Recent developments in tandem mass spectrometry for lipidomic analysis

a n a l y t i c a c h i m i c a a c t a 6 2 7 ( 2 0 0 8 ) 62–70

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Review article

Recent developments in tandem mass spectrometry for lipidomic analysis Nicole Zehethofer a,b , Devanand M. Pinto a,b,∗ a b

Institute for Marine Biosciences, National Research Council of Canada, 1411 Oxford St., Halifax, NS, Canada B3H 3Z1 Chemistry Department, Dalhousie University, Halifax, NS, Canada B3H 4J3

a r t i c l e

i n f o

a b s t r a c t

Article history:

This review will focus on the role of mass spectrometry in the emerging field of lipidomics.

Received 4 March 2008

Particular emphasis will be placed on recent developments in the use of tandem mass spec-

Received in revised form

trometry methods in lipid analysis using low-energy collision induced dissociation (CID).

17 June 2008

After a brief discussion on ionization techniques, novel ion-activation methods that allow

Accepted 19 June 2008

for increased sensitivity and selectivity will be critically discussed. Examples will be drawn

Published on line 4 July 2008

from the analysis of higher order lipids, specifically triacylglycerols (TAGs) and glycerophospholipids, as the numerous positional isomers and head groups present in these classes of

Keywords:

lipids continue to pose a significant analytical challenge to the field of lipidomics. The role

Lipidomics

of bioinformatics in the development of lipidomics will also be discussed.

Diacylglycerols

Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved.

Triacylglycerols Tandem mass spectrometry Ion activation

Contents 1. 2.

3. 4. 5. 6. 7.

∗

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ionization techniques for lipidomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Atmospheric pressure chemical ionisation—variations on a theme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. MALDI—an emerging role in lipidomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ion activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Ozone-induced dissociation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The state of the art in lipidomics of DAGs and TAGs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Lithiated TAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Novel scan modes—increasing selectivity through tandem MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Corresponding author. Tel.: +1 902 426 0558; fax: +1 902 426 9413. E-mail address: [email protected] (D.M. Pinto). 0003-2670/$ – see front matter. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.06.045

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1.

Introduction

Lipidomics, the systematic study of lipids and their function in biological systems, has emerged as an active area of interest and, despite the fact that it is often considered as a branch of metabolomics, it is unique in terms of the analytical and bioinformatic techniques employed. For general discussions on the emerging field of lipidomics, the reader is directed to recently published reviews [1,2]. In recent years, other reviews focused on lipidomics in the context of their structural role in biological membranes [3], exosomes [4] and cytoskeletal dynamics [5] or their functional roles in pain modulation [6], signaling [7] and toxicology [8] have been published. The traditional role of lipids has been one of passive ‘commodity’ molecules with primary responsibilities consisting of energy storage and cell structure. As our understanding of these molecules has grown, so have their roles evolved to now being considered as powerful and active regulators of the complex signaling pathways that govern cell biology. It is now clear that lipids are integral components of cell signaling pathways and that lipids act as ligands and mediators of protein–protein interactions [9,10]. In addition to the well known link between defects in lipid metabolism and cardiovascular disease, lipidomic analysis has been used to demonstrate the importance of lipids in the pathogenesis of diseases such as nonalcoholic fatty liver disease (NAFLD) [11], neurological disorders [12], and cancer [13]. The growth of lipidomics is primarily a result of technological advances in mass spectrometry. Lipid analysis is traditionally performed by gas chromatography–mass spectrometry (GC–MS), however, the development of electrospray ionization (ESI) [14] and matrix-assisted laser desorption/ionization (MALDI) [15,16] has significantly expanded the range of lipids that can be analyzed by mass spectrometry and the coupling of ESI-MS to liquid chromatography (LC) has greatly increased the number of lipid classes that can be analyzed in a single experiment. Furthermore, instruments capable of performing tandem mass spectrometry (MS/MS) provide the detailed structural information necessary for characterization of novel lipids and the selectivity required for the determination of individual lipid species present in complex mixtures. Tandem mass spectrometry data is also being used to develop quantitative methods for targeted lipidomic analysis in complex samples. This review will focus on advances in the use of tandem mass spectrometry for lipid analysis over the last five years. Particular emphasis will be placed on recent developments in the use of tandem mass spectrometry methods using lowenergy collision induced dissociation (CID). While low-energy CID is currently the most popular ion-activation method for lipid analysis using LC-MS/MS, high-energy CID has been shown to be valuable for lipid characterization as well [17]. Novel ion-activation methods that allow for increased sensitivity, selectivity or that expand the range of lipids that can be analyzed by LC-MS/MS will be critically discussed. Examples will be drawn from the analysis of higher order lipids, specifically triacylglycerols (TAGs) and glycerophospholipids as the numerous positional isomers and head groups present

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in these classes of lipids continue to pose a significant analytical challenge in the field of lipidomics.

2.

Ionization techniques for lipidomics

Although the focus of this review is tandem MS of lipids, a brief discussion on recent developments in ionization techniques is warranted, as these techniques have vastly improved the sensitivity and range of lipids that can be analyzed by tandem MS. As with any chemically heterogeneous mixture of analytes, the study of lipids requires the use of a variety of ionization techniques for comprehensive analyses. Lipidomics is most commonly performed using ESI, followed by atmospheric pressure chemical ionization (APCI) and MALDI. These ionization techniques, and variants thereof, are capable of producing molecular ions, radical cations and various adducts. Enhancements in ionization efficiencies can be made through derivatisation of the lipids before analysis to make them more amenable to conventional ionization processes such as ESI and APCI. Derivatisation has been used extensively in lipid analysis by GC–MS but less so in lipid analysis by ESI-MS. For lipidomics analysis, derivatisation can be used to improve the ionization efficiency, shift analytes into an m/z range of decreased chemical noise and provide a unique mass signature of a particular lipid or lipid class [18]. Despite the variety of ionization techniques and derivatisation methods available, complete ionization of all lipid classes using a single ionization technique remains an unmet challenge due to the significant class-specific differences, such as functional groups and polarity.

2.1. Atmospheric pressure chemical ionisation—variations on a theme Even though the majority of lipid analyses are performed using conventional ionization techniques, the desire for higher sensitivity has resulted in numerous modifications thereof. For example, the utility of APCI in lipidomic analysis can be increased by labelling the lipids of interest with electron capturing groups, such as the pentafluorobenzyl (PFB), followed by electron capture APCI analysis [19]. Electron capture APCI is comparable to electron capture negative chemical ionization [20] but differs in that the possibility exists for the coupling to liquid chromatography. Briefly, electron capture APCI results in the decomposition of analyte molecules and the formation of anionic species, which can be measured by negative ion mode MS, resulting in a sensitivity increase of 2 orders of magnitude compared to conventional APCI. This allows for the analysis of trace amounts of lipids in biological samples. The interested reader is referred to a recent review of PFB derivatisation for targeted lipidomics analysis by Lee and Blair [19]. Ionization is also improved by modification of the mechanism responsible for ion formation. One such modification is atmospheric pressure photoionization (APPI). APPI can be thought of as a modified form of APCI, where ionization occurs through photoactivation of a dopant and resulting ionization of the lipid through ion–molecule reactions [21]. A manufacturer of an APPI source demonstrated improvements in

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sensitivity and linear dynamic range of APPI when compared to ESI and APCI [22]. However, this study used a limited range of lipids (free fatty acids, fatty acid methyl esters, mono-, diand triglycerides). Other researchers have found no significant differences in the performance of APPI and ESI if used for the analysis of edible oils, where the two ionization techniques were seen as complimentary [23]. As with other complex analytes, “true” lipidomic analysis will require the use of several ionization techniques in both positive and negative mode and the suitability of APPI for this application remains to be determined.

2.2.

MALDI—an emerging role in lipidomics

Lipid analysis by MALDI offers several advantages over ESI, such as speed of analysis, simplicity and stability of the ion source and higher tolerance to salts and other sample impurities [16]. However, for lipid analysis, these advantages are outweighed by the difficulty in coupling LC to MALDI-MS. Although instrumentation exists for LC–MALDI-MS analysis, it is not routine nor is the throughput comparable to that of LC–ESI-MS. Nevertheless, lipid analysis by MALDI-MS is an active area of research. Approaches to improve lipid analysis by MALDI-MS through modification of the matrix have resulted in significant improvements. In the area of TAG analysis, laser desorption/ionization time-of-flight mass spectrometry (LDITOFMS) has been used by Calvano et al., who applied the technique to the analysis of olive and other food oils [24]. The main advantage compared to MALDI is the absence of (possibly interfering) matrix peaks in the obtained spectra. However, only sodium and potassium adduct ions were produced by this method and, for certain classes of lipids, these adduct ions do not produce informative product ion spectra. This has been shown for the fragmentation of sodiated TAGs, which do not provide product ions suitable for detailed lipid characterization by either high-energy [25] or low-energy CID [26]. The latter only results in the neutral loss of fatty acids forming diacylglycerols. More recently, the use of 2,4,6trihydroyacetophenone was used as a matrix to study a variety of lipids including TAGs [27]. It was found that by judicious choice of salt, sodium, potassium and lithium adducts, which are potentially more useful for tandem MS analysis, could be formed. Traditionally, MALDI was coupled with time-of-flight (TOF) mass spectrometers and relatively few instruments existed for performing tandem mass spectrometry following MALDI. This limitation has been alleviated by the development of reliable, high-performance platforms for MALDI-MS/MS such as the MALDI-triple quadrupole linear ion trap (QqQ/LIT) [28,29], MALDI-QqTOF [30], and MALDI-TOF–TOF [31]. A unique advantage of MALDI over ESI is the ability to measure analytes as a function of position in a tissue section [32]. This “imaging” technique has been extensively applied to the lipidomics analysis of brain tissue [31]. The major ionization techniques used in lipidomics have been discussed above and although others exist, they are not as extensive in terms of use or utility and will not be discussed for the sake of brevity. The same is true for geometries of mass analyzers. Although no specific combination of ion

source and mass analyzer stands out as the method of choice for lipidomic analysis, the combination of LC with tandem mass spectrometry is generally sufficient for carrying out high quality lipidomics analysis.

3.

Ion activation

Following ionization, tandem MS requires the lipid of interest to be isolated by the first stage of MS, followed by ion activation to induce fragmentation. Analytes with complex structures, such as TAGs and glycerophospholipids, place enormous demands on the method used for ion activation as it must produce extensive fragmentation in order for information such as double bond position to be obtained. However, the method for ion activation must not introduce artifacts, such as migration of double bond position. Significant progress has been made in achieving these seemingly conflicting requirements and, when tandem MS is combined with efficient chromatography, lipidomics analysis of extremely complex mixtures of analytes is possible. In cases where standards are available, isomeric lipids can be distinguished based on retention time, fragment ion mass or, preferably, both. In cases where isobaric lipids co-elute or where standards are not available or are impractical to use, ion-activation methods that permit specific identification of isomers are necessary. Low-energy CID is currently the most popular ionactivation method for lipidomic analyses using LC-MS/MS. The tandem MS spectra of isomeric, polyunsaturated fatty acids obtained through low-energy CID of their dilithiated adducts often provides unique fragment ions that can be used to confidently identify isomers and, in many cases, assign double bond location(s) [33]. This is true despite the fact that fragmentation under these conditions does not produce fragments from the distal portion of the molecule (distal is defined as C1 to the Cn , where n is the position of the first double bond when counting from the methyl carbon end). Traditional low-energy CID fails to provide sufficient information for the analysis of higher order lipids. Therefore, several novel ion-activation methods have been developed to increase the information content of LC-MS/MS analysis in lipidomics. Liang et al. demonstrated that electron-transfer dissociation (ETD) provided unique fragmentation as compared to CID for the analysis of various glycerophosphocholine (GPC) lipids [34]. The use of a linear ion-trap mass spectrometer also allowed for MS/MS/MS (MS3 ) experiments employing a combination of ETD and CID thus providing rich tandem MS data for the study of GPCs. These initial results are promising; however, the sensitivity obtained was quite low so demonstrating the utility of the technique for lipidomic analysis will require improvements in sensitivity and a demonstration of the utility of the technique for a wide array of lipid classes. It has been shown that a new technique termed “covalent adduct chemical ionization” (CACI) can be used to determine the locations of conjugated and methylene-interrupted double bonds in fatty acid methyl esters (FAME) in the gas phase [35,36]. This is due to the formation of the (1-methyleneimino)1-ethynylium (MIE) ion during collisional activation of CH3 CN (Scheme 1) which reacts with the FAME double bond resulting in an adduct ion at [M+54]+ . After fragmentation of this adduct

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Scheme 1 – Proposed mechanism for the formation of the (1-methyleneimino)-1-ethynylium (MIE) ion during collisional activation of CH3 CN. (From [60] with permission from John Wiley and Sons Ltd.).

ion, highly abundant product ions formed by the cleavage of bonds allylic and vinylic to the double bond are observed, which can be used to locate the double bond position. Ions observed due to allylic and vinylic cleavage were termed ␣ and ␻ diagnostic ions, respectively. Xu and Brenna [37] combined CACI with atmospheric pressure chemical ionization creating a new technique termed atmospheric pressure covalent adduct chemical ionization (APCACI) to analyze TAGs. After showing that the MIE ion is formed during APCACI as well, the fragmentation behavior of a selection of model TAG adduct ions were studied. Fragmentation of these adduct ions resulted in the formation of characteristic product ions similar to the ions obtained for FAMEs. Figs. 1 and 2 show the fragmentation spectra obtained for monounsaturated and diunsaturated positional isomers. For monounsaturated TAGs, abundant ␣ and ␻ ions as previously reported for FAMEs were observed. Diunsaturated TAGs, containing conjugated or homoallylic double bonds, showed cleavages adjacent to the double bonds but not between double bonds. It should be noted, that hydrogen migration to the ␣ and away from the ␻ ions is observed in these spectra resulting in a difference of one mass unit in the spectrum compared to the expected mass after homolytic cleavage.

3.1.

Ozone-induced dissociation

Double bond positions in lipids can also be determined using tandem mass spectrometry involving the conversion of the double bonds to ozonides prior to analysis. As demonstrated by Harrison and Murphy, lipids can be converted to ozonides using an offline process where the lipids are deposited on a glass surface followed by exposure to a stream of ozone [38]. This method was modified for online analysis of glycerophospholipids in 2006 by Thomas et al. [39] who also showed the use of ozone-induced fragmentation for TAG characterization termed ozone electrospray ionization mass spectrometry (OzESI-MS) [40]. In this modification, the TAGs of

Fig. 1 – Product ion spectra obtained for isomeric TAG(18:1/18:1/16:0)-MIE precursor ions which are formed during APCACI. (A) TAG(11 Z-18:1/11 Z-18:1/16:0), (B) TAG(13 Z-18:1/13 Z-18:1/16:0) and (C) TAG(13 Z-18:1/13 Z-18:1/16:0). Note that the double bond position can be localized using these spectra as characteristic product ions are observed. Reprinted with permission from Anal. Chem., 79 (2007) 2525. Copyright 2007 American Chemical Society.

interest react with ozone during the ionization process forming ozonides, which further decompose forming an aldehyde. Reaction with the solvent (methanol in this case) resulted in a neutral loss and the formation of an ␣ hydroxyperoxide. The observed neutral losses are characteristic of the double bond position, which has been shown to be true for monoand diunsaturated fatty acids. The MS spectrum obtained for TAG(16:0/Z-18:1/16:0) is depicted in Fig. 3; the mechanism for the reaction of ozone with double bonds and the subsequent fragmentation reaction is shown in Scheme 2. Although not definitively demonstrated, the author inferred that fatty acids with different degrees of unsaturation could also be analyzed using this technique. In the study by Thomas et al., the ozone was added during the ionization process. However,

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Scheme 2 – Mechanism for the reaction of ozone with a double bond and subsequently fragmentation reaction resulting in the formation of an aldehyde and an alpha hydroxyperoxide. Reprinted with permission from Anal. Chem., 79 (2007) 5013. Copyright 2007 American Chemical Society.

the reaction can take place in an ion-trap as well resulting in ozone-induced dissociation (OzID) [41], which has been shown to be useful for characterization of a variety lipid classes in complex matrices such as human lens lipid extract and bovine kidney [41]. Despite these advances in lipid analysis using tandem MS, complete determination of TAGs remains a challenge; in addition to the numerous isobaric TAGs that may exist, extensive fragmentation of TAGs remains difficult. Therefore, several studies have attempted to assign structures based on relative intensities of the fragments that are produced. However, a careful analysis of this method has raised questions as to the validity of this method [42]. The significant variation between instrument types, ionization techniques and ion-activation methods employed makes broad applicability of this method an open question.

4. The state of the art in lipidomics of DAGs and TAGs Early work by Duffin et al. [25] using a triple quadrupole instrument showed that TAG ammonium adduct ions yield interesting fragmentation patterns suitable for characterization of the TAG under low-energy CID conditions (∼50 eV). The main product ions in the spectra observed are the protonated TAGs formed by the loss of ammonia and diglyceride ions formed by the loss of the fatty acid moieties. Using higher CID energy (∼130 eV) monoglycerides and acylium ions and carbon–carbon bond cleavage could be observed. The position of the double bond could not be determined based on these spectra due to double bond migration. Malone and Evans [43] investigated the fragmentation of TAGs using an ion-trap instrument. Diglycerides formed by the loss of fatty acid moieties were observed as well, but the

loss of the central fatty acid seemed to be less favored resulting in abundance differences. Interestingly, the loss of higher unsaturated fatty acids and long chain fatty acids was shown to be favored resulting in higher abundance product ions. The authors showed that it is possible to distinguish between isomeric TAGs by measuring the product ion intensities. However, without chromatographic separation, complex product ion spectra would be observed if TAGs with similar mass to charge (m/z) values are present. Reversed phase-high performance liquid chromatography (RP-HPLC) separation greatly reduced the overlap between TAGs of similar m/z values. However, the analysis of complex mixtures is still challenging due to the occurrence of isomeric TAGs, which cannot be resolved chromatographically. The authors suggested the use of MS3 experiments on the acylium ions obtained for higher CID energies as it has proven to be useful for characterization of fatty acids bound to TAGs [44,45]. Nevertheless, the main disadvantage of this approach is the low abundance of the obtained acylium ions, which is why improvement of the separation was suggested. Li and Evans have conducted a comprehensive study of various model TAGs with different degrees of unsaturation and chain lengths to develop an algorithm for fragmentation prediction and simplify positional analysis of TAGs. As observed by others, the main product ions were diglyceride ions formed by the loss of fatty acid moieties and protonated ions formed by the loss of ammonia. The product ions observed are summarized in Fig. 4. A linear relationship between the relative intensities of the products ions and the degree of unsaturation was observed as well as the favored loss of unsaturated fatty acids with double bonds close to the carboxylic group [46,47]. The use of relative intensities, rather than isomer-specific fragment ions, to identify isomers is an effective method even in cases where the mixtures are quite complex. However, the method relies on differences in relative intensity that can be

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Fig. 2 – Product ion spectra obtained for isomeric TAG(18:2/18:2/16:0)-MIE precursor ions which are formed during APCACI. (A) TAG(9,11 Z,Z-18:1/9,11 Z,Z-18:1/16:0), (B) TAG(10,12 Z,Z-18:2/10,12 Z,Z-18:2/16:0) and (C) TAG(9,12 Z,Z-18:2/9,12 Z,Z-18:2/16:0). Note that the double bond position can be localized using these spectra as characteristic product ions are observed. Reprinted with permission from Anal. Chem., 79 (2007) 2525. Copyright 2007 American Chemical Society.

as small as only a few percent. This implies that one must be aware and control for small changes in instrument parameters, such as source conditions, collision gas density and collision energy that affect the intensities of the observed peaks. This is in addition to instrument specific parameters, such as source and collision cell design that also significantly affect tandem MS spectra and are beyond the control of the user.

4.1.

Lithiated TAG

Hsu and Turk [48] studied the fragmentation behavior of lithiated TAGs using low-energy CID (∼50 eV). CID spectra of TAGs showed neutral losses of fatty acids and fatty acid lithium salts as well as peaks for lithiated fatty acids. Peaks for acylium ions, formed by loss of lithium hydroxide from the lithiated fatty acids and further loss of water were observed as well. It

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Fig. 3 – (a) Full scan spectrum of TAG(16 Z-18:1/16:0) showing molecular ion and ozone-induced product ions. Product ion spectra of the product ions are shown in (b) 740.9 m/z and (c) 788.5 m/z. Reprinted with permission from Anal. Chem., 79 (2007) 5013. Copyright 2007 American Chemical Society.

was also shown that the loss of the central fatty acid is not favored, this information can be used to determine the fatty acid position on the glycerol backbone (as seen for the ammoniated species). Interestingly, Hsu and co-workers observed that the unsaturation positions of the bound fatty acids could be determined by increasing the skimmer potential allowing the formation of dilithiated free fatty acid species. MS3 analysis of dilithiated fatty acid product ions allowed the determination of the unsaturation position as the fragmentation was similar to the fragmentation observed for dilithiated free fatty acids [33]. For monounsaturated and polyunsaturated fatty acids, closed-shell series separated by 14 m/z were observed that terminate at the carbon–carbon bond vinylic to the first carbon–carbon double bond. For polyunsaturated

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Fig. 4 – Product ions observed for the fragmentation of a model TAG which was ionized using ESI in the presence of ammonium ions. Main product ions are diglyceride ions (LO+ , LS+ , OS+ ) formed by the loss of fatty acid moieties (O, S, and L) and protonated TAGs (MH+ ) formed by the loss of ammonia. (From [41] with permission from John Wiley and Sons Ltd.).

fatty acids, abundant product ions formed by the cleavage of carbon–carbon single bonds between two double bonds were observed whereas no cleavage distal to the last double bond could be observed. Fragmentation sites for mono- and polyunsaturated fatty acids are summarized in Fig. 5.

5. Novel scan modes—increasing selectivity through tandem MS MS3 analysis provides an extensive catalogue of information for the characterization of structurally complex lipids; however, the length of the analysis precludes the use of MS3 for rapid screening purposes. Several rapid scan modes exist, such as neutral loss scanning, where the MS can be set to scan for neutral losses of fatty acids and fatty acid lithium salts [49].

Fig. 5 – Fragmentation sites observed for model dilithiated mono- and polyunsaturated fatty acids. Dilithiated species are formed during ESI if lithium ions are present.

This method has been further developed by Murphy et al. who used deuterium labelled fatty acids as internal standards and studied TAGs in RAW 264.7 cells [50]. This method for characterization of TAGs is rapid but requires further validation prior to routine use due to the inherent complexity in fragmentation of mixtures of TAGs. The majority of lipidomic analyses are preformed by obtaining fragment ion spectra using data dependent acquisitions, also known as shotgun methods [51]. Han and Gross developed a shotgun lipidomics technique termed “intrasource separation” [52]. This techniques uses modification of ESI parameters rather than chromatographic separation for qualitative and quantitative lipidomics analysis, specifically, ionization mode (positive/negative) and cationization agent (H+ or Li+ ) and scan type (MS and precursor ion scans) are used. The additional selectivity of multiple precursor ion scanning was crucial in deconvoluting the complex components of lipidomic samples. Precursor ion scanning is a well-developed tandem MS scan mode that is becoming more routine in lipidomics analysis. It is an excellent method for profiling of specific classes of lipids, especially when present in complex mixtures. Its combination with data dependent acquisition has recently been reviewed [51]. Another well-established technique is multiple reaction monitoring mass spectrometry (MRM-MS), where specific parent ion–fragment ion pairs that are indicative of a specific lipid are monitored rapidly, usually for a few tens of milliseconds. LC–MRM-MS, due to its wide availability, ease of use and excellent quantitative performance, is increasingly being used in lipidomics analysis. In many cases, isomeric lipids cannot

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Fig. 6 – Demonstration of the advantages of multiple precursor ion scanning using a QqTOF instrument (lower traces) versus lipidomic analysis using a single precursor ion. Reprinted with permission from Anal. Chem., 78 (2006) 62025. Copyright 2006 American Chemical Society.

be resolved chromatographically; however, when standards are available, identification can be made using fragment ions of proven selectivity using precursor ion or MRM approaches [53]. Alternatively, ion mobility techniques can be used separate isobaric lipids in the gas phase [54], which we expect will be a powerful technique for lipidomic analysis once coupled with instrumentation capable of tandem mass spectrometry measurements. The careful choice of a particular scan mode that is appropriate for the class of lipids being studied provides excellent selectivity. In some cases, this added selectivity can aid in the analysis of lipids that cannot be resolved chromatographically and, in some cases, altogether alleviate the need for chromatographic separation. However, the disadvantage of using specific scan modes is that several novel lipids can escape detection if an inappropriate scan mode is selected.

6.

Bioinformatics

The increase in analytical capabilities described above and the increase in sample complexity has resulted in the production of large amounts of data that are no longer amenable to manual data analysis. As has been witnessed in the field of proteomics, there has been a significant lag between the development of efficient analytical methods for analysis of lipidomic samples and the development of suitable bioinformatic tools for automated data analysis. Nevertheless, significant progress has been made, such as the recent development of the Lipid Profiler software as described by Ejsing et al. for the analysis of glycerophospholipids [55]. This software, when combined with the ability of QqTOF mass

spectrometers to simultaneously perform multiple precursor ion scans, enabled the identification and quantitation of ∼200 lipids in a complex lipid extract from bovine heart (Fig. 6). Novel bioinformatic tools, such as the cognoscitivecontrast-angle algorithm and databases (COCAD) method, have been developed for the identification of lipids. This method makes use of databases of spectra and search algorithms that perform lipid identifications through comparison of liquid chromatography–ultraviolet-tandem mass spectrometry (LC–UV-MS/MS) spectra with those in a database and, where spectra are not available, through matching with spectra from a theoretical database [56]. This type of research is demonstrating both the complexity of lipidomic analyses and the insight that can be gained into the behavior of complex biological systems through the combination of advanced LCMS/MS analysis and efficient bioinformatic analysis [57,58]. The creation of a standardized classification system for lipids is perhaps a sign of the rapidly evolving size of the field of lipidomics and the range of potential applications of this important field [59].

7.

Concluding remarks

Lipidomics has benefited from a tremendous increase in importance as a result of recent development in tandem mass spectrometry, especially when coupled to chromatography. A critical component of any lipidomic analysis is the deployment of high performance analytical capabilities. As has been witnessed in other disciplines of systems biology, such as genomics, transcriptomics and proteomics, the creation of novel bioinformatic tools for lipidomic analysis has lagged

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the development of analytical techniques. Nor has there been cohesion in the development of lipidomics standards or even agreement on a standard nomenclature; much work remains to be done. In spite of these challenges, several impressive examples of lipidomic analysis were discussed above and it is hoped that the reader is left with an impression of the enormous potential of the field of lipidomics.

references

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