1. Methodology of lipidomics: Tandem mass spectrometry of ether phospholipids

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1 Transworld Research Network 37/661 (2), Fort P.O. Trivandrum Kerala, India Lipidomics: Sea Food, Marine Based Dietary Supplement, Fruit and Seed, 2012: 1-19 ISBN: Editor: 1. Methodology of lipidomics: Tandem mass spectrometry of ether phospholipids Chainon Neurotrophin Biotechnology Inc. San Antonio, Texas the United States of America Abstract. Tandem mass spectrometry of intact lipids is a highly specialized technique. It has been widely used in structural elucidation of molecular species of various ether phospholipid subclasses. All marine animals contain abundant ether phospholipid species. Although a combination of liquid and gas chromatography is the time-consuming in providing such structural information of a common phospholipid molecular specie, the characterization of ether bond linkage at the sn-1 position of an ether phospholipid species including plasmenyl (1-O-alk-l -enyl linkage) and plasmanyl (1-O-alkyl linkage) forms is difficult or impossible, especially the differentiation of phospholipid species isomers having an identical molecular weigh but containing different ether bonds and fatty acid chains esterified by using this approach. In the present Chapter, the application of tandem mass spectrometry for structurally analyzing molecular species of various ether phospholipids by the unique method, particularly in the differentiation of molecular species of plasmenyl and plasmanyl phospholipids in biological samples including the structural elucidation of isobaric species isomers, are reviewed. Correspondence/Reprint request: Dr., Vance Jackson Suite 721, San Antonio, TX the United States of America. su@chainonbio.com

2 Introduction Lipids play a variety of roles in biological systems. They are molecules that are insoluble in water and soluble in organic solvents. The diversity of lipids can be very high due to (i) the complexity of the hydrophobic acyl chains and (ii) the nature of the hydrophilic polar-head group. Thus, over a thousand different lipid molecular species can be theoretically devised which exhibit a wide polar range. Lipids can be divided into non-polar lipids and polar lipids based on the number of primary products formed after hydrolysis ( A glycerophospholipid (GPL) class consists of a mixture of molecular species. Their structural diversity is due to (i) a variety of fatty chains esterified at the sn-1 and sn-2 positions of the glycerol backbone, (ii) locations of the double bond(s) (between 1-6) within unsaturated fatty acid chains, which are usually located at the sn-2 position with a number of carbon atoms (between 14-24), and (iii) a polar-head group linked at the sn-3 position. For example, phosphatidylethanolamine (PE) or glycerophosphoethanolamine represents a group of molecular species having an ethanolamine polar-head group at the sn-3 position and different fatty acid chains esterified at the sn-1 and sn-2 positions of the glycerol backbone (Fig. 1a). Ether GPLs are usually present in various marine animals as major components [1], existing together with diacyl molecular species carrying the same polar head group (Fig.1-left). Ether GPLs have the two predominant types of ether bond linkages in the molecules. One form is represented by the plasmalogens (1-O-alk-1 -enyl fatty chain(s) linked to the sn-1 position of the glycerol backbone; also called plasmenyl GPLs), which is the most abundant subclass of GPLs in the most of tissues (Fig. 1-right). The other form is alkyl ether linkage (Fig. 1-central), called plasmanyl form that contains 1-O-alkyl fatty chain(s) linked to the sn-1 position of the glycerol backbone. It has been reported that plasmanyl GPLs are abundant in marine fish livers [1]. A lysophospholipid ether species contains only either 1-O-alk-1 -enyl chain (plasmenyl linkage) or 1-O-alkyl chain (plasmanyl linkage), which is linked to the sn-1 position of the glycerol backbone (also see Fig. 1). The function of GPLs in a number of biochemical processes are well documented and appear to be determined by both the polar-head and composition of fatty acyl chains, esterified either at the sn-2 position or at the sn-1 position. For examples, phosphatidylserine (PS) or glycerophosphoserine shows the effect on anti-inflammatory [2] and sport medicine [3], rather than phosphatidylcholine (PC) or glycerophosphocholine (the polar head selectivity); the Caco-2 cell barriers allow 16:0/22:6 PC (the code N:M/n:m PC, where N

3 Methodology of lipidomics: Tandem mass spectrometry of ether phospholipids 3 Figure 1. Chemical structures of diacyl PE (left), 1-alkyl-2-acyl PE (central) and 1-alkenyl-2-acyl PE or 1-O-alk-1 -enyl-2-acyl PE (right). is the total number of carbon in the sn-1 fatty chain, M is the total number of double bond(s) in the fatty chain esterified at the sn-1 position; n is the total number of carbon in the sn-2 fatty chain, m is the total number of double bond(s) in the fatty chain esterified at the sn-2 position; and PC means PhosphatidylCholine; the code is often used for indicating the nature of the position and unsaturation of the fatty chains esterified in the glycerol backbone and the polar-head moieties of GPLs) and 16:0/20:5 PC species to pass through, whereas 16:0/18:2 PC species are blocked (the fatty acid chain selectivity) [4]; endothelial lipase prefers to catalyze PE, as well as DHA PC and DHA PE species, rather than PS, ether GPLs, and other species (selectivity of both the polar head and composition of fatty acid chains) [5]. Conventional strategies for analyzing various GPLs and their molecular species including ether GPLs require many steps. The first step is to extract a total lipid fraction from a biological sample, followed by isolating individual GPL class from other lipids by either silica thin-layer chromatography (TLC) or normal-phase high performance liquid chromatography (HPLC). Subsequent steps include the removal of the GPL head-group, derivatization of the sn-3 position of glycerol backbone, and then separation by normal- and reversed-phase HPLC. Individual GPL class (separated by TLC or/and

4 4 normal-phase HPLC) can further be separated into their molecular species by reversed-phase HPLC. After species are collected from liquid chromatography, samples are then saponified and esterified to prepare fatty acid methyl esters and dimethylacetals that can be analyzed by gas chromatography (See the Chapter 5 in this book). It is clear to see that these prior separations and manipulations are labor-intensive and time-consuming, resulting in suffering from reduced recovery and selective losses of certain GPL molecular species up to 50%. The analysis of unprocessed total lipid extract by HPLC coupled to electrospray ionization (ESI) tandem mass spectrometry (LC/ESI-MS/MS) or direct flow-injection ESI and tandem mass spectrometry (ESI-MS/MS; or called shotgun lipidomics [6]) bypasses many of these analytical problems because it i) requires minute amounts of biological samples, ii) separates GPL molecular species by employing a soft ionization procedure that produces mostly singly charged molecular ions of mass spectrometryisolated GPL species by molecular weight differences of the lipids, iii) produces reproducible mass spectra with much less overlapping molecular ions of GPL species, (iv) has the advantage of analyzing bioactive minor GPL species, such as various ether GPLs. Tandem mass spectrometry of intact lipids is a highly specialized technique. It has been widely used in structural elucidation of molecular species of various GPLs and their ether subclasses. The major advantage of the powerful technique is to enable to generate informative fragments, corresponding to the characteristics of (i) the composition and location of fatty chains esterified at the sn-1 and sn-2 positions of the glycerol backbone, (ii) the double-bond linkage positions in the unsaturated fatty acids at the sn-2 position, and (iii) a polar head group linked at the sn-3 position, for determining the chemical structure of an individual GPL species. Although a combination of liquid and gas chromatography is the time-consuming in providing such structural information of a common GPL specie, the characterization of ether bond linkage at the sn-1 position of an ether GPL species including plasmenyl (1-O-alk-l -enyl linkage) and plasmanyl (1-Oalkyl linkage) forms is difficult or impossible, especially the differentiation of GPL species isomers having an identical molecular weigh but containing different ether bonds and fatty acid chains esterified by the combination. In the present Chapter, applications of tandem mass spectrometric approach for structurally analyzing molecular species of various ether GPLs by the unique method, particularly in the differentiation of molecular species of plasmenyl

5 Methodology of lipidomics: Tandem mass spectrometry of ether phospholipids 5 and plasmanyl GPLs in biological samples including the structural elucidation of isobaric species isomers, are reviewed Tandem mass spectrometric analyses of ether GPL species (i) Precursor selection of intact ether GPL species for the MS/MS ESI/MS-generated protonated and deprotonated precursors of intact GPLs for MS/MS: Ether GPL species are significantly abundant in marine animals. Figure 2 shows the positive-ion ESI mass spectra of 1-acyl-2-DHA PC (Fig.2a; purified from monkfish liver), 1-O-alk-1 -enyl-2-dha PC (Fig.2b; made by DHA aceylation with purified bovine heart LysoPC), and 1-O-alkyl-2-DHA PC (Fig.2c; purified from shark liver). It is clear to see that due to the diversity of the chemical structure, the molecular weight of plasmenyl (16:0)/DHA PC (at m/z 790 in Fig.2b) and plasmanyl (16:1)/DHA Figure 2. Positive-ion ESI mass spectra of DHA-containing diacyl PC(a), plasmenyl PC (b) and plasmanyl PC (c).

6 6 Figure 3. Chemical structures of plasmanyl (16:0)/DHA PC (a) and plasmenyl (16:0)/DHA PC (b). Figure 4. ESI mass spectra of diacyl and (ether bond linkage) molecular species of shark liver PC (a) and PE (b). PC (at m/z 790 in Fig.2c), as well as plasmanyl (16:0)/DHA PC (at m/z 792 in Fig.2c) are less than 16 and 14 Da, respectively, compared with 16:0/DHA PC (at m/z 806 in Fig.2a). For another example, an ion at m/z 834 in Fig. 2a is due to 18:0/DHA PC; peaks at m/z 818 (834-16) in Fig. 2c and m/z 818 (834-16) in Fig. 2b correspond to plasmanyl (18:0)/DHA PC and plasmenyl (18:0)/DHA PC species, respectively. The question is that whether it is possible to identify ions at m/z 790 shown in Fig.1b and Fig.1c, which

7 Methodology of lipidomics: Tandem mass spectrometry of ether phospholipids 7 correspond to plasmenyl (16:0)/DHA PC and plasmanyl (16:1)/DHA PC, respectively, as well as the peaks at m/z 818 in Fig.1b and Fig.1c, which related to plasmenyl (18:0)/DHA PC and plasmanyl (18:1)/DHA PC, respectively, if they are present in a GPL mixture. Figure 4 shows the mass spectra of PC (a) and PE (b) extracted from shark liver, mixtures of diacyl, plasmenyl and plasmanyl species. Based on above methods, the differentiation of natural GPL [M + H] + /[M H] - and ether GPL species [(M+H) -14/-16] + /([M-H) - 14/-16] - can be made readily. However, is it possible to distinguish plasmenyl and plasmanyl linkage GPL species in a mixture by tandem mass spectrometry? The answer is Yes. This just shows the first step to carry out tandem mass spectrometric analyses of various GPL species. The rule can be also used for the differentiation of lysophospholipid species and their ether bond linkage molecules. (ii) ESI/MS-generated adduct precursor ions of ether GPL species Because choline GPLs including PC and LysoPC contain a quaternary nitrogen moiety in the molecules, the negative-ion ESI mass spectra of the lipids contain no abundant deprotonated molecules [M - H] - for the MS/MS analyses of PC and LysoPC species. Experiments have shown that lithiated adducts of PC and LysoPC species are readily formed and collision-induced dissociation (CID) MS/MS of their [M + Li] + precursors [7-8] yields mass spectra that provide informative fragments for the differentiation of 1-O-alk- 1 -enyl and 1-O-alkyl linkage ether GPL species. Studies also found that the MS 3 experiments of [M + CL] - [9] and [M + HCOO] - [10] precursors of ether PC species performed with negative-ion ESI coupled to multiple-state linear ion-trap mass spectrometry yields informative fragments that can identify not only the fatty acid chains at the sn-2 position of the glycerol backbone and but also plasmenyl and plasmanyl linkage forms of the ether PC species. Figure 5 shows the positive-ion and negative-ion mass spectra of the lithiated (a) and chlorided (b) adducts of plasmenyl DHA PC species (see figure 2b). The ions can be used as precursors in MS/MS analyses of ether PC species. Ether linkage molecular species of phosphatidylinositol (PI), phosphatidic acid (PA) and phosphatidylglycerol (PG) also can be ionized with ESI/MS to form deprotonated molecules [M - H] - for MS/MS analyses of ether bond linkage GPL molecular species (See below).

8 8 Figure 5. Positive-ion ESI mass spectra of lithiated (a) and cholorided (b) 1-O-alk-1 - enyl-2-dha PC (plasmenyl PC). (iii) Structural elucidation of intact ether GPL species by tandem mass spectrometry (MS/MS) Ether phosphatidylethanolamine (PE): The positive-ion ESI-MS 2 of the [M+H] + and ESI-MS n of the [M+2Li] + precursors: The structural identification of plasmenyl PE species using the CID of the protonated molecules [M + H] + ion generated by ESI/MS has been described [11]. The fragmentation processes (see the Schemes 1 and 2) observed following CID of the protonated molecules of plasmenyl PE species generate unique informative ions for identifying the fatty acid characterization esterified at the sn-2 position (see F1 fragment in the Scheme 1) and 1-O-alk-1 -enyl bond linkage (plasmenyl PE) at the sn-1 position (see F2 fragment in the Scheme 2) of the glycerol backbone. For example, it is possible to detect the presence of a plasmenyl (18:0) fatty chain at the sn-1 position (Scheme 1), by the fragment at m/z 392, and a DHA at the sn-2 position, by the fragment at m/z 385 (Scheme 2), of a plasmalogen PE species using the CID of the precursor ion [M + H] + at m/z 776 [11]. If the performance of a precursor ion scan of m/z 392 can be carried out, it is possible to detect all molecular species containing a plasmenyl

9 Methodology of lipidomics: Tandem mass spectrometry of ether phospholipids 9 Scheme 1. PE positive-ion fragmentation process for the sn-2 fatty acid identification. Scheme 2. PE positive-ion fragmentation process for the plasmenyl linkage identification. (18:0) chain at the sn-1 position of the ether PE. This method should be useful in identifying unique plasmenyl PE species in biological samples. Thus, differentiation of plasmenyl PE from plasmanyl PE by this method using MS 2 product ion mass spectra is not readily available. The plasmenyl PE species can also be identified by the MSn of the [M + 2Li] + precursors on ion trap mass spectrometry [12]. The negative-ion ESI/MS 3 : The further differentiation of plasmenyl PE species and plasmanyl PE molecules has been described [13]. The identification was performed using multiple-stage linear ion-trap mass spectrometry (ITMS), and the MS 3 product ion spectra of the ESI/MSgenerated deprotonated molecules [M H] - [M H R 2 CH = C=O] - fragment ions yield informative product ions for distinguishing 1-O-alk-1 - enyl (plasmenyl) and 1-O-alkyl (plasmanyl) linkage, as the following fragmentation processes (see the Schemes 3 and 4). The differentiation of the two ether linkages is based on the presence or absence of abundant alkenoxide anions [O-CH=CH-(CH 2 )nch 3 ] - (n = 12 20), which is derived from lysophosphatidic acid fragments [M - H - R 2 COOH - polar-head group]-, glycerol phosphoethanolamine fragment at m/z 196 [CH 2 -CHO-CH 2 -PO 4 -CH 2 CH 2 -NH 2 ] -

10 10 Scheme 3. PE negative-ion fragmentation process for the sn-1 plasmenyl linkage identification. (F3 in the Scheme 3) and phosphoethanolamine fragment at m/z 140 [HPO 4 - CH 2 CH 2 -NH 2 ] - (F4 in the Scheme 3), in the MS 3 product ion spectra of plasmenyl PE compounds. Additional diagnostic ions for distinguishing ether PE from diacyl PE are due to losses of the free polyunsaturated fatty acids at the sn-2 position only, rising abundant anions. Furthermore, DHA, EPA and arachidonic acid (AA) anions can be further break to form the ions of [(DHA/EPA/AA) 44] - at m/z 283 (327 44), or m/z 257 (301-44) or m/z 259 (303-44), respectively [14,6]. Scheme 4. PE negative-ion fragmentation process for the sn-1 plasmanyl linkage identification.

11 Methodology of lipidomics: Tandem mass spectrometry of ether phospholipids 11 Figure 6. The MS 2 (a) and MS 3 (b) product ion spectra of p18:0/epa PE, e16:0/dha PE and e16:1/dpa PE (Experiments were carried out on a quadruple-multiple-stage linear ion-trap mass spectrometer (AB Sciex 5500)). Figure 6a shows the MS 2 product ion spectrum of the ion at m/z 748 (see also figure 4b). Anions at m/z 301, 327 and 329 correspond to EPA (20:5), DHA (22:6) and DPA (22:5) fatty acids esterified at the sn-2 position of the glycerol backbone. Fragments at m/z 464, 438 and 436 were formed by the loss of the (R 2 CH-C=O) group from the deprotonated molecule at m/z 748, supporting identification of the fatty acid chains at the sn-2 position. Furthermore, the MS 3 product ion spectrum also further assisted in the differentiation of plasmenyl and plasmanyl linkages in the lipid molecules. The fragments at m/z 403, 267, 196 and 140 suggested that plasmenyl (18:0) linkage exists in the lipid (see F1, F2, F3 and F4 fragments in the Scheme 3), alone with the two co-exist plasmanyl linkages (16:0 and 16:1) species, evidence by fragments at m/z 438 (plasmanyl (16:0) LysoPA H 2 O - H) and m/z 436 (plasmanyl (16:1) LysoPA H 2 O - H). The PE species due to m/z 748 are structurally elucidated as p18:0/epa PE or plasmenyl (18:0)/EPA PE, e16:0/dha PE or plasmanyl (16:0)/DHA PE and e16:1/dpa PE or plasmanyl (16:1)/DPA PE. Figure 7 shows MS 2 (a) and MS 3 (b) product ion spectra of the precursor at m/z 750. Plasmanyl linkage PE can be characterized by the absence of abundant anions corresponding to alkenoxide anions, which is derived from

12 12 Figure 7. MS 2 (a) and MS 3 (b) product ion spectra of e18:0/epa PE, e16:0/dpa PE and 17:0/EPA PE; (Experiments were carried out on a quadruple-multiple-stage linear ion-trap mass spectrometer (AB Sciex 5500)). the Lysophosphatidic acid fragments (F1 fragment in the Scheme 3), glycerolphosphoethanolamine anion (F3 fragment in the Scheme 3) and phosphoethanolamine fragment (F4 fragment in the Scheme 3), in their MS 3 product ion spectra (See the Scheme 4). An ion at m/z 269 is due to a 17:0 fatty acid esterified at the sn-1 position [1]. The peak at m/z 750 corresponds to e18:0/epa PE (plasmanyl (18:0)/EPA PE), e16:0/dpa PE (plasmanyl (16:0/DPA PE) and 17:0/EPA PE. Ether Lysophosphatidylethanolamine (LysoPE): LysoPE negative-ion fragmentation process is shown in the Scheme 5. Plasmenyl and plasmanyl linkage LysoPE can be identified by the MS 2 of their deprotonated molecules [M H] - based on the presence of abundant alkenoxide anions [O-CH=CH- (CH 2 ) n CH 3 ] - (n = 11-19) (F1 fragment in the Scheme 5), glycerol phosphoethanolamine fragment at m/z 196 [CH 2 -CHO-CH 2 -PO 4 -CH 2 CH 2 - NH 2 ] - (F2 fragment in the Scheme 5) and phosphoethanolamine fragment at

13 Methodology of lipidomics: Tandem mass spectrometry of ether phospholipids 13 m/z 140 [HPO 4 -CH 2 CH 2 -NH 2 ] - (F3 fragment in the Scheme 5), which should be present only in the MS 2 product ion spectra of plasmenyl LysoPE species. Plasmenyl linkage LysoPE can be also identified by the MS 2 experiments of their protonated molecules [M + H] + precursors based on the LysoPE fragmentation process Scheme 6 [15]. Scheme 5. LysoPE negative-ion fragmentation process for the sn-1 plasmenyl linkage identification. Scheme 6. LysoPE positive-ion fragmentation process for the sn-1 plasmenyl linkage identification. Ether phosphatidylcholine (PC): The positive-ion ESI/MS 2 of [M + Li] + precursors of ether PC species: The product ion mass spectra of the [M + Li] + adduct precursors provide informative fragments for distinguishing various PC species (see the Scheme 5)[8,16]. The differentiation of plasmenyl and

14 14 plasmanyl PC species in their MS 2 spectra is based on the presence of a prominent ion [CH 2 =CH-O-CH=CH-(CH 2 ) n CH 3 ] + (n = 11 19), arising from the dissociation of [M + Li - 189] + fragment (see the Scheme 7) from protonated plasmenyl PC species precursors. This ion is the absence in the product ion spectra of the [M + Li] + precursors of plasmanyl PC species. For example, Figure 8 shows the product ion spectrum of plasmenyl (16:0)/DHA PC (a) and plasmanyl (16:1)/DHA PC. It is clear to see the difference on the Figure 8. MS 2 product ion spectra of (a) plasmanyl (16:1) PC and (b) plasmenyl (16:0) PC species). Scheme 7. PC positive-ion fragmentation process for the sn-1 plasmenyl linkage identification.

15 Methodology of lipidomics: Tandem mass spectrometry of ether phospholipids 15 presence of a fragment at m/z 279 [M + Li R 2 COOH] + in the product ion spectrum of the former, and the absence of this ion in the spectrum of the latter compound. Ether Lysophosphatidylcholine (LysoPC): Figure 9 shows MS 2 product ion spectra of the [M + Li] + precursors of fish liver plasmanyl 16:0 LysoPC (a), plasmenyl 16:0 LysoPC (b), plasmanyl 18:1 LysoPC (c) and plasmanyl 18:0 LysoPC (d). Only plasmenyl linkage form of LysoPC species gives a fragment at m/z 187 (Hsu et al. 2003), which corresponds to [M + Li 59 R 1 -CH=CH-OH] +. The scheme 8 shows LysoPC positive-ion fragmentation process for the sn-1 plasmenyl linkage identification [16]. Figure 9. MS 2 product ion spectra of [M + Li] + precursors of Plasmanyl 16:0 LysoPC (a), Plasmenyl 16:0 LysoPC, plasmanyl 18:1 LysoPC (c) and Plasmanyl 18:0 LysoPC (d); (Adapted from Chen and Li, J. Agri.. Food Chem. 2007). Scheme 8. LysoPC positive-ion fragmentation process for the sn-1 plasmenyl linkage identification.

16 16 The Negative-ion ESI/MS 3 of the [M + CL] - and [M + HCOO] - precursor ions of ether PC and ether LysoPC species: A LC-negative-ion ESI/MS/MS method has been reported for identifying plasmenyl ether PC species [17] based on the presence of an anion due to a single free fatty acid esterified at the sn-2 position by the MS 3 experiment of [M - HCOO] - [M HCOO - R 2 CH-C=O] - precursors. Furthermore, a new approach has been developed for the differentiation of plasmenyl and plasmanyl PC/LysoPC species by the negative-ion ESI/quadruple multiple-stage ITMS of the [M + CL] - precursors of ether PC and ether LysoPC species in our laboratory. The identification is based on the presence of alkenoxide anions, which is derived from plasmenyl-lysophosphatidic acid fragments, and the glycerol phosphocholine fragments in the MS 3 spectra of plasmenyl PC species and the MS 2 spectra of plasmenyl LysoPC molecules. The major advantage of this new approach is to enable to further differentiate plasmenyl and plasmanyl choline ether GPLs by LC/MS n. A detailed report on the new method and its application in structurally elucidating plasmenyl and plasmanyl PC/LysoPC species from biological samples will be published in a separated paper in the near future. Ether phosphatidylserine (PS): The negative-ion ESI/MS 3 of [M H] - precursor ions of plasmanyl PS species provides informative fragments for the structural identification [13]. An ion at m/z 135 arising from further loss of the 1-O-alkyl group (see the Scheme 9) is the diagnostic fragment. However, the negative-ion MS 3 product ion spectra of plasmenyl PS species have not been studied yet in details [13]. Thus, we further investigated the negative-ion fragmentation process of plasmenyl PS on a quadruple-multiplestage linear ion-trap mass spectrometer, and a detailed description regarding the differentiation of plasmanyl and plasmenyl PS species by this approach will be reported in a separated paper in the near future. Ether phosphatidylinositol (PI), phosphatidic acid (PA) and phosphatidylglycrol (PG): Deprotonated molecules [M H] - of ether PI, PA and PG species can be generated readily by negative-ion ESI, and the MS 2 product ion mass spectra of PI and PA provide informative fragments for the differentiation of their ether molecules and their diacyl molecular species [13]. Schemes 3, 4 and 9 show the fragmentation processes for the sn-1 plasmenyl and plasmanyl linkage identification generated by the ESI/MS 3 experiments. Significantly abundant peaks corresponding to [ether LysoPA H 2 O H] - is the diagnostic anions. Based on the MS 3 experiments of [M H]- [ether LysoPA H 2 O H] - of ether PE and PS, plasmenyl and plasmanyl species of PI and PA could be identified by the presence (plasmenyl linkage) and absence (plasmanyl linkage) of alkenoxide anions [O-CH=CH-(CH 2 ) n CH 3 ] - (n = 11-19).

17 Methodology of lipidomics: Tandem mass spectrometry of ether phospholipids 17 Scheme 9. PS negative-ion fragmentation process for the sn-1 plasmanyl linkage identification. (iv) Structural elucidation of ether GPL species by Ozone-induced dissociation tandem mass spectrometry Ozone-induced dissociation/tandem mass spectrometry is a new method to identify plasmenyl and plasmanyl linkage ether GPL species [18]. Gasphase ion molecule reaction between double bond(s) in unsaturated fatty chain(s) in PL species and ozone has been found to generate two primary product ions for each carbon-carbon double bond in the species. Plasmenyl linkage species can be differentiated from plasmanyl linkage GPLs by the diagnostic of the unsaturation in the double bond(s) at the sn-1 position that is different in the ether GPL species with plasmenyl and plasmanyl linkages [18]. The major advantage of this approach in the differentiation of plasmenyl and plasmanyl linkage GPL species is that the more than one double-bonds in plasmenyl PL species can be detected compared with the MS 2 or MS 3 analyses of intact ether GPLs species. By using this method, ether GPL molecular species extracted from human and animal lens have been structurally elucidated [18]. However, this approach cannot be directly applied for the LC-ESI/MS n analyses of various intact ether GPL species, compared with the LC/MS/MS analyses of intact ether GPL molecular species [19].

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