Probing phospholipid dynamics by electrospray ionisation mass spectrometry

Progress in Lipid Research Progress in Lipid Research 46 (2007) 200–224 www.elsevier.com/locate/plipres

Review

Probing phospholipid dynamics by electrospray ionisation mass spectrometry q Anthony D. Postle

a,c,*

, David C. Wilton b, Alan N. Hunt a, George S. Attard

c

a

b

School of Medicine, University of Southampton, Southampton SO17 1BJ, UK School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK c School of Chemistry, University of Southampton, Southampton SO17 1BJ, UK

Received 23 January 2007; received in revised form 30 March 2007; accepted 4 April 2007

Abstract Recent advances in electrospray ionisation mass spectrometry (ESI-MS) have greatly facilitated the analysis of phospholipid molecular species in a growing diversity of biological and clinical settings. The combination of ESI-MS and metabolic labelling employing substrates labelled with stable isotopes is especially exciting, permitting studies of phospholipid synthesis and turnover in vivo. This review will first describe the methodology involved and will then detail dynamic lipidomic studies that have applied the stable isotope incorporation approach. Finally, it will summarise the increasing number of studies that have used ESI-MS to characterise structural and signalling phospholipid molecular species in development and disease. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Phospholipid molecular species; Electrospray ionisation mass spectrometry; Stable isotopes; Phospholipid synthesis; Disease

Abbreviations: Adomet, S-adenosylmethionine; AZ, Alzheimer’s disease; BTHS, Barth syndrome; DSPC, disaturated PC; CD, cell determinant; CSF, cerebrospinal fluid; DRM, detergent resistant microdomains; DMSO, dimethyl sulfoxide; ESI-MS, electrospray ionisation mass spectrometry; ER, endoplasmic reticulum; FCS, fetal calf serum; FT-ICR, Fourier transform ion cyclotron resonance mass spectrometry; GLC, gas liquid chromatography; GC–IRMS, gas chromatography isotope ratio mass spectrometry; HPLC, high performance liquid chromatography; LBPA, lysobisphosphatidic acid; LDL, low density lipoprotein; lysoPA, lysophosphatidic acid; lysoPC, lysophosphatidylcholine; NE, nuclear envelope; PH, pleckstrin homology; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; 32Pi, 32P-labelled phosphate, phosphotidylinositol; PIP, phosphatidylinositol monophosphate; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PPAR, peroxisomal proliferator receptor; PS, phosphatidylserine; PLA2, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D; PAF, platelet activating factor; QToF, quadrupole:time of flight; TAZ, tafazzin gene; TLC, thin layer chromatography; TAG, triacylgycerol. q The composition of phospholipid species are designated as PXa:b/c:d where X represents the headgroup (choline, ethanolamine, etc.) and the fatty acyl groups are identified by number of carbon atoms (a and c) and numbers of unsaturated double bonds (b and d). For example, PI18:0/20:4 is sn-1-palmitoyl-2-archidonoyl-glycerophosphoinositol. Species with ether-linked fatty acids are designated by inclusion of the letter a; for example PC16:0a/16:1 is sn-1-hexadecyl-2-hexadecenoyl-glycerophosphocholine. * Corresponding author. Address: School of Medicine, University of Southampton, Southampton SO17 1BJ, UK. Tel.: +44 2380796161; fax: +44 2380796378. E-mail address: [email protected] (A.D. Postle). 0163-7827/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.plipres.2007.04.001

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Historical perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI-MS analysis of phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Tandem MS/MS analysis of phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Analysis of phospholipid synthesis by ESI-MS/MS using stable isotopes . . . . . . . . . . . . . . . . . . . . . 2.2.1. Phosphatidylcholine synthesis by the CDP:choline pathway. . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Phosphatidylcholine synthesis by the phosphatidylethanolamine N-methylation pathway . . . . Molecular specificity of phospholipids and cell differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phospholipid molecular species in health and disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Lung surfactant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Brain function and disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Barth syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Analysis of oxidised phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. Analysis of specific sub-cellular membrane fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.1. The endonuclear compartment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.2. The nuclear envelope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.3. Intracellular lipid droplets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.4. Detergent resistant membranes/lipid rafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8. Analysis of low abundance natural phospholipids: signalling lipids . . . . . . . . . . . . . . . . . . . . . . . . . ESI-MS of membrane phospholipids: the state of the art and future directions . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

201 201 202 204 204 205 205 207 208 208 210 211 211 212 212 213 213 214 214 214 216 219 219

Contents 0. 1. 2.

3. 4.

5.

0. Introduction The application of electrospray ionisation mass spectrometry (ESI-MS) to lipid analysis has rapidly become established over the past decade as the methodology of choice for the detailed characterisation of molecular compositions of a wide range of classes of lipid. It is beyond the scope of this review to address all aspects of what has become known as Lipidomics, and a number of excellent reviews have detailed many of the technical and methodological aspects [1–3]. Instead it will concentrate on dynamic aspects of phospholipid composition, including the analysis of their synthesis and turnover using stable isotopes, cell-type molecular specificities of phospholipids and their modulation during disease processes and finally the application of ESI-MS technologies to the analysis of lipid signalling and oxidation processes. 1. Historical perspective The history of lipid biochemistry, as with most other aspects, of biochemistry has been intimately linked with the development of new analytical methods. In earlier times the use of paper chromatography followed by the use of silicic acid impregnated paper allowed the separation and analysis of classes of phospholipids that, in conjunction with radioactive labelling experiments, started to provide insights into lipid biosynthesis and turnover. A now classic example of this approach stems from the early work of the Hokins in identifying the ‘‘phospholipid effect’’, that is the rapid incorporation of 32P-labelled phosphate (32Pi) into phospholipids of pancreatic slices following cholinergic stimulation [4]. The subsequent ability of chromatography to separate classes of phospholipid allowed the identification of phosphotidylinositol (PI) along with phosphatidic acid (PA) as the phospholipids showing the largest increase in incorporation of 32Pi following stimulation (reviewed in [5]). These original observations in the 1950s provided the foundation for the multitude of studies on phosphoinositide turnover and cell signalling.

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The development of thin layer chromatography (TLC) by Kirchner and Miller and by Stahl (reviewed in [6]) provided a rapid and reproducible technique for the separation of all lipid classes and became the basis for much of the lipid biochemistry of the 1960s and onwards. Over a similar time scale, the development of gas liquid chromatography (GLC) (reviewed in [6]) allowed the rapid and quantitative analysis of the fatty acid composition of both triacylgycerols (TAG) and phospholipids providing a considerable archive of information about the fatty acid composition of both diets and tissues. Our understanding of the importance of unsaturated fatty acids in terms of cell function and human health relied on such analyses. The development of high performance liquid chromatography (HPLC) methods led to their use in lipid biochemistry for the separation of lipid classes from which evolved systems that resulted in the separation and detection of individual phospholipid molecular species within classes of phospholipids, differing in their fatty acid compositions [7]. This technology has been used to determine the molecular-species specificity of rat hepatic phosphatidylethanolamine N-methyl transferase [8] and the specificity of phosphatidylcholine (PC) synthesis in human fetal lung [9]. The analysis of molecular species present in mixtures of PC and phosphatidylethanolamine (PE) isolated from rat liver confirmed the acyl chain specificity of human cytosolic phospholipase A2 (cPLA2) compared with the pancreatic secreted enzyme [10]. 2. ESI-MS analysis of phospholipids The use of magnetic sector and GC–mass spectrometry for lipid analysis has had a long and distinguished career [11]. The development of ESI-MS, however, has been a watershed in detailed phospholipid analysis, due in part to its ability to couple an HPLC effluent directly to the mass spectrometer but more importantly because it is an efficient soft ionisation technique that enables ionisation of intact molecular species of phospholipid. Coupled with the inherent exquisite sensitivity of the technique, these advances have dramatically improved separation and detection of individual phospholipid molecular species. ESIMS has provided the foundation for the emerging discipline of Lipidomics or phospholipid profiling of cells where, in theory, all phospholipid molecular species can be identified and quantified. This is the primary goal of the LIPID MAPS consortium in the United States (http://www.lipidmaps.org), a grouping of laboratories with the defined aims of (1) separating and detecting all the lipids in a specific cell and to discover and characterise any novel lipids that may be present, (2) quantifying each lipid metabolite present and the changes in their levels and location during cellular function and (3) defining the biochemical pathways for each lipid and develop lipid maps to describe interaction networks. One important outcome of this project will be the establishment of a data standard catalogue that will provide a hierarchical scheme covering all lipids found in eukaryotic and prokaryotic cells [12]. Hopefully this catalogue will form the basis of Bioinformatics for Lipidomics by providing a fundamental common reporting scheme for experimental data sets. Early work from the laboratories of Gross and co-worker involved the analysis of the human erythrocyte plasma membrane [13], the starting point for many developments in lipid and membrane biochemistry, and where they first experienced the remarkable sensitivity of the technology for phospholipid analysis. This technique would allow the structural determination of picomoles amounts of phospholipid via electrospray ionisation tandem mass spectrometry [14]. The demonstration of changes in phospholipid composition as a result of a physiological response was first seen with platelet phospholipids during thrombin stimulation [15]. A subsequent paper provided information on the sphingomyelin molecular species in isolated pancreatic islets and provided evidence that sphingomyelin hydrolysis is not involved in the IL-1 signalling pathway that results in overproduction of nitric oxide [16]. During this early period the power of ESI-MS analysis was also applied to other polar lipids including acylcarnitines [17], lipid sulfates [18] and sulfatides [19], again demonstrating the sensitivity and selectivity of the technique. Single quadrupole ESI-MS analysis preferentially detects PC and sphingomyelin in positive ionisation conditions and acidic phospholipids and PE in negative ionisation. While a considerable improvement over previous analytical approaches, ESI+ and ESI scans only provide information based on molecular mass, which is a severe limitation given that most ion peaks will be comprised of multiple components. Additionally, the variable formation of adducts, such as sodium (+22) or potassium (+35) in positive ionisation, considerably

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complicate molecular-species assignments from such complex spectra. Various groups added NaOH or LiOH in attempts to generate complete sodiation [13,20] or lithiation [1,21]. Formation of lithium adducts has proved particularly useful to increase detection of neutral PE species in negative ionisation [22], but assigning definite molecular identities to individual ion peaks is not possible with single quadrupole MS. This problem is especially important for isobaric molecular species of the same phospholipid class, which have identical mass but differ in combination of acyl chains. One additional approach to provide more detailed analysis without resorting to tandem MS/MS is exact mass measurement. Such analysis is based on the observation that the individual atoms within a biological sample will have masses that are not exact integers. This is especially relevant for hydrogen, with a mass of 1.008. Consequently species with the same nominal mass but comprised of different numbers of H, O or 13 C atoms will have mass differences of 0.1 or less. Separating such small mass differences is beyond the maximal resolution of triple quadrupole or QToF instruments (typically 20,000 to 30,000), but is possible using Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry [23]. FT-ICR can have resolution up to 1,000,000 and resolve at 1 ppm, but generally only at a narrow band pass of 10–20 amu. The resolving power of such an approach is immense, as shown in Fig. 1. This spectrum illustrates a portion of the FT-ICR spectrum of PE from Caenorhabditis elegans, which unusually contains a high proportion of odd carbon number acyl chains and demonstrates clearly the resolution of a diacyl species of mass 752.5230 (PE17:0/20:5) from an alkyl–acyl species (containing one less oxygen atom) of mass 752.5587 (PE18:0alk/20:5).

Fig. 1. Mass spectrum of C. elegans PE by nano-ESI-FTICRMS. Mass peaks of diacyl PE molecular species containing odd numbers of carbon atoms in their fatty acyl chains were effectively resolved and identified from even numbered alkyl acyl ether PE species, despite having the same nominal molecular mass (reproduced with permission from Ishida et al. [23]).

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Table 1 Diagnostic ESI-MS/MS scans for the major phospholipid classes Lipid class

Diagnostic scan (native)

Synthesis substrate

Diagnostic scan (synthetic)

Phosphatidylcholine (PC) Phosphatidylethanolamine (PE) (diacyl species) Phosphatidylserine (PS) Phosphatidylinositol Phosphatidic acid (PA) & phosphatidylglycerol (PG) Phosphatidylinositol-4,5-bisphosphate (PIP2)

P184+ NL141+ NL87 P241 P153 P401

(methyl-d9) Choline Cl Ethanolamine-d4 Serine-d3 myo-d6-Inositol Glycerol-d5 myo-d6-Inositol

P193+ NL145+ NL90 P247 P158 P407

Precursor (P) and neutral loss (NL) scans are indicated for both native phospholipid species and those with the incorporated appropriate deuterium-labelled substrate. While alternative scans are possible for some of these phospholipid classes (for instance negative precursor scan of m/z 196 for PE), the identified diagnostic scans provide the most abundant ions for detection of labelled substrates.

2.1. Tandem MS/MS analysis of phospholipids The development of a wide array of diagnostic tandem MS/MS analyses have employed triple quadrupole or quadrupole:time of flight (QToF) mass spectrometers to provide a variety of diagnostic precursor or neutral loss scans for the different phospholipid classes. These methodologies are summarised in Table 1 and have recently been extensively reviewed [1,2]. They provide both more detailed structural information and greater sensitivity by reducing baseline noise. There are now well established tandem MS/MS methodologies for quantifying the most common classes of phospholipid, but these are not without inherent problems. All ESI-MS spectra require correction for the contribution of the [M+2]+ ion of species 2 amu lower, due to the presence of molecules containing two 13C atoms [15], but tandem MS/MS results must also be corrected for the effect of differential fragmentation. This is due to the molecular mass dependence of collision gas induced fragmentation, such that the ion response decreases with increasing mass. This effect can be readily corrected using algorithms constructed from authentic standards [24,25], but great care must be taken to maintain constant mass spectrometry conditions and to employ dilute lipid concentrations no greater than 1 lM for individual components in the reconstituted extract being measured [26]. Ion suppression and insource lipid aggregation effects become significant at high sample concentrations, leading to differential responses on the basis of acyl unsaturation and chain length. Fragmentation of glycerophospholipid molecular ions in negative ionisation generates ions derived from their constituent fatty acyl chains. Moreover, under appropriate experimental conditions, fatty acyl fragments from the sn-1 and sn-2 positions generate fragment ions of different abundance and this can be used to assign not only fatty acyl composition but also their regiospecificity [13]. The plasmanyl and plasmenyl ether phospholipids are exceptions to this rule, as no fatty acid fragment is formed from the ether-linked component. Product ion scanning to determine the molecular-species components of individual ion peaks works well for acidic phospholipids, but is more problematic for PC. The intact PC molecule cannot acquire a negative charge due to the positively charged quaternary nitrogen in the choline headgroup. PC will ionise under negative conditions by loss of a methyl group ([M 15] ) or formation of a chloride adduct ([M+35] ), which in turn can be fragmented to generate the fatty acyl ions. This process also has problems, as it is often difficult to control the negative ionisation of PC and consequently multiple ions will be generated from a single molecular species resulting in overlapping fatty acyl fragments. Addition of LiOH to form lithiated adducts, followed by neutral loss scans of fatty acyl fragments under positive ionisation is one approach to overcome the problem of PC species assignment [21,27]. Finally, molecular-species assignment of a complex biological mixture of phospholipids is time consuming as individual product scans are required for each precursor ion. This has been addressed by multiple precursor scanning using a QToF instrument. This elegant approach selects sequential masses in the quadrupole, fragments them in the collision cell and then uses the rapid response of the ToF component to monitor the fatty acyl fragment region for each precursor mass in the sample [28,29]. 2.2. Analysis of phospholipid synthesis by ESI-MS/MS using stable isotopes Traditional approaches for metabolic flux analysis using substrates labelled with radioactive or stable isotopes underpin all the basic concepts of metabolic pathways. Early studies based on the incorporations

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of deuterium or tritium from respectively deuteriated or tritiated water provided the means to determine absolute rates of fatty acid and sterol synthesis, while incorporations of [14C]-acetate and related precursors were central to the determination of mechanisms of lipid biosynthesis. While such approaches have proved invaluable for the analysis of the synthesis of phospholipid classes, they have inherent limitations for characterising the synthesis of individual lipid molecular species. Some degree of molecular specificity for PC synthesis by the Kennedy or PE N-methylation pathways was provided by HPLC of intact [30] or derivatised [31] PC species coupled to radioactivity detection. Such approaches defined the basic principles for the molecular specificity of PC synthesis by rat liver and developing fetal lungs [32], but individual species identification was reliant on a combination of HPLC retention time and fatty acid analysis [33]. The combination of using stable isotopes coupled to gas chromatography isotope ratio mass spectrometry (GC–IRMS) has been applied to clinical studies in vivo of lipid metabolism [34]. While providing enhanced product identification, GC–IRMS requires extensive sample preparation and derivatisation and, as few intact lipid species can be resolved by GC, provides relatively little molecular-species data. This experimental approach has been used to quantify rates of synthesis of lung surfactant phospholipid fatty acids in preterm infants following incorporations of 13C-glucose, 13C-palmitate, 13C-acetate and deuteriated water [35–38]. ESI-MS/MS has provided a very powerful alternative approach that harnesses the technique’s great sensitivity and specificity to provide detailed information about phospholipid synthesis in terms of individual molecular species. In principle, instead of analysing the accurate deuterium or [13C] enrichment within an isolated lipid fraction [39,40], synthesis analysis by ESI-MS relies on quantification of the relative ion intensities of a subset of intact lipid species labelled with the relevant isotope. This in turn is dependent on the formation by tandem MS/MS of diagnostic fragment ions containing the stable isotope. The overall sensitivity of the approach is then a function of the number of isotope atoms within this diagnostic fragment. In practice, three or more isotope atoms are required to overcome the effect of the M+1, M+2, M+3 isotope due to the approximately 1% natural abundance of [13C], and is best exemplified by the incorporation of (methyl-d9)-choline into PC molecular species [41–44]. 2.2.1. Phosphatidylcholine synthesis by the CDP:choline pathway Incorporation of (methyl-d9)-choline by the CDP:choline Kennedy pathway will generate PC species 9 mass units higher than the corresponding native species (Fig. 2a). Although this [M+9]+ ion can be readily resolved by ESI-MS, its abundance cannot be distinguished from that of the native ion of the same mass. However, tandem MS/MS fragmentation of the ion will generate a choline phosphate product ion of m/z +184 for native (Fig. 2b) and m/z +193 for newly synthesised PC (Fig. 2b and c). Precursor scans of m/z +184 (Fig. 2d) and +193 (Fig. 2e) will provide a direct comparison of the patterns of native and newly synthesised PC species (Fig. 2c). Quantification of the ion intensities of these two precursor scans will thus provide an estimate of the fractional enrichment of each PC species with the (methyl-d9)-choline substrate. Measurement of the enrichment of (methyl-d9)-choline in the intermediate choline phosphate pool can then be used to calculate absolute rates of PC synthesis and turnover. Additionally, in liver, for instance, PC is synthesised de novo with PC16:0/ 18:2 as the predominant component and the equilibrium composition is then established by sequential actions of phospholipase and acyltransferase or transacylase enzymes [30,44,45]. Comparison of the modification of the pattern of incorporation of (methyl-d9)-choline with time in comparison with the molecular species composition of the endogenous native PC will give an indication of the extent and details of such acyl remodelling. 2.2.2. Phosphatidylcholine synthesis by the phosphatidylethanolamine N-methylation pathway In addition to the CDP:choline pathway, PC can also be synthesised by sequential N-methylation of PE using S-adenosylmethionine (Adomet) as the methyl donor [46,47]. PE N-methylation in yeast, quantified by incorporation of the (methyl-d3)-methionine precursor of Adomet into PC, generated a different selection of molecular species than those synthesised by the CDP:choline pathway, determined by incorporation of choline-d13 into PC [43]. These results were interpreted as evidence for acyl remodelling of PC synthesised by both pathways, involving a combination of phospholipase and acyl transferase activities. In mammals, the two isoforms of phosphatidylethanolamine N-methyltransferase (PEMT) enzymes derived from a single gene are effectively only expressed in hepatocytes [48]. The importance of this pathway has been clearly demonstrated in the PEMT / mouse, which survives under normal dietary conditions but dies within a few days of being

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E. Precursor of 193 819.9

791.9

767.9

100

769.9 795.9

%

743.8 845.8

0

D. Precursor of 184 760.8

100

%

786.8 758.8 734.8

810.8

782.8

836.8

0

m/z

720 100

740

760

780

800

820

184.0

840 184.0

100

%

100

C.

%

B. 760.6

193.0 0 180 190 200

788.6

m/z

810.5

786.6

0 180 190 200

m/z

782.5

%

A. ESI+ scan 808.6

758.6

813.6 734.6 806.6

834.5 838.5

0

720

740

760

780

800

820

840

m/z

Fig. 2. The incorporation of (methyl-d9)-choline into PC by cultured human lymphocytes. Panel A shows that for the PC16:0/18:1 species (native mass m/z 760.8) any newly synthesised PC at m/z 769.8 cannot be distinguished by single quadrupole MS. The fragmentation spectrum of the m/z 760.8 ion in Panel B shows as single product ion at m/z +184, corresponding to choline phosphate. Fragmentation of m/z 769.8 generated in addition a small proportion of a (methyl-d9)-choline phosphate product at m/z +193 (Panel C). Precursor scan of m/z +184 reconstructed the native PC species (Panel D) while comparable scans of m/z +193 revealed the newly synthesised PC species (Panel E). The spectra in Panels D and E have each been normalised to the largest ion peak on display for direct comparison. In reality, the fractional incorporation of deuteriated label in this analysis was less than 0.3%, and the sensitivity of this methodology is easily an order of magnitude lower.

fed a choline free diet [49]. In addition to providing an alternative route for PC synthesis that is subject to differential regulation, for instance increasing dramatically in late gestation in the pregnant rat [30], PE N-methylation is the only mechanism for choline synthesis within the body. A complex recycling pathway regulates choline flux from the liver to peripheral tissues, especially the brain, and prevents generation of neurotoxic concentrations of choline in the blood [50]. This is evidenced by infusion of (methyl-d9)-choline into human volunteers, which is followed by a transient enrichment of the label in blood choline that is totally cleared after 6 h [51]. Excess dietary choline is rapidly converted to betaine by choline oxidase in the liver, with efficient salvage of methyl groups into Adomet via homocysteine. A portion of these methyl groups are then recycled back into choline by PE N-methylation. The flux through this pathway has been estimated as the major demand out of all the 50 and more methylation reactions in vivo [52]. This metabolic network has provided an elegant means to quantify hepatic PC synthesis by both CDP:choline and PE N-methylation pathways [41] in mammals. Rat hepatocytes incubated with (methyl-d9)-choline

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rapidly metabolised a substantial proportion to (methyl-d3)-betaine and subsequently (methyl-d3)-Adomet. PE N-methylation then generated PC with one or two methyl-d3 moieties in the phosphorylcholine headgroup of PC, which were monitored by precursor scans of m/z +187 and m/z +190 respectively. The very small proportion of PC that incorporated three methyl groups would of course be indistinguishable from that synthesised directly from (methyl-d9)-choline. Because they employed incubation times of 24 h, only minor differences were apparent for the molecular-species composition of PC synthesised by either pathway. This methodology, however, is ideally suited to determination of the molecular specificity of PC synthesis and acyl remodelling in vivo. Incorporation of (methyl-d9)-choline has been applied to studies of PC acyl remodelling in yeast [43] and of PC synthesis in cultured transformed and primary cells [41,42]. The enhanced sensitivity of this experimental approach has facilitated the characterisation of the saturated nature of PC synthesis within the matrix of the cell nucleus [42,53], the increased turnover of arachidonoyl-containing PC species in human blood neutrophils compared with lymphocytes from the same blood sample [54], and mechanisms of PC synthesis by rat hepatocytes [41]. This stable isotope incorporation is ideally suited to quantification of synthetic fluxes through other phospholipid synthesis pathways, not just PC synthesis. Incorporation of myo-d6-inositol has been shown in a preliminary study to quantify the flux and molecular specificity of PI synthesis [55]. While comparable methodologies are feasible to study phosphatidylserine (PS) and PE synthesis and PS decarboxylase specificity, so far such studies have relied on tracing the deuteriated acyl distributions of phospholipids [56]. 3. Molecular specificity of phospholipids and cell differentiation Cell membrane phospholipids are maintained in vivo in a cell type-specific distribution which extends to different cells within the same tissue. ESI-MS analysis has clearly shown such variation for neuronal grey matter compared with non-neuronal white matter in brain [57] and between peripheral blood mononuclear cells and neutrophils [58]. Additionally, tissue specificity does not necessarily apply to all phospholipid classes. For instance, PC composition of total lipid extracts of mouse tissues demonstrates a range from highly saturated (lungs, brain), through di-unsaturated (pancreas) to highly polyunsaturated (liver, plasma) molecular species. By contrast PI from the same tissues is in all cases principally the single molecular species PI18:0/20:4 [59]. Such diversity of composition is not seen for cells maintained under conventional culture conditions with fetal calf serum (FCS) as the major lipid source. PC from transformed cell lines is typically monounsaturated with only a residual memory from the tissue origins. PI is either monounsaturated (e.g. HL60, Cos cells) or PI18:0/20:4 (m/z 885) is replaced substantially with PI18:0/20:3 (m/z 887) containing the 20:3n-9 Mead acid. Use of fetal calf serum is certainly a major factor in producing this relatively homogenous composition of cells in culture, and different batches of FCS will readily modify lipid profiles of cells. Recognition of this influence means that all long-term studies of lipid compositions in cultured cells, such as that in the LIPID MAPS project, must use a constant source of FCS. In addition removal from the dynamic lipid supply experienced by cells in vivo together with loss of differentiation-specific factors all contribute to a marginal state of essential fatty acid deficiency experienced by most cells in long-term culture. This deficiency has no major apparent consequences for cell growth and survival in vitro, although adding 30 lM 20:4/albumin as a lipid supplement over six passages acted as growth promoter for HL60 cells with some 80% of PC species containing 20:4 [58]. The effects of this deficiency on intracellular lipid signalling have largely been ignored, but it is potentially significant that polyphosphoinositides signalling has mostly been studied in cells lacking the physiological PIP2 substrate 18:0/20:4. One consequence of these discrepancies in phospholipid composition is to complicate interpretation of studies of the molecular specificity of phospholipase C (PLC) and phospholipase D (PLD) signalling. The suggestion that polyunsaturated DAG species are generated by activation of PLC and monounsaturated PA species by activation of PLD [60] is attractive and may well apply for instance to haematopoietic cells in vivo but not to cells with a high proportion of polyunsaturated PC species in their membrane lipids. Differentiation exerts a potent effect on cell membrane phospholipid compositions and primary cultures of hepatocytes and type II lung epithelial cells readily lose their characteristic phospholipid profiles as one of the earliest manifestations of de-differentiation. The reverse process, investigating the extent to which differentiation in culture modifies cell membrane phospholipid composition, has not been systematically studied. One

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760.8

Lymphocyte

PC16:0/18:1 PC18:0/18:2 734.8

703.8

732.8

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Fig. 3. Composition of PC composition of cultured and primary cells. These three positive ionisation spectra compare the PC composition of U937 promonocytic cells with corresponding precursor of m/z +184 scans of human monocyte and lymphocyte PC. While all three spectra are dominated by monounsaturated PC16:0/18:1 (m/z 760.8), the distribution of shorter chain PC species was very different. Monocyte and lymphocyte PC in this region was enriched principally in PC16:0/16:0 (m/z 734.8) in contrast to the higher proportion of PC16:0/16:1 (m/z 732.8) in U937 cells.

limitation here has been the lack of suitable, purified differentiated cells ex vivo to act as comparators. For instance, differentiation of promonocytic U937 cells with dimethyl sulfoxide (DMSO) reportedly increased 20:4-containing PC species from 6.4 ± 2.3% to 26.8 ± 2.7% and decreased sn-1palmitoyl-containing PC species from 75.7 ± 0.2% to 45.3 ± 2.7% [61]. However, the physiological relevance of this observation was not established as no comparison was included with either blood monocytes of monocyte-derived macrophages. Human blood monocyte PC, measured in our laboratory by ESI-MS, indeed contains a comparable proportion of 20:4-containing species (29.2 ± 1.1%, n = 3), but this is predominantly sn-1palmitate (78.4 ± 0.8%) rather than sn-1stearate. Consequently, while DMSO differentiation can modulate U937 cell PC, it does not achieve a characteristic monocyte composition. The ratio of PC16:0/16:0 (m/z +734) to PC16:0/16:1 (m/z +732) is particularly interesting here and may prove to be a sensitive marker of a differentiated phospholipid composition, at least for haematopoietic cells. This ratio was 0.64 and 0.76 for undifferentiated and differentiated U937 cells respectively but 7.72, 7.0 and 6.02 respectively for monocytes, lymphocytes and neutrophils [54] and, in these blood cells, PC16:0/16:0 can readily comprise 10% of total PC (Fig. 3). 4. Phospholipid molecular species in health and disease 4.1. Lung surfactant Pulmonary surfactant is the lipid:protein complex secreted by the type II epithelial cell of the lung alveolus. It spreads as a monolayer at the air:liquid interface in the lungs and, by opposing surface tension forces, prevents alveolar collapse on exhalation. The major component of human lung surfactant is the PC molecular-species dipalmitoyl PC (PC16:0/16:0). It has long been thought that PC16:0/16:0 was responsible for the surface tension lowering properties on lung surfactant, with a variety of acidic phospholipid and hydrophobic peptides assisting its rapid adsorption to the air:liquid interface in the lungs. The application of ESI-MS to the analysis of lung surfactant has dramatically altered this simplistic view of surfactant phospholipid composition with considerable implications for understanding of its mechanism of action. Historically, surfactant PC16:0/16:0 has been analysed as disaturated PC (DSPC) by the residue after oxidative destruction of unsaturated lipids [62]. Although having the virtue of providing a straightforward result for clinical applications, DSPC analysis includes a considerable number of PC species in addition to PC16:0/ 16:0. Initially HPLC analysis [63] but more recently ESI-MS [20] has shown a more complex composition of

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Phosphatidylcholine (mole % total)

lung surfactant phospholipid. Comparison over a large number of different animal species [64] has demonstrated a wide variation of surfactant PC with PC16:0/16:0 not always being the dominant molecular species. Heterothermic mammals and marsupials that undergo periods of hibernation or torpor with reduced body temperature have surfactants based on the monounsaturated species PC16:0/16:1, while surfactant from the Tasmanian Devil for instance contains predominantly the plasmanyl ether species PC16:0alk/16:1. Even for animals with a high content of surfactant PC16:0/16:0 in the adult, such as rat [66] or pig [67] surfactant PC composition changes dramatically in the postnatal period with increased content of the alternative disaturated palmitoylmyristoyl PC (PC16:0/14:0). This adaptation has been linked either to postnatal alveolarisation [65] or to the concomitant increased respiratory rate [67] in these neonatal animals. The link with parameters of respiratory physiology is particularly intriguing and holds over a wide range of animals from the pigmy shrew that breathes at 900 breaths/min to larger animals breathing at less than 100 breaths/min (Fig. 4). These comparative studies highlight a common theme that decreased PC16:0/16:0 in these surfactants is always compensated for by increased contents of a limited number of shorter chain PC species, predominantly PC16:0/14:0 and PC16:0/16:1. The contents of longer chain and polyunsaturated PC species characteristic of cell membranes, such as PC16:0/18:1 and PC16:0/20:4, are invariably low except in disease states. This correlation holds even for fetal human alveolar type II epithelial cells differentiated by hormone treatment in culture [68]. Differentiation in these cells is accompanied by the appearance of intracellular lamellar storage bodies and secretion of functionally active surfactant, but again the major PC species in secreted surfactant from these cells are PC16:0/14:0 and PC16:0/16:1 rather than PC16:0/16:0 [69]. Overall, the combination of animal and cell differentiation studies suggest that selection criteria for acquisition of surfactant PC species are probably based on molecular size rather than the disaturated nature of the phospholipid. The study of (methyl-d9)-choline incorporation into sputum PC in human volunteers [51] using ESI-MS analysis supports this conclusion. Besides demonstrating both the feasibility of performing such analysis in human subjects in vivo, this paper provides important insights into the molecular dynamics of both lung and liver PC synthesis. Appearance of labelled PC in plasma was rapid, peaked at 24 h and was then elevated for up seven days, indicating substantial recycling of the choline headgroup. Appearance of label in sputum PC was more delayed, reflecting transit from the alveolus to the airways but was also reasonably elevated at seven days. Importantly, the pattern in sputum of secreted newly synthesised PC in sputum at early time points varied considerably from the equilibrium native PC composition, an observation consistent with acyl remodelling mechanisms previously demonstrated for experimental animals in vivo. These studies suggest that PC is initially synthesised on the endoplasmic reticulum of the type II epithelial cells with a high content of PC16:0/18:1 and PC16:0/18:2 and is then modified by phospholipase-mediated acyl remodelling and intracellular transport over a period of hours into mature surfactant characteristically enriched in a combination of PC16:0/14:0, PC16:0/16:1 and PC16:0/16:0. The molecular-species composition depends on animal species,

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Fig. 4. Composition of surfactant PC is related to breathing frequency. The concentration of PC16:0/16:0 in lung surfactant from a variety of animal species and developmental ages was inversely related to breathing frequency. For all animals studied, the decreased content of PC16:0/16:0 was replaced by increased contents of a combination of PC16:0/14:0 and PC16:0/16:1 (modified from Bernhard et al. [67]).

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stage of development and respiratory rate. Moreover, they also indicate the lack of any ordered sequence of temporal maturation of lamellar bodies followed by secretion of mature surfactant. Instead, newly synthesised PC is rapidly incorporated into lamellar bodies and, when these are secreted, appears in alveolar surfactant initially in the unmodified form. The final composition of functional surfactant is then the net outcome of acyl remodelling in type II cells, refinement by surface pressure at the air–liquid interface in the lungs, catabolism by alveolar macrophages and re-uptake and recycling by the type II cell. While many of the fundamental questions remain uncertain, such as the molecular mechanisms responsible for phospholipid selection and acyl remodelling, the surfactant system of the lungs remains one of the best example of how tissues regulate their phospholipid compositions. 4.2. Brain function and disease The extensive membrane structures characterised by neuronal networks necessitates that the brain is particularly enriched in a wide variety of lipid structures, with some 70% of the dry weight of the brain being lipid. The essential requirement of the brain for phospholipids, especially those containing n-3 polyunsaturated fatty acids, is readily demonstrated by numerous studies in human infants and animal models of impaired neurological development in response to fetal insufficiency of n-3 fatty acid supply following preterm delivery [70]. The role of docosahexaenoyl-containing phospholipids (22:6n-3) has been a major focus of phospholipid research in brain development, function and neurological disorders [71]. Dietary restriction of 22:6n-3 and its a-linolenic acid precursor (18:3n-3) in rats over two generations resulted in replacement of 22:6n-3 in hippocampal PC, PE, PE plasmalogen and PS molecular species with a combination 22:5n-6 and 22:4n-6 and with a significant decrease in the fractional content of PS [72]. This decreased PS may then promote apoptosis by reducing the membrane translocation of Akt [73]. Exposure of fetal rats to ethanol in pregnancy caused a specific decrease in hippocampal PS18:0/22:6, together with decreased PC and increased PE [74], responses possibly linked to brain dysfunction in the fetal alcohol syndrome. Comparable deficits of 22:6n-3 in PC and PE molecular species in fetal guinea pig brain together with severe neurological dysfunction were all partially remedied by supplementation of maternal diet by fish oil in pregnancy [75]. While most studies of phospholipid abnormalities in neurological conditions such as Alzheimer’s Disease, schizophrenia and Down’s Syndrome have relied on fatty acid analysis of surrogate tissues and, increasingly, on 31P MRI imaging techniques [76–78], a number have used ESI-MS approaches to quantify phospholipids in post mortem brain and cerebrospinal fluid. Han and co-workers employed the sensitivity inherent in ESI-MS to probe small samples of post mortem brain and distinguished alterations to phospholipid compositions of white and grey matter in early Alzheimer’s disease (AZ) subjects [57]. White matter PE plasmalogen was comprised predominantly of the 18:1/18:1 species and was decreased in both cerebral and cerebellar samples by 40 mol% for all AZ patients. In contrast, grey matter PE plasmalogen was highly enriched in PUFA-containing species and was lost from cerebral but not cerebellar samples with disease progression. Given the antioxidant role of plasmalogens [78,79], the authors suggested that oxidative damage may be the cause of this plasmalogen deficit and may be linked to the neurological pathology of AZ. By contrast, Murphy et al. found a generalised decreased phospholipid content of both cerebral and cerebellar grey matter in brains from older people with Down’s syndrome, which was greatest for PI and plasmalogen species and concluded this to be a direct effect of trisomy 21 not any accompanying AZ disease [80]. A generalised deficit of all major phospholipid species was observed in samples of cerebral cortex from patients with neuronal-ceroid lipofuscinosis, a recessive storage disease that leads to progressive encephalopathy in children. Infants with the more severe form of the disease exhibited a dramatic decrease in the proportion of PUFA-containing phospholipid species, with proportionally more monounsaturated species and an increased content of lysobisphosphatidic acid (LBPA) [81,82]. Apo-lipoproteins synthesised in the brain and found in the cerebrospinal fluid (CSF) but excluded from the circulation by the blood brain barrier include ApoA1, ApoD, ApoJ and ApoE and have been postulated to have an important role for transfer of lipids within the brain (for review see [83]). PC in CSF was unchanged in either composition or concentration in patients with AZ disease, but with a lower lysoPC concentration [84], while platelet activating factor (PAF) was elevated in CSF of patients with relapsing multiple sclerosis [85]. Intriguingly, PAF in multiple sclerosis was the 18:0 species in CSF but the 16:0 species in plasma, suggesting

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a different metabolic source. In contrast to the decreased lysoPC, concentrations of other metabolites of PC, including glycerophosphocholine, phosphocholine and choline were all significantly increased in patients with AZ disease [86], possibly mediated by elevated PLA2 activity. Analysis of phospholipids and other lipids, particularly sulfatides and oxidised sterols, in CSF offers considerable potential as biomarkers of neurological disease. 4.3. Barth syndrome One of the best characterised ‘‘lipidomic’’ derangements [87], Barth syndrome (BTHS), is a rare cause of congestive heart failure in infants [88]. Its original description showed a typical presentation of a multisystem disorder involving cardiomyopathy, cyclic neutropenia, growth retardation, proximal and distal muscle weakness and elevated excretion of 3-methylglutaconic acid [89]. BTHS is an X-linked, inherited disease involving derangement of the tafazzin gene (TAZ) [90–93]. TAZ is remarkable in sharing considerable sequence homologies with phospholipid acyltransferases [94]. Consequently, aside from those few cases with no recognised TAZ mutations, BTHS likely reflects a defect in phospholipid metabolism. The principal molecular defect in TAZ-associated mutation is reduced cardiolipin (CL) content in cells [95,96]. While recognition of CL involvement is relatively recent, intensive subsequent studies with a strong focus on ESI-MS/MS-based phospholipid analyses have delineated much of the molecular basis of BTHS [97–100]. Since CL is a dominant mitochondrial lipid, it is most abundant in cells with large numbers of mitochondria such as cardiac and skeletal myocytes [101]. Indeed, 15–20% of mammalian heart phospholipid comprises CL [101] localized almost exclusively to the inner mitochondrial membranes where it has a function in myocardial electrical conductivity [102]. Tetralinoleoyl-CL and trilinoleoyl-oleoyl-CL are the major mitochondrial CL molecular species [103]. Reduced transfer of linoleic acid into CL of BTHS fibroblasts [95] and absence of tetralinoleoyl-CL in platelets [96,97], fibroblasts [98], and muscle tissue [96] of BTHS patients is consistent with an acyltransferase deficiency. However, CL molecular species are altered even in lymphoblasts that do not contain any tetralinoleoyl-CL, suggesting a more general impairment of fatty acid trafficking from and to CL [104] and possibly other lipids. Indeed, lipidomic analyses of other BTHS phospholipids reinforce this view, with increased content of linoleate in PC and, to a lesser extent, in PE at the expense of 16:0/20:4 species [99]. This may have functional implications, for example in eicosanoid-dependent signal transduction in BTHS hearts. All data suggest a misdirection of fatty acids between CL, PC and PE. How lipid abnormalities relate to the various phenotypes and genotypes remains uncertain. Links between tetralinoleoyl-CL deficiency and TAZ mutation suggests involvement in the transfer of linoleoyl groups to CL though some mutations could retain partial acyltransferase activity rather than resulting in total enzyme ablation. CL lipidomic analyses are potentially a valuable diagnostic tool to identify and differentiate those cardiomyopathies reflecting TAZ mutation. Diagnostic CL analysis in platelets is amenable to use of a rapid mass-spectrometric technique [97] a potential routine screen in the evaluation of all children with cardiomyopathy in whom X-linked inheritance is suspected. Therapeutic correction of the CL derangement by lipid supplementation with 18:2 aimed at reinstating tetralinoleoyl-CL species and based upon its ability to restore levels in BTHS fibroblasts in vitro, has also been suggested but not yet evaluated clinically. 4.4. Diabetes Turk and co-workers presented a comprehensive ESI-MS molecular-species analysis of PE and PC in a range of adult rat tissues, with particular emphasis on isolated pancreatic islets [105]. They used a combination of ESI and ESI+ scans with and without the addition of LiOH, together with product, precursor and neutral loss scans. They demonstrated a preponderance of arachidonoyl-containing species in both rat islet PC and PE (77% total PE) and confirmed by acid hydrolysis of the vinyl bond that over 50% of this PE fraction were plasmalogen ether species. Analysis of isolated insulin secretory granules demonstrated a comparable enrichment of arachidonoyl-plasmenylethanolamine species, and liberation of arachidonate by action of PLA2 was proposed as one mechanism for membrane fusion in the secretion process by formation of inverted micellar structures. Human islet PE was very similar to that of the rat, but with a lower content of arachidonoyl species

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(64%) in agreement with observations that rodent phospholipid in general are enriched in arachidonate. Comparison of rat islet PE with other rat tissues clearly illustrated the extent of tissue specificity. Rat brain PE contained a similarly high proportion of plasmenylethanolamine, but this comprised predominantly docosahexaenoyl rather than arachidonoyl species. By comparison, almost 50% of rat liver and heart PE contained arachidonate, but plasmalogen species were virtually absent from liver and much lower in heart than in either pancreatic islet or brain. Further analysis of rat heart showed dramatic alterations to phospholipid molecular species in response to streptozotocin-induced diabetes that were substantially ameliorated by insulin administration [106]. PI and PE plasmalogen concentrations were both increased by almost 50%, while that of the diacyl species PE18:0/20:4 was decreased by 22%. By contrast, no changes were observed to either composition or concentration of PC. There was extensive alteration to rat myocardial triacylglycerol (TAG); tripalmitin content increased 5-fold while the sum of all TAG species containing at least one polyunsaturated fatty acid decreased by some 60%. Intriguingly, in contrast to the diabetes-induced changes to rat heart phospholipid, these alterations to TAG species concentrations were not corrected by insulin treatment. It is, of course, not clear the extent to which these changes in chemically induced diabetes have implications for human diabetes patients but they suggest one mechanism that may contribute to insulin resistance in muscle from diabetic patients. 4.5. Nutrition Mammals are unable to synthesis n-6 and n-3 polyunsaturated fatty acids de novo and consequently all the tissue-specific distributions outlined above must be dependent on an adequate dietary intake principally of the precursors linoleate and a-linoleneate. Given this dependence, it is perhaps surprising that no comprehensive ESI-MS study has yet been published either in human subjects or experimental animals of the effects of dietary lipid modulation on plasma and cellular compositions of individual phospholipid molecular species. Duffin et al. addressed the nutritional aspects of phospholipid species distribution in essential fatty acid deficient mice [107]. Balb/C mice were fed either adequate amounts of corn oil-based diet or an essentially fatty acid depleted diet containing virtually no n-6 or n-3 unsaturated fatty acids for at least 8 weeks following weaning. The study was somewhat limited, in that compositions were only reported of liver and peritoneal macrophage PC and diacyl PE, while plasmalogen PE species would not have been detected by the neutral loss scan of m/z 141 used to determine PE and no attempt was made to distinguish relative contributions of isobaric species. Nevertheless, the changes observed were very significant, especially in comparison to the effective essential fatty acid deficiency of most cultured cells described above. Liver PC and PE in the fatty acid sufficient mice both contained a high proportion of arachidonoyl and docosahexaenoyl-containing species, which were virtually absent in the fatty acid deficient mice and replaced with monounsaturated species. 4.6. Analysis of oxidised phospholipids There is increasing evidence that oxidatively modified phospholipids play a crucial role in the development of atherosclerosis and other diseases (reviewed in [108]). This phospholipid oxidation may occur extracellularly as part of the lipoprotein particles or intracellularly as part of the cell membrane. The chemical site of oxidation is the sn-2 position of the phospholipid that contains the polyunsaturated fatty acids, normally linoleate and arachidonate. Further metabolism can produce a variety of bioactive molecules. Selective removal of the oxidised fatty acid will produce lysophosphatidylcholine (lysoPC) and also lysophosphatidic acid (lysoPA), both of these molecules being involved in signalling processes. LysoPA is a potent growth factor and has been reported as the endogenous ligand for PPARc [109]. The oxidised fatty acids that are released may also function as ligands for PPAR systems. The cleavage of the oxidised fatty acid to produce derivatives such as 2-oxovaleroyl groups at the sn-2 position can result in adduct formation with the lysine groups of proteins. Shortened fatty acids at the sn-2 position following oxidation will generate PAF-like lipids. This broad scenario of metabolic events following phospholipid oxidation can thus impact on a variety of regulatory functions within the cell and highlights the need to identify and quantify oxidised phospholipids and metabolites within cells and tissues.

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ESI-MS analysis of oxidised phospholipids has highlighted a number of novel compounds with biological activity. Most studies have looked at products of in vitro Cu2+-mediated oxidation either of individual polyunsaturated phospholipid molecules or of various lipoprotein preparations, which produces an extremely large number of potential oxidation products. The relevance of these extreme conditions to the much more modest modifications to LDL in atherosclerosis has not been clearly demonstrated. Additionally, such studies have generally concentrated on a relatively restricted number of oxidation products, and it is by no means certain that these are the most biologically active molecules generated in vivo. Oxidation of LDL-phospholipid where the sn-1 position is an ether bond results in formation of products with inflammatory PAF-like activity. ESI-MS identified production of 1-O-hexadecyl-2-(butanoyl or butenoyl)-PC which exhibited PAF activity that was only 10-fold less potent than PAF itself, but which was 100-fold more abundant in oxidised LDL [110]. The uncontrolled production of PAF-like molecules resulting from phospholipid oxidation could have serious physiological consequences and has been reviewed [111]. The structural identification by ESI-MS of oxidised phospholipids in minimally oxidised LDL revealed the presence of three products that induced monocyte adhesion to human aortic endothelial cells in bioassays. Two products, 1-palmitoyl-2-(5-oxovaleryl)-PC (m/z 594.3) and 1-palmitoyl-2-glutaryl-PC (m/z 610.2) were unambiguously identified and confirmed by chemical synthesis [112] and was also produced by the oxidation of PC16:0/20:4. The third product (m/z 828.5) was subsequently identified as an epoxyisoprostane phospholipid with the structure 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-PC [113] and is another example of a biologically active isoprostane. The role of oxidised phospholipids in promoting LDL uptake by the macrophage scavenger receptor CD36 is an important pathway in atherogenesis and 1-palmitoyl-2-(5-oxovaleryl)-PC has been implicated in this process [114]. Recently, a novel family of oxidised phospholipids has been identified by LC/ESI/MS/MS that serve as ligands for the macrophage scavenger receptor CD36. The family of compounds resulted from the oxidation of either 1-palmitoyl-2-arachidonyl-PC or 1-palmitoyl-2-linoeyl-PtdCho using a myeloperoxidase–H2 O2 –NO2 system of monocytes. The active phospholipid derivatives that bound to CD36 contained an sn-2 acyl group that incorporates a terminal c-hydroxy (or oxo) -a, b-unsaturated carbonyl. Referred to as oxPCCD36, these oxidised phospholipids are formed during LDL oxidation by multiple distinct pathways as demonstrated by LC/ESI/MS/MS [115] and were confirmed by chemical synthesis. The authors also use LC/ESI/MS/MS to demonstrate that oxPCDC36 are generated in vivo and are enriched in atherosclerotic lesions [116]. 4.7. Analysis of specific sub-cellular membrane fractions The sensitivity of ESI-MS has provided a unique opportunity to analyse the phospholipid composition of defined sub-cellular components. Early work involving the human erythrocyte plasma membrane [13], platelets membranes [15] and the membranes of pancreatic islets [16,105] have been described above. These studies have pointed to a considerable degree of segregation of phospholipid molecular species even between the membranes of adjacent sub-cellular compartments, but obviously the purity of such compartments is critical for interpretation of the results. 4.7.1. The endonuclear compartment ESI-MS analysis supports the existence of an endonuclear pool of phospholipid, in association with the nuclear matrix and distinct from the nuclear envelope (NE). The distribution of phospholipid classes within this endonuclear compartment is unremarkable in overall composition and is present at typically 4–10% of total cell phospholipid. However, in molecular-species terms there is remarkable enrichment and retention of saturated PC within endonuclear PC and studies on PC biosynthesis by this endonuclear system have highlighted in situ biosynthesis from compartmentalised Kennedy pathway enzymes [42]. This endonuclear region was enriched in molecular species of PC where both fatty acids were saturated, comprising 60.3% of total endonuclear PC as compared with 16.8% for whole cells. In contrast PC species containing a polyunsaturated fatty acid, particularly 20:4 and 22:6, comprised 27.9% of whole cells but were below the level of detection (<0.5%) for the endonuclear pool. Biosynthetic studies involving (methyl-d9)-choline indicated that there was a progressive remodelling of newly synthesised endonuclear PC to more saturated species within the nuclear matrix and it has been proposed that endonuclear PC synthesis may regulate periodic nuclear

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accumulations of PC-derived lipid second messengers [42,53]. While it has not been consistently possible to identify endonuclear membranous systems by conventional EM visualisation methods [117], these typically rely on reacting OsO4 with lipid species containing unsaturated fatty acids. As such unsaturated species are only resent at low abundance within the nucleus, any nuclear membrane structures would in effect be essentially invisible using these techniques. 4.7.2. The nuclear envelope The double bilayer membrane that envelopes the nucleus is spatially distinct from the endonuclear lipid pool, and its phospholipid is synthesised on the endoplasmic reticulum (ER) rather than within the nucleus [118,119]. Analysis of myocardial nuclear envelope 119[124] showed that PUFA-containing molecular species comprised more than 50% of PC and virtually all of the PE pool. This composition is strikingly different from that of endonuclear phospholipid and the abundance of unsaturated species in the nuclear envelope is most likely related to the proximity of lipases, cycloxygenases and lipoxygensases that generate eicosanoids at both inner and outer bilayer [120]. The presence so close to the genomic material of such oxidisable lipid species together with their oxidation products may provide a teleological rationale for the existence of the newly recognised NE-associated antioxidant systems [121]. Similarly, the presence of saturated lipid within the nucleus may act to prevent the dissemination of oxidant species across the nucleic acid material [42,117]. 4.7.3. Intracellular lipid droplets Lipid droplets within the cell have been shown to have a surface monolayer of phospholipid [122]. The ESIMS analysis of this monolayer revealed PC with a specific fatty acyl composition. Thus, in the lipid droplet the peak of m/z 786.4 corresponding to a diacyl 36:2 resolved at high ionisation voltage to primarily two fatty acid chains of 18:1 (PC18:1/18:1) whereas the same parent ion from the rough ER resolved into a mixture of both 18:0 and 18:2 (PC18:0/18:2). Similarly, m/z of 758.4 resolved into a mixture of 16:1 and 18:1 in the lipid droplet sample [122]. Thus the composition of the lipid droplet surface was distinct from the rough ER membrane while separate analyses revealed its composition to be different from the sphingolipid/cholesterol-rich microdomain of cells identified as a Triton-X-100-insoluble floating fraction. The precise function of this specialised phospholipid monolayer surrounding the lipid droplet has yet to be determined. 4.7.4. Detergent resistant membranes/lipid rafts One of the problems inherent in all lipid analyses of sub-cellular fractions is the degree to which this reflects lipid distributions within the membrane in situ. For instance, a quantitative analysis of phospholipids in detergent resistant microdomains (DRM) from RBL-2H3 mast cells using ESI-MS resolved over 90 different phospholipid species with a high degree of saturation compared with total cellular lipids [123]. Significant difference in headgroup distribution between DRM and plasma membrane vesicles isolated as a result of chemically induced blebbing was demonstrated, with the vesicles exhibiting a degree of phospholipid saturation intermediate between DRM and total cellular phospholipids. Analysis of plasma membrane lipids in our laboratory also demonstrated that the plasma membrane was enriched in saturated PC species compared with the whole cell. Moreover, the composition of plasma membrane PC was very different from that of endonuclear PC from the same cells with an unexpected enrichment of alkyl species (Fig. 5). While it is attractive to propose that this high degree of saturation promotes a liquid-ordered structure in the membrane, presumably required for biological function, caution must be exercised with such studies to eliminate the possibility that measured compositions can reflect individual lipid solubilities in detergent rather than membrane structures [124]. The changes in phospholipid composition in RBL-2H3 cells following degranulation were determined by ESI-MS [125], resolving more than 130 different phospholipids. The effects of adding externally phospholipases (PLA2, PLC, PLD) on phospholipid profiles were determined, as all these enzymes initiated degranulation. These phospholipases showed a preference for PC with long polyunsaturated alkyl chains as substrates while PA was generated by treatment with PLC and PLA2 in addition to PLD. Degranulation paralleled the percentage of PA produced by the phospholipase treatment. Such studies should help define specific functions for particular phospholipid molecular species required in cell functions such as regulated exocytosis.

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A study looked at the changes in the lipid turnover, composition and organisation as sphingolipid-enriched membrane domains in rat cerebellar granule cells developing in vitro. They used ESI-MS to look at PC composition of sphingolipid-enriched fractions during cell development in culture and observed that this fraction was enriched in PC16:0/16:0 and this represented the main glycerophospholipid species in the membrane fraction [126]. The analysis of other phospholipid classes by ESI-MS was not reported. This is another example of the involvement of disaturated PC species in specific membrane environments, presumably related to overall membrane function.

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Fig. 5. Compositions of PC from sub-cellular fractions of HL60 cells. PC molecular species from (A) whole cells, (B) the endonuclear compartment of the nuclear matrix and (C) the plasma membrane were determined by precursor scans of m/z +184. Saturated diradyl species (the diacyl species PC16:0/14:0 and PC16:0/16:0 and the 1-alkyl-2-acyl species PC16:0a/16:0) were enriched in endonuclear PC compared with whole cells. While saturated PC species were not enriched in plasma membrane, both endonuclear and plasma membrane fractions were selectively depleted of PC species containing polyunsaturated fatty acids. Sphingomyelin species (16:0SM, 24:1SM and 24:0SM) were also considerably enriched, especially in the plasma membrane fraction where they became the most abundant component. As plasma membrane was purified by partitioning into a biphasic polyethylene glycol/dextran mixture [141] without involvement of membrane modifying detergents, a major potential source of artefactual manipulation of membrane composition was eliminated from this analysis.

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Lipid rafts are specialised plasma membrane domains that have an important role in cell signalling and metabolic regulation. There has been considerable controversy about the structure and composition of lipid rafts, largely due to the inherent difficulty of their analysis in the absence of detergent extraction. A recent consensus definition is that they are ‘‘small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalise cellular processes. Small rafts can sometimes be stabilised to form larger platforms through protein–protein and protein–lipid interactions’’ [127]. Caveolae are related specialised domains associated with cell signalling events. The distinct identity of caveolae is associated with the expression of the structural protein caveolin-1 in the caveolae. Caveolin-1 is a cholesterol-binding integral membrane protein that also appears to be a negative regulator of cell growth. The precise relationship between these two domains is not clear, however, lipid rafts from cells that lack caveolae contain a similar complement of signalling proteins to those that express the protein. Thus, caveolae and lipid rafts are thought to be specialised regions of the plasma membrane that facilitate signal transduction by mechanisms that remain to be fully elucidated. To fully understand the signalling function of caveolae and lipid rafts, an analysis of the phospholipid composition is essential. The ESI/MS analysis of lipid rafts from caveolin-1 expressing and non-expressing cells, isolated by sequential density gradient centrifugation in the absence of detergent, has revealed that this phospholipid composition differed from that of the plasma membrane fraction [128]. The most striking difference was that lipid rafts were enriched in plasmenylethanolamines, particularly those containing arachidonic acid. The enrichment of arachidonic acid in these domains implies that these domains may represent a localized pool of substrate for the generation of free arachidonic acid in response to cell activation. PS is also substantially enriched in lipid rafts but these domains are relatively depleted in PI. Detergent resistant microdomains from the same cells showed a higher cholesterol content than the lipid rafts, but were depleted of anionic phospholipids and arachidonoyl species, emphasising once more the caution required for interpretation of detergent extraction procedures. The expression of caveolin-1 in otherwise caveolin1-negative cells induces the formation of caveolae and increases the content of cholesterol in the lipid raft fraction but does not substantially change the phospholipid composition of these domains. Recently, Bru¨gger et al. have reported the ESI-MS analysis of the lipid composition of HIV-1 particles derived from infected MT-4 lymphocyte cells [129]. Compared with cell phospholipid compositions, HIV particles were enriched in sphingomyelin species, especially 16:0 dihydrosphingomyelin, cholesterol, plasmenylethanolamines, PS and cholesterol, and the reduced content of PC was enriched in short chain saturated species. As HIV-1 particles are thought to bud from lipid raft structures, this elegant work provides an independent approach to support the concept that the properties of lipid rafts are dramatically different from those of the surrounding plasma membrane and this is likely to contribute to the specialised functions of these lipid domains [128]. 4.8. Analysis of low abundance natural phospholipids: signalling lipids Perhaps the greatest challenge facing ESI-MS technology for lipid analysis is to quantify mass and compositional changes of signalling lipids in response to agonist stimulation. Analysis of diacylglycerols is relatively straightforward by HPLC–MS [130], although distinguishing species generated by PLC-mediated hydrolysis of membrane phospholipid from that of bulk cellular DAG remains problematical. Analysis of signalling phosphatidate by MS of phosphatidylalcohols formed by PLD-dependent transphosphatidylation is in contrast more specific [130,131]. However, the analysis of intact molecular species of polyphosphoinositides is much more problematic but critically important to determining a wide range of signalling mechanisms. The ability of phosphoinositides to bind to specific protein domains is the basis for the targeting of signalling proteins to specific cell locations as a result of cell stimulation by growth factors and hormones. These phosphoinositides include the well-known species that are phosphorylated at the D-4 and D-5 positions of the inositol ring (PIP2). However, more recently, the phosphorylation of the D-3 position by phosphoinositide 3-kinase produces a further range of phosphoinositides including phosphatidylinositol-3,4,5-trisphosphate (PIP3) (reviewed in [132]). The binding of specific protein domains such as the Pleckstrin Homology (PH) domain, the FYVE domain and the Phox Homology domain to these phosphoinositides result in the targeting of these proteins to specific membrane surfaces while the association of particular proteins at the membrane surface as

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a crucial event in cell signalling (reviewed in [133,134]). Therefore the location of specific phosphoinositides within cell membrane domains and the change in levels as a result of cell stimulation is a major challenge in understanding the molecular events associated with cell signalling. The selective and specific analysis of intact molecular species of polyphosphoinositides is a very clear example of both the strengths and weaknesses of ESI-MS approaches to determining the dynamics of phospholipase-mediated signalling. PI molecular species are readily analysed by ESI-MS by precursor scan of m/z 241, a common dehydrated cyclic inositol phosphate fragment [24], and comparable methodologies have been established to characterise phosphatidylinositol monophosphate (PIP) (precursor scan of m/z 321) and PIP2 standards (precursor scan of m/z 401) [135]. These studies have shown that the PI-3-P and PI-4-P monophosphoinositide isomers generate different CID-induced fragment ions, raising the possibility of establishing diagnostic precursor scans to distinguish between these two isomers. So far, however, no comparable diagnostic scan has been proposed that would distinguish between intact PIP2 species with phosphates at the 3,5 rather than at the 4,5 positions. However, although methods for pure standards can be readily established, many groups have experienced severe problems translating such methodologies to the analysis of phosphoinositides in biological systems. Establishing a robust, sensitive and discriminatory assay for phosphoinositide isomers has become a major focus for many MS-based research groups. Examination of the problems inherent in the development of ESI-MS assays for phosphoinositides is instructive as an extreme paradigm for the wider application of ESI-MS to analysis of signalling lipids generally present at low abundance in cells. 1. Depending on ionisation conditions, PIP2 can generate either the [M 1] or the double deprotonated [M 2]2 in variable proportion, so maintenance of constant MS conditions becomes critical. Moreover, the intensity of the PIP2 molecular ion is proportionally considerably lower than that of PI, probably due to charge repulsion effects when adjacent phosphates on the inositol ring are ionised. 2. Extraction of polyphosphoinositides from biological matrices is beset by difficulties. Protonation at low pH is essential for their extraction into organic solvents, but the acyl groups are very labile under acidic conditions. This can be observed, for instance, by formation of deacylated lyso derivatives of added internal standards [136]. 3. Ion suppression can often mask the signal from very low abundance species. 4. Polyphosphoinositides bind avidly to glass and metal surfaces, which can severely limit their recovery both in extraction and in the mass spectrometer itself. 5. Adherence of other components of organic extracts of cells, such as proteolipid, can also provide additional binding sites for phosphoinositides. This can be clearly seen for sequential direct injections of a lipid extract in this case from Jurkat T-cells which initially contained measurable quantities of PIP2 (Fig. 6). The phosphoinositide response decreased progressively with repeated injection even if authentic standard PIP2 was added to the sample. The hydrophobic nature of phosphoinositides means that ion exchange chromatographic approaches widely used to resolve deacylated isomers are not applicable to separation of intact species. A number of approaches have been reported that overcome some of these problems, and some show considerable promise for application to biological questions. Use of piperidine as a phase modifier during extraction with acidified chloroform and methanol and subsequent direct injection [137] improved signal responses for both PIP and PIP2, but did not overcome the fundamental problems of binding to surfaces and acid lability. A two step extraction, initially with neutral and then with acid solvent [138], increased recovery and sensitivity by the removal of less acidic components during the first extraction and enabled detection of PIP3 as well as PIP2 and PIP species. While a significant improvement, phosphoinositides extracted by this approach were still potentially susceptible to acid hydrolysis and the direct injection ESI-MS analysis precluded resolution for instance of PI-3-P from PI-4-P. Very recently, extraction at low pH using a citrate buffer gave more efficient recovery with much less hydrolysis and, coupled with normal phase LC–MS or MS3 analysis, enabled the resolution of isomers of both PIP and PIP2 and detection of PIP3 [136]. This analytical approach is still technically demanding and the authors caution about variabilities in extraction efficiency and LC resolution that can seriously impair the quality of the procedure.

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18:1/18:1

1045.8 1047.8 1043 1023.8

1021.8

a

1 st injection

b

5 th injection

c

20 th injection m/z 980

1000

1020

1040

Fig. 6. The effect of multiple injections on the ESI-MS/MS signal response to PIP2. These precursor ion spectra (precursor of m/z 401) show the response to Jurkat cell PIP2 molecular species (a) for the initial sample following rigorous cleansing of the ESI source and capillary tubing, (b) following five previous sample injections and (c) following 20 previous sample injections. There is complete loss of signal by 20 injections even if a measurable quantity of authentic PIP218:0/20:4 (m/z 1045.8) is added to the extract just prior to analysis.

Even with these prototype analytical procedures, a number of tentative conclusions can be inferred about phosphoinositide molecular-species compositions in mammalian cells. First, although PI composition in most cells is predominantly the 18:0/20:4 species, this specificity is generally lost in cultured cells for which PI is generally monounsaturated. For all cell types analysed so far, the molecular species composition of PIP2 closely reflected that of PI, suggesting little molecular specificity of the responsible lipid kinases. Typical mass spectra of PI and PIP2 are shown for a mouse/T-lymphocyte hybridoma in Fig. 7 as respectively a precursor scan of m/z 241 and a neutral loss scan of 98 (loss of phosphate). Currently, there is too little information available to formulate a clear idea of any potential selectivity in the formation of PIP3. Analysis of platelets showed little selectivity at the peak of PIP3 generation two minutes after thrombin stimulation, although this may possibly have been influenced by the essentially monospecific 18:0/20:4 composition of the substrate PIP2 [136]. Analysis of PIP3 in stimulated RAW 264.7 macrophages suggested differential species generation, but the PIP2 composition of these cells was not physiological [138]. More definite conclusion here awaits improvements in the robustness and reproducibility of analytical techniques to enable their application to a wider range of primary and cultured cells in response to a number of different agonists. Addressing this important goal of resolving phosphoinositide species and isomers remains a major task in lipidomics research and, as a PIP2

PIP218:1/18:1

1021.4 1023.6

100

PIP216:/16:0 int. std.

NL98: ES -ve

1045.4

PIP218:0/20:4

995.5

%

969.5 1071.6

0

m/z 975

1000

PI

1025

861.5

100

1050

1075

PI18:1/18:1

PI16:0/16:0 int. std. 863.4

PI16:0/18:1

%

P241: ES -ve

PI18:0/20:4 885.4

835.4 809.4

911.5 0 800

m/z 820

840

860

880

900

920

Fig. 7. The composition of PIP2 from B3Z hybridoma T-lymphocytes. The lower panel shows PI species determined by precursor ion scan of m/z 241 (dehydrated phosphoinositol) and the upper PIP2 species determined by neutral loss of m/z 98 (phosphate). The upper spectrum has been offset by 160 mass units to align PI and PIP2 responses. PIP2 always reflects the very variable composition of PI in cultured cells, suggesting little acyl specificity for its synthesis.

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crucial aspect of their function is their specific location within the cell, final analysis will require the measurement of defined sub-cellular membranes in conjunction with a variety of imaging techniques. 5. ESI-MS of membrane phospholipids: the state of the art and future directions The analysis of membrane and signalling lipids by ESI-MS is a new but rapidly expanding field of research, supported by considerable technological innovations. Advances in mass spectrometry technology, especially the development of hybrid instruments such as QToF, quadrupole:linear ion trap and linear ion trap:Orbitrap mass spectrometers, are providing platforms for increasingly sophisticated analyses, while newer interfaces such as automated chip-based nanoelectrospray systems potentially offer true high throughput analysis compatible with the information provided by genomics and proteomics [139,140]. Finally, considerable effort is being put into the bioinformatic aspects and the classification system for lipids proposed by LIPID MAPS [12] provides an internationally agreed platform for establishing experimental data standards. The review of the use of ESI-MS has highlighted the crucial role played by phospholipids in lipid signalling and cell targeting. The most important issues relate to (a) being able to analyse the phospholipid composition of defined sub-cellular membrane structures or domains and (b) being able to quantify and identify the minor phosphoinositides that are now seen to play a central role in cell signalling, providing the structural basis for the targeting of specific cell signalling proteins to the membrane surface. As a cell responds to environmental changes, these phosphoinositides are now seen to play a central role in regulating the adaptive response. The analysis of oxidised phospholipid continues to be another important area directly related to clinical disorders. The completion of various genome projects, and hence the identification of all possible expressed proteins, has resulted in attention being switched to the function of systems in real time within the cell. The expression of genes within a cell under particular conditions using DNA arrays to identify message and the subsequent identification of the proteins expressed by theses systems and their regulation using proteomics are now the cornerstone to providing a holistic approach to explaining cell function at the molecular level (integrative biology) and for identifying new drug targets. The critical role of membrane and signalling lipids in regulating these processes highlights the importance of detailed lipidomic analysis. In addition, true integration of lipidomic with genomic and proteomic approaches will require dynamic as well as static lipid analyses. Using ESI-MS/MS to provide lipid pathway fluxes from incorporation rates of substrates labelling with stable isotopes is an exciting approach to progress in this area of research. References [1] Han X, Gross RW. Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry: a bridge to lipidomics. J Lipid Res 2003;44:1071–9. [2] Pulfer M, Murphy RC. Electrospray mass spectrometry of phospholipids. Mass Spectrom Rev 2003;22:332–64. [3] Forrester JS, Milne S, Ivanova PT, Brown HA. Computational lipidomics: a multiplexed analysis of dynamic changes in membrane lipid composition during signal transduction. Mol Pharmacol 2004;65:813–21. [4] Hokin MR, Hokin LE. Enzyme secretion and the incorporation of P32 into phospholipides of pancreas slices. J Biol Chem 1953;203:967–77. [5] Hokin LE. Receptors and phosphoinositide-generated 2nd messengers. Ann Rev Biochem 1985;54:205–35. [6] Fontell K, Holman RT, Lambertsen G. Some new methods for separation and analysis of fatty acids and other lipids. J Lipid Res 1960;1:391–404. [7] Patton GM, Fasulo JM, Robins SJ. Separation of phospholipids and individual molecular species of phospholipids by HPLC. J Lipid Res 1982;23:190–6. [8] Ridgway ND, Vance DE. Specificity of rat hepatic phosphatidylethanolamine N-methyltransferase for molecular species of diacyl phosphatidylethanolamine. J Biol Chem 1988;263:16856–63. [9] Caesar PA, Wilson SJ, Normand ICS, Postle AD. A comparison of the specificity of phosphatidylcholine synthesis by human fetal lung maintained in either organ or organotypic culture. Biochem J 1988;253:451–7. [10] Burdge GC, Creaney A, Postle AD, Wilton DC. Mammalian secreted and cytosolic phospholipase A2 show different specificities for phospholipid molecular species. Int J Biochem Cell Biol 1995;27:1027–32. [11] Hankin JA, Murphy RC. Lipid mediators and mass spectrometry: an historical perspective. Int J Mass Spectrom 2000;200:201–17. [12] Fahy E, Subramaniam S, Brown HA, Glass CK, Merrill Jr AH, Murphy RC, et al. A comprehensive classification system for lipids. J Lipid Res 2005;46:839–61.

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