MNa]+ ions show lossf trimethylamine (59 Da), loss of polar head HPO4(CH2)2N(CH3)3−183 Da), loss of sodiated polar head NaPO4(CH2)2N(CH3)3−205 Da), loss of fatty acyl chains placed at sn-1 (R1COONa and1COOH) and sn-2 (R2COONa and R2COOH), the product ion at/z 147 (cyclophosphane group), and acyl product ions R1CO+

nd R2CO+ (Han and Gross, 1995; Domingues et al., 1998; Hsund Turk, 2003). PC phospholipids have a positive charge in theitrogen and thus there are few mass spectrometry studies donen the negative mode. Analysis in the negative mode showsegative ions [M−CH3]− and collision-induced decomposition ofM−15]− anions characterizes both the identity and substituentosition of radyl groups (Zirrolli et al., 1991; Pulfer and Murphy,003).

Tandem mass spectra of plasmalogen phosphocholines shows,esides the fragment ions involving loss of polar head previ-usly described for diacyl PC, fragment ions resultant from theoss of alkenyl or alkyl chains (sn-1 or sn-2 fatty acids) as alco-ols and ketene/acid (R1OH and R2 C O/R2COOH) (Hsu et al.,003; Hsu and Turk, 2007). The higher relative abundance of theroduct ions resultant from loss of sn-2 acyl chains (1-acyl-2-lyso-hosphatidylcholine), which is a more favorable fragmentationathway, allows identifying the relative position of fatty acids (Hsut al., 2003; Hsu and Turk, 2007; Pulfer and Murphy, 2003).

.1.2. Identification of short chain oxidation products of PCMass spectrometry in the MS mode was used to identify

xidized phosphocholines containing short chains with terminalarbonyl and carboxylic groups, including (un)saturated alde-ydes, (keto)hydroxy-aldehydes, (un)saturated carboxylic acids,

laiw2

Physics of Lipids 156 (2008) 1–12

keto)hydroxy-carboxylic acids and lyso-phospholipids (Itabe et al.,996; Frey et al., 2000; Ishida et al., 2004; Harrison et al., 2000;egli and Russo, 2008; Ravandi et al., 2004). Because of the lim-

ted number of possibilities for a given m/z value, this approachllows reliable identification of these oxidation products. Also, thenalysis of LC–MS data, which allows to separate isomeric shorthain products, enables correlating the retention times with theerminal functional group thus providing aid in identifying theseroducts (Hoff et al., 2003; Khaselev and Murphy, 2000b; Podrezt al., 2002b; Reis et al., 2005).

Short chain products were identified using tandem mass spec-rometry either in positive or negative modes or, in some studies,sing LC–MS/MS. However, most of the published work onlyeported the ion at m/z 184 for the [MH]+ ions as means of identify-ng the phospholipid class. The MS/MS spectra usually shows loss ofn-2 residues, and occasionally loss of sn-1 and loss of the polar head−183 Da) (Hoff et al., 2003; Reis et al., 2004a; Gopfert et al., 2005).ther product ions are formed by cleavages near the terminal groupf the short fatty acyl chain (Reis et al., 2004a). These product ionsnclude cleavage of the �-bond near the carboxylic acid and the �-ond near the aldehyde group, providing significant information.he presence of aldehydic and carboxylic acids with hydroxy andeto groups increases the fragmentation observed in ESI tandemass spectra. This allows identifying product ions resulting from

leavage of the �-bond near the hydroxy and of the �-bond nearhe keto group, and recognizing the substituted carbon atom in the

odified acyl chain (Reis et al., 2004a).MS/MS analysis of negative ions [M−CH3]− was used in identify-

ng short chain products with terminal aldehyde and carboxylic acidith C5 and C9 chain lengths, from arachidonic acid at sn-2. These

dentifications resulted from observing the product ions resultantrom loss of sn-2 fatty acyl chain, as ketene, and the carboxylatenions of the unmodified R1COO− and of the shortened R′

2COO−

atty acids (Kayganich-Harrison and Murphy, 1994; Harrison et al.,000; Marathe et al., 2000; Gopfert et al., 2005; Subbanagoundert al., 2002a). In oxidized phosphocholines with a terminal car-oxylic group, under collision induced dissociations, the transferf a methyl group from the trimethylamine group to the carboxy-ate can occur (Kayganich-Harrison and Murphy, 1994). Thus, lossf acyl residue is observed in the negative mode with a 14 Da massncrement. The loss of terminal aldehyde or carboxylic groups wasot observed in the fragmentation pattern of negative ions.

Phospholipid hydroxyalkenals (�-hydroxy, �, �-unsaturatedldehydes) were identified by LC–MS and LC–MS/MS analysis inoth positive and negative mode. These resulted from lipid per-xidation of arachidonate or lineolate at the sn-2 position in LDLHarrison et al., 2000; Hoff et al., 2003; Podrez et al., 2002a,b) andn endothelial cells (Subbanagounder et al., 2002a).

The oxidative modifications of plasmenylPC leads to shorthain products, originating sn-1 lysoPC, either because of lossf entire sn-1 alkenyl chain and formation of 1-formyl deriva-ives or oxidation in the neighboring of vinylic bond at sn-1Berry and Murphy, 2005). Other products with terminal alde-ydes and hydroxyalkenal or carboxylic terminal in sn-2, resultrom modifications on the sn-2 unsaturated fatty acyl (Berry and

urphy, 2005). These products were identified by ESI-MS/MSnd LC–ESI-MS/MS in negative mode, analyzing the fragmenta-ion pathway of the [M−CH3]− ion (Berry and Murphy, 2005;haselev and Murphy, 2000a). Fragmenting these short chainroducts by tandem mass spectrometry, produces an ion from

oss of modified sn-2 acyl chain as ketene (–R′2C = C = O) as well

s the sn-2 carboxylate R′2COO−. This is similar to the behav-

or of diacylPC negative ions which allowed identifying productsith terminal aldehyde and hydroxyalkenals (Berry and Murphy,

005).

y and Physics of Lipids 156 (2008) 1–12 5

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M.R.M. Domingues et al. / Chemistr

.1.3. Identification of long-chain oxidation products of PCLong-chain oxidation products include products resulting from

nsertion of oxygen atoms or hypohalous acid (HOX, X = Cl or Br)olecules, originating products with higher molecular weight such

s hydroxy-, epoxy-, keto-, hydroperoxy-, polyhydroxy- and halo-ydrin derivatives. The following section will discuss the mostommon oxidation methods used in mass spectrometry studies,nd the identification of long-chain PC oxidation products.

The first studies on PC oxidation products using mass spectrom-try, were performed interpreting negative tandem mass spectra ofxidized fatty acids obtained from saponification of oxidized phos-holipids (Nakamura et al., 1997, 1998; Hall and Murphy, 1998a,b;

nouye et al., 1999; Khaselev and Murphy, 2000a). However, thisethod needs sample manipulation and can difficult the iden-

ification. In this review we will focus on the direct analysis ofnderivatized oxidized PC by mass spectrometry.

Oxidation products formed by insertion of oxygen atoms weretudied using diverse oxidation methods. These include differ-nt nonenzymatic methods, like hydroxyl radical generated from2O2/Fe2+ or H2O2/Cu2+ systems, t-butylhydroperoxide or other

adical generators or, in aerobic conditions, by radiation, autox-dation, and oxidation promoted by presence of Cu2+. Oxidationf PC by enzymatic methods using lipoxygenase or myeloperoxi-ase to mimic in vivo conditions was also studied. This is a processhat occurs naturally mainly in phagocytes and neutrophils, whereyeloperoxidase reduces H2O2 to water while oxidizing various

ubstrates by the adding hypohalous acid. This occurs when in theresence of a halogen X, which is responsible for the generation ofOX.

.1.3.1. Oxidation promoted by the presence of hydrogen peroxide anderivatives. Long-chain PC oxidation products were identified byass spectrometry, using ESI-MS and LC–MS in the positive mode.

hese products were generated by radicalar process (hydroxyl rad-cal) in the presence of H2O2/Fe2+ or Cu2+ (Reis et al., 2004b,005, 2007; Megli and Russo, 2008) or t-butylhydroperoxideerivative (Spickett et al., 1998, 2001). These long-chain prod-cts are usually identified based on the observation of 16 Da mass

ncrements (n × 16 Da). Its presence in the mass spectra allowsroposing the presence of hydroxy (mono, di, tri or polyhydroxy),ydroperoxide (mono, di and tri hydroperoxy) derivatives as well asydroxy–peroxy derivatives (Spickett et al., 1998; Reis et al., 2005,007; Megli and Russo, 2008). Epoxy and keto derivatives were alsobserved in long-chain PC oxidation products (Reis et al., 2004b,005, 2007; Megli and Russo, 2008). FAB-MS and MS/MS in positiveode was used in identifying hydroxy, peroxy and hydroxy–peroxy

erivatives from soybean PCs after exposure to hydroxyl radicalFacino et al., 1996). Different type of oxidation products were alsodentified using data from ESI-MS/MS of [MH]+ or [MNa]+, andC–MS/MS of [MH]+ experiments (Reis et al., 2005, 2007). Mainroduct ions observed correspond to loss of neutral molecule ofhe polar head (183 Da), confirming that the oxidation product isf PC class. Loss of the fatty acids at the sn-1 and sn-2 positions,orming lyso-phosphocholines ions, allowed identifying the fattycyl chain that undergoes oxidative modifications. Other fragmentons, not observed in tandem mass spectra of unmodified PC, suchs loss of water (−H2O, −18 Da), loss of hydrogen peroxide (−H2O2,34 Da) or combine loss of two or three water molecules (−2 orH2O, −36 or 48 Da), have been used to propose the presence of

ydroxy, peroxy and di-hydroxy or polyhydroxy groups on oxidized

hosphocholines (Spickett et al., 2001; Adachi et al., 2004a,b, 2005;eis et al., 2004b, 2007). Loss of 34 Da is a typical fragmentation pat-ern of PC monohydroperoxides and was first noted by Adachi et al.,n PCOOH obtained from photoxidation (Adachi et al., 2004a). Otherroduct ions result mainly from C–C cleavages of the �-bond near

fits1T

ig. 1. Product ion spectrum (ESI-MS/MS) of the [MH]+ ion of the hydroxy deriva-ive of PC (16:0, 18:2). Cleavage in the vicinity of the hydroxy group produces theragment ions that allow the oxidation site to be assigned.

he functional group, involving hydrogen rearrangements. Theseragmentations occur away from the charge location (polar head)nd are named charge remote fragmentation in analogy with theypical fragmentation of the fatty acid chains (Cheng and Gross,000; Jensen and Gross, 1987). Also, the fragmentation pathwaysllowed inferring the carbon atoms where the oxidative modifica-ion occurred in the fatty acid chain. These were observed in eitherMH]+ and [MNa]+ ions of oxidized long-chain PC, namely PC (16:0,8:1), PC (16:0, 18:2) and PC (16:0, 20:4) (Reis et al., 2004b, 2007).igs. 1 and 2 show these informative pathways, using as an exam-le the hydroxy and peroxy or dihydroxy oxidation products of PC16:0, 18:2). Fig. 1 shows the ESI-MS/MS spectrum of the [MH]+ ionf the PC–OH, allowing to identify several isomers with the hydroxyroup placed at C9, C10, C12, and C13. Fig. 2 shows the (A) LC–MS/MSass spectra of the peroxy derivative of PC (16:0, 18:2), and the (B)ass spectrum of the dihydroxy derivative. We also show in this

gure the structures of the product ions that were used to assign

he position of the functional groups (Reis et al., 2007). The MS/MSpectra of [MNa]+ precursor ion of long-chain oxidized PC (16:0,8:1), PC (16:0, 18:2) and PC (16:0, 20:4) shows the ion at m/z 163.his fragment ion corresponds to the sodiated polar head with an

6 M.R.M. Domingues et al. / Chemistry and Physics of Lipids 156 (2008) 1–12

F e of Pv oxidat

o(

ms2otahvpAca(d(Ica

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ig. 2. Product ion spectra (LC–MS/MS) of the [MH]+ ions of the (A) peroxy derivativicinity of the peroxyl or hydroxy group produces the fragment ions that allow the

xygen, leading to conclude that oxidation of polar head occursReis et al., 2004b).

Separation and characterization of functional and structural iso-ers of OxPC (16:0, 18:2) by LC–MS/MS has been done, although,

eparation of the positional isomers was not feasible (Reis et al.,007). This approach allows a more accurate identification of thexidized PC. Difficulties exist in interpreting LC–tandem mass spec-ra of oxidized phosphocholines with a high number of oxygentoms because of the presence of unresolved components. Also,igher numbers of oxygen atoms present in OxPC leads to higherariability of oxidation products which increases the number ofroduct ions in the tandem mass spectra. Using an LC–MS/MS,dachi et al. identified in the plasma of alcoholic patients PC long-hain products, including monohydroperoxides, epoxyhydroxy, oxond trihydroxy derivatives of PC (16:0, 18:2) and PC (18:0, 18:2)Adachi et al., 2006). Using a similar approach monohydroxides, oxoerivatives, and trihydroxides of PC (16:0, 18:2), PC (18:0, 18:2). PC18:1, 18:2) were observed in rat heart (Adachi et al., 2004b, 2005).n these works, long-chain products of PC were identified using thehromatographic retention time and observing the losses of H2Ond/or HOOH in the tandem mass spectra.

PlasmenylPC oxidation induced by the AAPH radical generatoread to several oxidation products that were identified by nega-ive mode tandem mass spectrometry of the [M−CH3]− precursorons. The main oxidation products were lyso or short chain prod-cts. The long-chain products identified were hydroxy and peroxyerivatives with the functional groups at the vinylic carbon of sn-1hain (Khaselev and Murphy, 2000b; Murphy, 2001).

.1.3.2. Oxidation promoted by photooxidation/radiation. The firsteport on the study of PC photooxidation by mass spectrometryas performed using thermospray LC–MS (Zhang et al., 1995).

his allowed to identify monohydroperoxides of PC (18:0, 18:2)

i(P3

C (16:0, 18:2) and of the (B) dihydroxy derivative of PC (16:0, 18:2). Cleavage in theion site to be assigned.

nd PC (18:2, 18:2). Mass spectrometry (ESI-MS) of PC (16:0, 18:2)xidation product after exposition of UV radiation, allows the iden-ification of mono, di and tri-peroxy derivatives and trace amountsf hydroxy derivatives and PC-epoxyisoprostane (confirmed withntibodies). The authors of this report propose that UV irradia-ion originates specific oxidation products (Gruber et al., 2007).xidation by �-irradiation of PC (16:0, 18:2), and LC–MS analy-

is allows the identification of epoxy, hydroxy and peroxy (monond diperoxy and hydroxyperoxy) derivatives, although MS/MSharacterization was not done (Vitrac et al., 2004). Also, the anal-sis of plasmenylcholine photooxidation products by MALDI–MSllows identifying hydroxy and hydroperoxy derivatives as long-hain products (Thompson et al., 2003).

.1.3.3. Oxidation promoted by Cu2+. Long-chain productsbtained by Cu2+ oxidation of LDL particles were identifiedy LC–MS/MS in positive (Harrison et al., 2000) and negativeodes (Subbanagounder et al., 2002b). The identification of

xidation products formed by addition of one oxygen atom toC (16:0, 18:1) and PC (18:0, 18.1) from LDL was made observingn the precursor ion scan of the product ion of m/z 184 (cholineolar head) ions with 16 Da increments on the m/z values of

ntact PC (Harrison et al., 2000). Also, oxidation products of PC16:0, 20:4), from in vitro samples or extracted from oxidizedDL, were analyzed by LC–MS/MS in negative mode. This analysisllows to identifying epoxyisoprostane and epoxycyclopentenonehospholipids as the bioactive oxidation products of PC (16:0,0:4) (Subbanagounder et al., 2002b).

PC from soybean were oxidized by Cu2+, and analyzed by ESI-MSn positive and negative modes and by ESI-MS/MS in negative modeIshida et al., 2004). The identification of peroxides of PC (34:3) andC (34:2) was made observing in high resolution FTICR mass spectra2 Da increments on the intact PC. ESI-MS/MS of the [M−H]− ions

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M.R.M. Domingues et al. / Chemistr

llowed to identify the oxidation products as peroxy derivatives ofC (34:3) (C16:0 and C18:3-OOH) and PC (34:2) (C16:0 and C18:2-OH) (Ishida et al., 2004).

.1.3.4. Autoxidation. Hydroperoxides PC–OOH formed by PC (16:0,8.1) and PC (18:0, 20:4) autoxidation were analyzed by LC–MSnd structurally characterized by MS/MS of the [M+Ag]+ ions, afterddition of a silver salt to the mobile phase (Milne and Porter,001). MS/MS mass spectra of the [M+Ag]+ ions show product

ons formed by fragmentation between the carbon with the per-xyl group and the adjacent carbon distal to the carboxylic group.hese ions, originated by cleavage near the peroxy groups, areenerated, presumably, by the Hock rearrangement of the silverydrogen peroxide complexes, and thus are catalyzed by the pres-nce of Ag+. Also, these fragmentation pathways allowed locatinghe OOH group in the fatty acyl chain.

.1.3.5. Enzymatic oxidation. Certain enzymes catalyze the oxida-ive modification of phospholipids, yielding specific oxidationroducts, in contrast with nonenzymatic oxidative reactions.nzymes like lipoxygenase are used in in vitro tests to inducepecific oxidative modifications in PC molecules. These products,ormed during LDL oxidation, were analyzed and quantified byC–MS and MS/MS (Milne et al., 2005; Podrez et al., 2002a,b). PCxidation products formed by stimulated phagocytes were detectedy MS. This approach allowed identifying long-chain products, withredominance of epoxyisoprostane PC and mono and di-peroxyerivatives of PC (16:0, 20:4) (Jerlich et al., 2003). These authorsroposed that oxidative modifications can be induced by radicalpecies produced from NADH oxidase rather than an enzymaticrocess. Further studies are necessary to confirm this hypothe-is. Identification of eicosano-phospholipids in LDL was previouslyone using MS/MS and LC–MS (Watson et al., 1999).

.1.3.6. Addition of hypohalous acid. There are only some few stud-es that report the characterization by mass spectrometry ofalogenated phospholipids, formed under oxidative conditions.ong-chain oxidized phosphocholines are formed in the presence ofypohalous acid. These are the result of the addition of hypohalousolecules to the unsaturated bonds of fatty acyl chains and are

amed halohydrins (chlorohydrins after addition of HOCl). Chloro-ydrins and bromohydrins were identified by ESI-MS (Spickett etl., 2001; Messner et al., 2006; Jerlich et al., 2000) and MALDI–MSArnhold et al., 2001, 2002; Lessig et al., 2007; Panasenko et al.,007). The identification was based on observing mass incrementsf 52 and 96 Da in MS spectra, which corresponds to the additionf, respectively, HOCl or HOBr. LC–ESI-MS was also used to identifyhlorohydrins in human LDL treated with myeloperoxidase (Jerlicht al., 2000). PC with more than one unsaturation may uptake morehan one HOX molecule. In fact, mono, bis, tris and tetrakis choro-ydrins were identified by MALDI–MS in phosphocholine withrachidonic acid (Arnhold et al., 2002). PC with more than one dou-le bond, in presence of H2O2/myeloperoxidase/halogen systems,enerate lysoPC but there is no evidence of this oxidation systematalyzing peroxidation (Jerlich et al., 2000; Arnhold et al., 2002;anasenko et al., 2007). Also, it seems that chlorine in PC moleculeromotes the formation of lysoPC (Panasenko et al., 2007).

PlasmenylPC is also a target of myeloperoxidase and addition ofypohalous acid was identified by MALDI–MS (Lessig et al., 2007)nd LC–ESI-MS and LC–ESI-MS/MS (Messner et al., 2006). Attack of

OCl to plasmenylPC starts in the vinylic bond leading to lysoPC.his seems to be a preferential route when compared with additionf HOCl to sn-2 (Messner et al., 2006; Lessig et al., 2007). Thus, inhe mass spectra it is possible to see a more abundant ion, assignedo lysoPc, and other less abundant ion, assigned to chlorohydrin

taadt

Physics of Lipids 156 (2008) 1–12 7

ysoPC, which was produced by addition of HOCl to the lysoPCMessner et al., 2006). MS/MS of [MNa]+ ions shows loss of HCl andtypical product ion produced by loss of 95 Da, corresponding to

he combined elimination of −59 Da, typical of fragmentation of PCead, plus the loss of HCl (−36 Da). MS/MS data of the negative ionM+Cl]− shows the carboxylate anion of the fatty acid bearing theCl molecule produced by addition in the double bond (Messner etl., 2006).

Phagocyte cells produce chlorinated lipids which seem to haveiological significance since they occur during inflammation pro-ess. This subject is nowadays an emerging field of researchSpickett, 2007).

.1.4. Identification of PC adductsThe high chemical stability of oxidized short chain phospho-

holines with terminal carbonyl groups allows further reaction,y cross-linking reactions, with amino groups present in peptides,roteins and aminophospholipids (PE), with formation of Schiffases or Michael adducts. Interest on this class of compounds haspecially increased since they were detected in oxLDL particles andound to play a role in the pathogenesis of atherosclerosis (Bramet al., 2004). The work devoted on the use of mass spectrom-try for identifying and characterizing oxidized phosphocholinedducts is rare and only applied to identify phosphocholine alke-al with N-acetylcysteine (Hoff et al., 2003) and phosphocholinelka(e)nal with leucine enkephalin (Reis et al., 2006). Tandemass spectrometry of GPC alkanal-peptide Schiff adducts and GPC

lkenal-peptide Schiff and Michael adducts [MH]+ ions, shows typ-cal fragmentation pattern consisting from losses characteristic oflycerophosphatidylcholines, loss of the peptide residues, cleav-ges in the peptide chain and cleavages characteristic of fatty acids.hese provide information on both the phosphocholine and theeptide components (Reis et al., 2006). The fragmentation pat-ern described for alka(e)nal PC-peptide Schiff and Michael adductshows significant differences when compared with the fragmenta-ion pattern described for lipid-peptide adducts (Carini et al., 2004;enaille et al., 2003). These lipid-peptide adducts fragment onlyy cleavage of peptide chain. MS/MS analysis of phosphocholinelkenal with N-acetylcysteine shows typical fragmentation of PC,amely the product ion at m/z 184 and others because of loss ofn-1 and loss of sn-2 acyl residues (Hoff et al., 2003).

.2. Glycerophosphoethanolamine

Phosphoethanolamines are, together with PC, major con-tituents of cell membranes and lipoproteins. Most of thetudies on identification by mass spectrometry of glycerophos-hatidylethanolamine (PE) oxidation product use ESI as ionizationethod, but some report the use of MALDI. In the ESI mass spec-

ra, usually [M+H]+ ions are detected when working in the positiveode, and [M−H]− ions are detected when working in the negativeode (Hsu and Turk, 2000a,b; Pulfer and Murphy, 2003). The ions

etected in the positive mode MALDI mass spectra of PE are [M+H]+,M+Na]+ and [M−H+2Na]+ ions (Estrada and Yappert, 2004).

Under collision induced dissociations, [M+H]+ ions undergooss of 141 Da from the precursor ion which corresponds to lossf the polar head (Jensen et al., 1986; Kayganich and Murphy,992; Pulfer and Murphy, 2003). Because of the absence of othernformative product ions in the [M+H]+ product ion spectra ofiacyl-ethanolamine phospholipids, other authors have induced

he formation of lithiated adducts ([M+Li]+ and [M−H+2Li]+ (Hsund Turk, 2000a). The fragmentation pathway of these ions include,part from loss of 141 Da from the precursor ion, loss of aziri-ine (−43 Da, –CH2CH2NH), as well as loss of 147 Da identified ashe lithiated polar head. Besides these, the product ion spectra of

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M.R.M. Domingues et al. / Chemistr

M+Li]+ ions also shows loss of sn-1 and sn-2 acyl chains as fatty acid[M+Li–RCOOH]+) or as ketene ([M+Li–RC O]+) in diacyl PE, andoss of sn-1 as an alcohol (R1COH) for plasmenyl PE (Kayganich and

urphy, 1992). Ions produced from loss of sn-1 fatty acid are morebundant than ions arising from loss of sn-2 fatty acid, identifyinghe positions of the fatty acid in the glycerol backbone.

Fragmentation of [M−H]− ions of diacylPE (Hsu and Turk,000b; Kayganich and Murphy, 1992) leads to abundant carboxy-

ate anion of sn-1 and sn-2 acyl residues ([R1COO]−). The relativebundance of R2COO− is usually higher than the R1COO− anion.ther product ions corresponding to neutral loss of sn-1 and sn-fatty acyl residues (RCOOH and R C O) are also detected (Hsu

nd Turk, 2000b). The product ion produced by loss of 141 Da isbsent. Fragmentation under MALDI–PSD of negatively chargedons are the most informative, leading to carboxylate anions, withR2COO]− > [R1COO]−. Positively charged ions only show loss of41 Da, typical of PE phospholipids (Fuchs et al., 2007).

The work available on the use of mass spectrometry for iden-ifying oxidized PE is insufficient. In fact, only few publicationseport the direct analysis of oxidized PE by MS using electrosprayr describe the short- and long-oxidation products. Khaselev andurphy (1999) reported the results of the oxidation (Fenton reac-

ion) of PE from bovine brain. They found that oxidizing plasmenylE with monounsaturated fatty acids produce 1-lyso-2-acyl plas-enyl PE with monounsaturated sn-2 acyl residues, which results

rom cleavage of the vinyl ether group of sn-1. They also foundhat plasmenyl PE with polyunsaturated fatty acids degrade andhat diacyl PE with polyunsaturated fatty acids produces oxida-ion products with the insertion of one and two oxygen atoms.onfirmation of the oxidation products was done by tandem masspectrometry, identifying the acyl carboxylate anion formed underS/MS. In addition, the authors also suggest that plasmenyl PE

ndergo oxidation much easier than the diacyl PE.In a recent study, Maskrey et al. (2007) aimed specifically to rec-

gnize the role of oxidation products of arachidonic acid esterifiedo glycero-phosphatidylethanolamines as contributors or media-ors in inflammatory response. Through liquid chromatography andrecursor ion scanning of the ion at m/z 319, assigned as the hydroxyicosatetraenoic acid (HETE), they detected in human platelets fourydroxy phosphatidylethanolamines produced via lipoxygenase.urther HPLC–MS/MS and MS3 of the hydroxy PE [M−H]− ions (m/z19), confirmed the identity of 18:0/15-HETE diacyl PE, 18:0/15-ETE plasmenyl PE, 18:1/15-HETE plasmenyl PE, and 16:0/15-HETElasmenyl PE.

Gugiu et al. (2006) using multiple reaction monitoring (MRM)dentified short chain products produced during ethanolaminehospholipids oxidation using three different oxidation systems.he systems were autoxidation in presence of Cu(II), UV radiationnd myeloperoxidase (MPO) (Gugiu and Salomon, 2003; Gugiu etl., 2006). Based on the short-chain products with terminal alde-yde and carboxylic acid identified from 1-palmitoyl-2-docohexa-noyl-sn-glycero-3-phosphatidylethanolamine (PDPE), 1-palmit-yl-2-arachidonoyl-sn-glycero-3-phosphatidylethanolaminePAPE) and 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidyl-thanolamine (PLPC) the authors inferred that C4 products pre-ominant in PDPE, C5 in PAPE and C9 in PLPE. Among these, therincipal oxidation products were �-oxobutyryl-PE for OxPDPE,-oxovaleroyl-PE for OxPAPE and �-oxononanoyl-PE for OxPLPE.

n this study, the oxidation induced by UV gave the highest yieldf oxPE and MPO the lowest yield, although the same oxidation

roducts were observed. The search for specific oxidized PE in ratetina allowed observing that PLPE lead to the majority oxidizedpecies, rather than the PAPE and PDPE. This is significant sinceLPE is not the most abundant neither the most unsaturatedhospholipid present in retina (Gugiu et al., 2006).

R1

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Physics of Lipids 156 (2008) 1–12

The presence of the amino group in the polar head of phospho-ipids justifies the possible reaction of these phospholipids, withther compounds such as the carbonyl products formed by oxi-ation of lipid components in phospholipids (Bacot et al., 2003,007; Zamora and Hidalgo, 2003). Although, carbonyl–PE adductsre not the primary products of oxidized PE, they have a significantresence in the overall oxidation products of PE and were found inuman blood platelets (Bacot et al., 2007), in retina of diabetic ratsBacot et al., 2003) and in rats brain (Bacot et al., 2007). There is arowing interest on the possible role of this products since it haseen suggested that these n-acyl-ethanolamine adducts may haveiological roles. For example, they can be important in the signal-

ng oxidative stress and in platelet dysfunction (Bacot et al., 2007).chiff base adducts of PE with 4-hydroxy-nonenal (Stadelmann-ngrand et al., 2004) and with 4,5-epoxy-heptenal (Zamora andidalgo, 2003) were detected by ESI-MS analysis. Also, adduct for-ation between PE and �-ketoaldehydes (isoketals, which are an

rachidonic acid oxidation products), as pyrrole and Schiff basedducts were detected using LC–MS analysis in the negative mode.he structures were confirmed by LC–MS/MS, observing the pres-nce of ions produced by loss of sn-2 (R2C O and R2COOH), asell as the presence of carboxylate anions (R1COO− and R2COO−)

Bernoud-Hubac et al., 2004). Observation of other fragmentationathways, involving the loss of modified polar head, allows con-rming the presence of the pyrrole and Schiff base PE adductsBernoud-Hubac et al., 2004).

A different approach, based on the identification by LC–MS/MSf N-hexanoyl ethanolamine (modified head group), after phos-holipase hydrolysis of oxidized erythrocytes and LDL, allowed

dentifying adducts produced by reaction of PE with lipid hydroper-xides (Tsuji et al., 2003).

In recent works, a correlation has been found between shorthain oxidation products of PE and retina degeneration (Gugiu etl., 2006). Also, oxidized PE was found to promote prothrombinasectivity and platelet aggregation (Zieseniss et al., 2001; Maskreyt al., 2007). Considering PE oxidation products known biologi-al roles in cellular apoptosis, atherosclerosis and inflammation,ore efforts are needed in identifying and evaluating their possible

mplications on physiopathological processes.

.3. Glycerophosphoserine

Glycerophosphoserine, also called phosphatidylserine (PS)ccurs usually in low concentrations in nature. PS and proba-ly oxidized PS play a role in the apoptotic signaling serving asn important recognition signal for macrophages (Kagan et al.,004). Unlike the two classes of phospholipids discussed above, the

iterature published focusing or studying oxidized glycerophospho-erines (PS) by mass spectrometry is rare (Tyurina et al., 2004, 2008;ayir et al., 2007). This phospholipid class may ionize either in theositive mode, with formation of [M+H]+, [M+Alk]+, [M−H+2Alk]+

ons (Alk = Na, K, Li,), or in the negative mode, with formationf [M−H]− or [M−2H+Alk]− ions. Negative ions are preferentiallyormed (Hsu and Turk, 2005; see also correction). Under collision-nduced dissociations in the positive mode, phosphatidylserinesypically undergo loss of polar head (−185 Da), loss of the sn-1nd sn-2 acyl residues from the precursor ion and the formationf acylium ions (R1 CO+ and R2 CO+). In the negative mode, theM−H]− PS product ion spectra typically shows major loss of ser-ne group (−87 Da). Observing carboxylate anions (R1COO− and

2COO−) allows discovering structural features of PS (Jensen et al.,986; Hsu and Turk, 2005).

Studies from Kagan and collaborators report the PS oxidativeodifications analyzed by mass spectrometry (Bayir et al., 2007;

yurina et al., 2004). Monitoring PS oxidation induced by an azo

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M.R.M. Domingues et al. / Chemistr

nitiator (AAPH) by negative mode ESI-MS allows identifying theresence of mono and di-hydroperoxide derivatives of PS (Shadyrot al., 2004a,b; Tyurina et al., 2004). The presence of hydroperoxideS derivatives in traumatic brain injury using controlled corticalmpact were also proposed (Bayir et al., 2007). Mono hydroper-xy derivatives of PS produced by �-irradiation induced intestinalnjury oxidation, were characterized by ESI-MS and ESI-MS/MS ofM−H]− ions (Tyurina et al., 2008). Cytochrome c/H2O2 systemeads to PS hydroperoxides, as well as hydroxy derivatives. Theseroducts were identified by tandem mass spectrometry in nega-ive mode (Tyurina et al., 2004). Product ions formed by [M−H]−

ragmentation include the loss of serine head and formation of1COO− and modified R2COO−. MS3 of the R2COO− of PS-OOH,ield the product ions with loss of H2O, CO2, and the cleavage of13–C14, showing the linkage of hydroperoxy to C-13 of docosa-exaenoic fatty acyl chain. MS3 of the R2COO− of PS-OH, showshe ions formed by cleavage of the linkages C13–C14 and C10–C11.his suggests linkage of the hydroxy group to C-13 or C-11. Theseleavages near the functional group are informative, yielding detailsbout the location of hydroxy or peroxy groups. This behavior, alsoescribed for long-chain PC, reveals the potential of tandem masspectrometry for the detailed analysis of oxidized phospholipids.

The number of studies dedicated in identifying oxidized phos-hoserines is explainable considering the low abundance of suchhospholipid class in biological samples, which is usually lesshan 10% of phosphorus lipids (Yorek, 1993). Recent findings ofxidized phosphoserines in the external leaflet of plasma mem-ranes, despite the knowledge that PS phospholipids are found inhe inner leaflet (Yorek, 1993), allows proposing an influence ofxPS in membrane scrambling, which might trigger early apoptoticvents (Kagan and Quinn, 2004). Also, PS play a significant role inell signaling, namely during macrophage recognition of cells thatndergo apoptosis (Kagan et al., 2004). This data clearly justifiesn effort towards characterizing oxidized PS to better understandheir biological role.

.4. Cardiolipin

Cardiolipin (CL) are complex phospholipids formed by a 1,3-isphosphatidyl-sn-glycerol group with four fatty acyl chains andifferent degrees of unsaturation. In result of the molecular com-lexity of cardiolipins, the analysis by mass spectrometry is notn easy task. Nonetheless, identifying their structural features isf relevance considering that cardiolipins are important compo-ents of mitochondrial membrane and a major target of oxidativeodification in neuronal and brain injuries (Bayir et al., 2007).Mass spectrometry analysis was used for characterizing cardi-

lipins, either by ESI (Beckedorf et al., 2002; Valianpour et al., 2002;su et al., 2005; Hsu and Turk, 2006a,b) or MALDI (McDonald-arsh et al., 2006; Wang et al., 2007) as ionization methods. In

hese studies, positive mode analysis produce the [M−2H+3Na]+

ons (Beckedorf et al., 2002; Hsu et al., 2005; Hsu and Turk, 2006a,b)nd the negative mode the [M−H]−, [M−2H]2− and [M−2H+Na]−

ons. The ions ([M−2H]2− and [M−2H+Na]−) are usually the mostbundant in the negative mode mass spectrum (Beckedorf et al.,002; Valianpour et al., 2002; Hsu et al., 2005; Hsu and Turk,006a,b).

The typical fragmentation pattern of [M−2H+3Na]+ ions underollision induced conditions shows the loss of one diacylglyc-rol group, leaving the charged substituted di-phosphatidyl ion

s the main product ion. This product ion undergoes further lossf sodiated glycerol phosphatidic acid (−136 Da), loss of one andwo different fatty acid residues and loss of phosphoacylglycerolBeckedorf et al., 2002; Hsu and Turk, 2006a). Similarly, fragmenta-ion of the [M−2H+Na]− ions resemble the fragmentation pathways

pwMsu

Physics of Lipids 156 (2008) 1–12 9

bserved for the positive ions, yielding abundant product ions fromoss of monoacylphosphatidylglycerol and diacylglycerol groups,nd loss of fatty acyl chains (Beckedorf et al., 2002; Hsu and Turk,006a). Fragmenting [M−H]− ions, the carboxylated anions fromn-1 and sn-1′ show higher relative abundance than the fragmentons from sn-2 and sn-2′. Relative abundances are the opposite

hen selecting the precursor ion [M−2H]2−. Ions formed from lossf diacylglycerol groups show different relative abundances allow-ng to infer their position in central glycerol (Hsu and Turk, 2006a).

Mass spectrometry studies of oxidative modifications on cardi-lipin are rare. One study reported the cardiolipin oxidation afterraumatic brain injury (Bayir et al., 2007). In this study, cardiolipinrom mitochondria of rat brain was extracted and analyzed by two-imensional thin-layer chromatography. The collected fractionsere used to study by ESI-MS the oxidative modifications on car-iolipin. Cardiolipin species containing one to five peroxyl groupss well as hydroxy and hydroxy–peroxy products were found bybserving increments of n × 16 Da (n = number of oxygen atoms)n the m/z value (Bayir et al., 2007). Tandem mass spectrometryas not used in confirming the proposed groups and their location

long the fatty acyl chains of the cardiolipin molecules.Kagan and collaborators verified that CL is oxidized by �-

rradiation induced intestinal injury (Tyurina et al., 2008). Thextracted CL included mono to tetra-peroxy derivatives of CL, iden-ified by ESI-MS in the negative mode. MS/MS of the [M−H]− ionsield the oxidized R2COO−, confirming the occurrence of the oxida-ive modifications in sn-2 fatty acyl chain. Negative mode MSn

nalysis of CL–OOH and CL–OH formed by cytochrome c/H2O2 sys-em allow identifying the position of the peroxyl group within theatty acyl chain. MS3 analysis of the R2COO− ion shows ions aris-ng from fragmentation near the peroxy or hydroxy groups whichllows gaining further information (Tyurina et al., 2008). Thus,he MS3 of the R2COO− of CL–OOH, yield the product ions fromleavage of C11–C12 and of C9–C10, suggesting the linkage of theydroperoxyl group to, respectively, C-13 and C-9 of linoleic fattycyl chain. MS3 of the R2COO− of CL–OH, showed the ions formedy cleavage of the linkages C13–C12 and C10–C9, showing the link-ge of the hydroxy group to C-13 or C-9. Using MALDI–MS Shadyrond co-workers showed that oxidative modification of cardiolipinnduced by �-radiation or iron, may lead to oxidation products byleavage on cardiolipin molecules yielding phosphatidic acid andiacylphosphatidyl-hydroxyacetone (Yurkova et al., 2005; Shadyrot al., 2004a,b).

.5. Other phospholipids

Sphingomyelins (SM) are, with glycerophosphocholines, amonghe most abundant class of phospholipids (Yorek, 1993). However,phingomyelins oxidative modifications have not been a subjectf interest by researchers. The study involving sphingomyelins haseen mainly on the role of sphingomyelins in preventing damagey reactive species to cholesterol (Sargis and Subbaiah, 2006) andhosphocholines (Oborina and Yappert, 2003).

Glycerophosphatidylinositols (PI) contain an inositol sugaresidue attached to the phosphono group at the polar head. Theyan be found in all tissues, but usually in residual amounts at thenner leaflet of membranes, although in mammalian mitochondriahey can also be found in both leaflets (Yorek, 1993). The presence ofsugar residue at the polar head of these phospholipids associates

hese molecules with recognition processes in signaling. Thus, it is

ossible to assume that modifications of the phospholipid structureould have major implications in their biochemical roles. ApplyingS/MS capabilities in identifying PI modifications under oxidative

tress could open new areas of research and could provide betternderstanding of the mechanisms involved in PI signaling events.

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. Future perspectives

Although there has been a rapid expansion in the use of tan-em mass spectrometry for the analysis of oxidized phospholipids,uch work remains to be done. Lipidomics has emerged as a tool for

rofiling phospholipids in tissue and fluids and has opened a neweld of research. This has brought new perspectives on outlininghospholipids composition in in vivo samples, and on evaluatinghe changes on phospholipids caused by oxidative stress. This is

ore important considering the role of oxidized phospholipids inignaling events. Detailed knowledge about the changes inducedo phospholipids under oxidative stress conditions, such as func-ional groups present and their position within the carbon chain isssential to understand differences in the biological and in phys-opathological activity of oxidized phospholipids.

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