BBA - Molecular and Cell Biology of Lipids (v.1811, #11)

Lipidomics and Imaging Mass Spectrometry by Robert C. Murphy; Alfred H. Merrill (635-636).

Lipid classification, structures and tools by Eoin Fahy; Dawn Cotter; Manish Sud; Shankar Subramaniam (637-647).
The study of lipids has developed into a research field of increasing importance as their multiple biological roles in cell biology, physiology and pathology are becoming better understood. The Lipid Metabolites and Pathways Strategy (LIPID MAPS) consortium is actively involved in an integrated approach for the detection, quantitation and pathway reconstruction of lipids and related genes and proteins at a systems-biology level. A key component of this approach is a bioinformatics infrastructure involving a clearly defined classification of lipids, a state-of-the-art database system for molecular species and experimental data and a suite of user-friendly tools to assist lipidomics researchers. Herein, we discuss a number of recent developments by the LIPID MAPS bioinformatics core in pursuit of these objectives. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► We established a comprehensive classification system for lipids. ► We created an extensive online database of over 30,000 lipid structures. ► We developed powerful search and display methods for a wide variety of lipidomic data. ► We created novel software for drawing and representing complex lipid structures. ► We developed online tools for predicting lipid structures from mass spectrometry experiments.
Keywords: Lipids; Lipidomics; Classification; Mass spectrometry; Bioinformatics;

Historically considered to be simple membrane components serving as structural elements and energy storing entities, fatty acids are now increasingly recognized as potent signaling molecules involved in many metabolic processes. Quantitative determination of fatty acids and exploration of fatty acid profiles have become common place in lipid analysis. We present here a reliable and sensitive method for comprehensive analysis of free fatty acids and fatty acid composition of complex lipids in biological material. The separation and quantitation of fatty acids are achieved by capillary gas chromatography. The analytical method uses pentafluorobenzyl bromide derivatization and negative chemical ionization gas chromatography–mass spectrometry. The chromatographic procedure provides base line separation between saturated and unsaturated fatty acids of different chain lengths as well as between most positional isomers. Fatty acids are extracted in the presence of isotope-labeled internal standards for high quantitation accuracy. Mass spectrometer conditions are optimized for broad detection capacity and sensitivity capable of measuring trace amounts of fatty acids in complex biological samples. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► We review quantitative analyses of fatty acids in biological material. ► Chromatographic separation of fatty acids is achieved by capillary GC. ► Mass spectrometry using soft ionization techniques enables sensitive detection. ► Isotope dilution method facilitates accurate and precise quantitation.
Keywords: Fatty acid; Fatty acid analysis; Lipidomics; Gas chromatography; Mass spectrometry;

L-3-Hydroxy fatty acids are unusual metabolites and rarely occur in significant quantities in normal human physiology. Genetic defects of both long-chain and medium-/short-chain mitochondrial L-3 hydroxyacyl coenzyme A dehydrogenases (LCHAD, M/SCHAD) have been identified as significant metabolic diseases in humans often with severe clinical phenotypes and pathophysiology that appears to differ from other defects of straight chain fatty acid oxidation. It is felt that accumulation of these atypical fatty acid species may play a role in this pathology. We have therefore developed an assay to measure these compounds in body fluids, and tissue culture medium to help in the diagnosis of these disorders and to better study the effects of 3-hydroxy fatty acid accumulation.We have developed a stable isotope dilution, selected ion-monitoring gas chromatography-mass spectrometric assay for the measurement of all 3-hydroxy fatty acids from chain lengths C6 to C18 using 1,2 13C-labeled internal standards for all species. Authentic patient samples were utilized to develop reference intervals for control subjects, for those associated with patient samples confirmed at the molecular level to have either LCHAD or M/SCHAD deficiency and for patients who did not have disease but were fasting or on diets high in medium-chain fatty acids. Likewise, skin fibroblasts were obtained from patients with confirmed disease for additional study. Samples were also obtained from the hadh (M/SCHAD) knockout mouse.The measurement of 3-hydroxy fatty acids in patient plasma is a valuable tool in the identification of defects of both enzymes. Severe starvation, prolonged fasting and increased medium-chain triglycerides in the diet produce a profile that is similar to that seen in M/SCHAD deficiency, making this a more difficult condition to diagnose but these biomarkers provide an important clue to the diagnosis, particularly in non-fasted, diet-controlled patients. Fibroblast studies in LCHAD deficiency demonstrate that long-chain 3-hydroxy fatty acid accumulation can be observed in cultured tissues. Incubation of cultured fibroblasts from LCHAD deficient patients with labeled fatty acids demonstrated a process of chain lengthening that has not previously been recognized.The measurement of body fluid and cultured cell 3-hydroxy fatty acids provides both diagnostic and pathogenic information regarding these genetic diseases of fatty acid oxidation in the mitochondrion. Presently, the measurement of medium- and short-chain species provides a major metabolic biomarker for the recognition of M/SCHAD deficiency. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► An assay for the measurement of 3-hydroxyfatty acids in biological fluids is presented. ► The assay is applied to the diagnosis of LCHAD and M/SCHAD deficiencies. ► The assay is valuable for long-term disease monitoring. ► The assay is also used in vitro to probe treatment for LCHAD deficiency with MCT's.
Keywords: Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency; Medium and short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency; 3-hydroxy fatty acids; Medium-chain triglycerides; Fatty acid oxidation;

Acyl-CoAs are intermediates of numerous metabolic processes in eukaryotic cells, including beta-oxidation within mitochondria and peroxisomes, and the biosynthesis/remodeling of lipids (e.g. mono-, di-, and triglycerides, phospholipids and sphingolipids). Investigations of lipid metabolism have been advanced by the ability to quantitate acyl-CoA intermediates via liquid chromatography coupled to electrospray ionization-tandem mass spectrometric detection (LC–ESI-MS/MS), which is presently one of the most sensitive and specific analytical methods for both lipids and acyl-CoAs. This review of acyl-CoA analysis by mass spectrometry focuses on mammalian samples and long-chain analytes (i.e. palmitoyl-CoA), particularly reports of streamlined methodology, improved recovery, or expansion of the number of acyl chain-lengths amenable to quantitation. This article is part of a Special Issue entitled: Lipodomics and Imaging Mass Spectrometry.► Methods for HPLC–UV analysis of acyl-CoAs are reviewed. ► Methods for HPLC–tandem mass spectrometry of acyl-CoAs are reviewed. ► Improvements in acyl-CoA extraction efficiency are reviewed.
Keywords: Acyl-coenzyme A; Acyl-CoA; HPLC–ESI-MS/MS; HPLC–UV; Tissue extraction; Solid-phase extraction;

Free carnitine and acylcarnitines play an important role in the metabolism of fatty acids. Sterols are structural lipids found in the membranes of many eukaryotic cells, and they also have functional roles such as the regulation of membrane permeability and fluidity, activity of membrane-bound enzymes and signals transduction. Abnormal profiles of these compounds in biological fluids may be useful markers of metabolic changes. In this review, we describe the subset of the lipidome represented by acylcarnitines and sterols, and we summarize how these compounds have been analyzed in the past. Over the last 50 years, lipid mass spectrometry (MS) has evolved to become one of the most useful techniques for metabolic analysis. Today, the introduction of new ambient ionization techniques coupled to MS (AMS), which are characterized by the direct desorbing/ionizing of molecules from solid samples, is generating new possibilities for in situ analysis. Recently, we developed an AMS approach called APTDCI to desorb/ionize using a heated gas flow and an electrical discharge to directly analyze sterols and indirectly investigate acylcarnitines in dried blood or plasma spot samples. Here, we also describe the APTDCI method and some of its clinical applications, and we underline the common complications and issues that remain to be resolved. This article is part of a Special Issue entitled: Lipodomics and Imaging Mass Spectrometry.► Acylcarnitines and sterols are useful markers of metabolic changes in mammals. ► We summarize how these compounds have been in the past analyzed by MS. ► We describe some ambient ionization - MS (AMS) techniques able to analyze in situ solid samples. ► We describe our AMS method for sterols and acylcarnitines analysis in dried blood or plasma spot. ► Direct analysis of DBS by AMS represents a new approach for in situ metabolic profiling.
Keywords: Sterol; Carnitine; Acylcarnitine; Ambient mass spectrometry; DBS; Lipid;

Mass spectrometry of fatty aldehydes by Evgeny V. Berdyshev (680-693).
Fatty aldehydes are important components of the cellular lipidome. Significant interest has been developed towards the analysis of the short chain α,β-unsaturated and hydroxylated aldehydes formed as a result of oxidation of polyunsaturated fatty acids. Multiple gas chromatography–mass spectrometry (GC/MS) and subsequently liquid chromatography–mass spectrometry (LC/MS) approaches have been developed to identify and quantify short-chain as well as long-chain fatty aldehydes. Due to the ability to non-enzymaticaly form Schiff bases with amino groups of proteins, lipids, and with DNA guanidine, free aldehydes are viewed as a marker or metric of fatty acid oxidation and not the part of intracellular signaling pathways which has significantly limited the overall attention this group of molecules have received. This review provides an overview of current GC/MS and LC/MS approaches of fatty aldehyde analysis as well as discusses technical challenges standing in the way of free fatty aldehyde quantitation. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► Fatty aldehyde analysis by gas chromatography–mass spectrometry. ► Fatty aldehyde analysis by liquid chromatography–mass spectrometry. ► Challenges during the analysis of free fatty aldehydes by mass spectrometry.
Keywords: Aldehyde mass spectrometry; Fatty aldehyde; Plasmalogen; 4-Hydroxynonenal; (2E)-Hexadecenal;

Compared to the arachidonic acid (C20:4) cascade, the oleic acid (C18:1) family comprises a handful known metabolites. The pathophysiology of oleic acid and its oxidized and nitrated metabolites, i.e., cis-9,10-epoxyoctadecanoic acid (cis-EpOA) and the two vinylic nitro-oleic acids cis-9-nitro-oleic acid (9-NO2-OA) and cis-10-nitro-oleic acid (10-NO2-OA), is only little investigated and little understood. cis-EpOA, 9-NO2-OA and 10-NO2-OA have been detected in plasma of healthy and ill human subjects by means of gas chromatography–tandem mass spectrometry (GC–MS/MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) techniques in their acid and esterified forms. cis-EpOA is formed from oleic acid by the catalytic action of various cytochrome P450 isozymes. In end-stage liver disease, cis-EpOA plasma concentration is lower than in healthy subjects suggesting liver as the main organ responsible for cis-EpOA synthesis. The origin of 9-NO2-OA and 10-NO2-OA and of other nitrated oleic acid metabolites is unknown. In vitro models, nitro-oleic acid species can be formed non-enzymatically from oleic acid and nitrogen dioxide. Thus, endogenous nitro-oleic acids could serve as biomarkers of fatty acid nitration by reactive nitrogen species. Synthetic 9-NO2-OA and 10-NO2-OA at concentrations of three orders of magnitude higher than their endogenous counterparts have interesting pharmacological features and are currently intensely investigated. The present article reviews and discusses currently available analytical methods for the quantitative determination of cis-EpOA, 9-NO2-OA and 10-NO2-OA in biological samples, notably in human plasma, and the potential biological significance of these oleic acid metabolites. Special emphasis is given to GC–MS/MS and LC–MS/MS methods utilizing the stable-isotope dilution technique. The sensitivity and specificity of the MS/MS approach make electron-capture negative ion chemical ionization (ECNICI) GC–MS/MS and negative electrospray ionization (NESI) LC–MS/MS methodologies indispensable in experimental and clinical settings on oxidative and nitrative oleic acid metabolism. These techniques are particularly suited to delineate the oleic acid cascade. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► Pentafluorobenzyl bromide derivatization of MUFAs and PUFAs. ► ECNICI GC-MS/MS of epoxidized and nitrated oleic acid and PUFAs. ► ESI LC-MS/MS of epoxidized and nitrated oleic acid and PUFAs. ► Tandem mass spectrometry identification of epoxidized and nitrated MUFAs and PUFAs. ► Quantification by selected reaction monitoring and reference values.
Keywords: Lipids; Nitration; Oxidation; Stable-isotope; Tandem mass spectrometry;

Quantification of endocannabinoids in biological systems by chromatography and mass spectrometry: A comprehensive review from an analytical and biological perspective by Alexander A. Zoerner; Frank-Mathias Gutzki; Sandor Batkai; Marcus May; Christin Rakers; Stefan Engeli; Jens Jordan; Dimitrios Tsikas (706-723).
The endocannabinoids anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2AG) are physiologically occurring, biologically active compounds on CB1 and CB2 receptors with multiple physiological functions. AEA and 2AG have been identified and quantified in many mammalian biological fluids and tissues, such as human plasma, adipocytes, tissues and tissue microdialysates, at concentrations in the picomolar-to-nanomolar range under basal conditions. In this article, recently published chromatographic and mass spectrometric analytical methods, i.e., HPLC with fluorescence or ultraviolet detection, LC–MS, LC–MS/MS, GC–MS and GC–MS/MS, are reviewed and discussed, notably from the quantitative point of view. We focus on and emphasize the particular importance of blood sampling, sample storage and work-up including solvent and solid-phase extraction and derivatization procedures, matrix-effects, and stability of analytes. As 2AG spontaneously isomerizes to its CB1/CB2 receptors biologically inactive 1-arachidonoyl glycerol (1AG) by acyl migration, this phenomenon and its particular importance for accurate quantification of 2AG are discussed in detail. Due to the electrical neutrality of AEA and 2AG their solvent extraction by toluene offers the least matrix-effect and minimum isomerization. LC–MS/MS is the most frequently used analytical technique for AEA and 2AG. At present, the utility of the GC–MS/MS methodology seems to be limited to AEA measurement in human plasma, bronchoalveolar liquid (BAL) and microdialysate samples. Despite great instrumental advances in the LC–MS/MS methodology, sampling and sample treatment remains one of the most crucial analytical steps in 2AG analysis. Extension of the LC–MS/MS methodology, for instance to microdialysate and BAL samples from clinical studies, is a big analytical challenge in endocannabinoid analysis in clinical settings. Currently available LC–MS/MS and GC–MS/MS methods should be useful to investigate the metabolism of AEA and 2AG beyond hydrolysis, i.e., by β- and ω-oxidation pathways. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► We provide an extensive review covering quantitative endocannabinoid analysis. ► It provides a broad overview of methodologies published in the last 30 years. ► The molecules anandamide, 2-arachidonoyl glycerol, virodhamine, 2-arachidonyl glycerol ether and arachidonoyl dopamide are discussed. ► Mass spectrometric approaches of endocannabinoid analysis are described in detail.
Keywords: Chromatography; Clinical studies; Plasma; Quantification; Stable-isotope dilution; Tandem mass spectrometry;

High-throughput lipidomic analysis of fatty acid derived eicosanoids and N-acylethanolamines by Darren S. Dumlao; Matthew W. Buczynski; Paul C. Norris; Richard Harkewicz; Edward A. Dennis (724-736).
Fatty acid-derived eicosanoids and N-acylethanolamines (NAE) are important bioactive lipid mediators involved in numerous biological processes including cell signaling and disease progression. To facilitate research on these lipid mediators, we have developed a targeted high-throughput mass spectrometric based methodology to monitor and quantitate both eicosanoids and NAEs, and can be analyzed separately or together in series. Each methodology utilizes scheduled multiple reaction monitoring (sMRM) pairs in conjunction with a 25 min reverse-phase HPLC separation. The eicosanoid methodology monitors 141 unique metabolites and quantitative amounts can be determined for over 100 of these metabolites against standards. The analysis covers eicosanoids generated from cycloxygenase, lipoxygenase, cytochrome P450 enzymes, and those generated from non-enzymatic pathways. The NAE analysis monitors 36 metabolites and quantitative amounts can be determined for 33 of these metabolites against standards. The NAE method contains metabolites derived from saturated fatty acids, unsaturated fatty acids, and eicosanoids. The lower limit of detection for eicosanoids ranges from 0.1 pg to 1 pg, while NAEs ranges from 0.1 pg to 1000 pg. The rationale and design of the methodology is discussed. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► We present a high-throughput methodology for eicosanoids and N-acylethanolamines. ► The rationale behind the method design is addressed. ► Quantitation for 100 eicosanoid and 33 N-acylethanolamine species. ► Limits of detections are determined. ► Biological application of the methodology is demonstrated.
Keywords: Eicosanoid; N-acylethanolamine; Lipidomics; LIPID MAPS; High-throughput methodology; Mass spectrometry;

Chiral lipidomics of E-series resolvins: Aspirin and the biosynthesis of novel mediators by Sungwhan F. Oh; Thad W. Vickery; Charles N. Serhan (737-747).
Control of the inflammatory response is of wide interest given its important role in many diseases. In recent years we identified novel mechanisms and lipid mediators that play an active role in stimulating the resolution of self-limited acute inflammation. These novel pro-resolving mediators include the essential fatty acid-derived lipoxins, resolvins, protectins and maresins. Members of each possess a unique pro-resolving mechanism of action; each limits neutrophilic infiltration, regulates local mediators (chemokines, cytokines) as well as stimulates macrophage-enhanced clearance of apoptotic PMN, cellular debris and microbes. Given this unique mechanism of action, resolvins have already been shown to play pivotal roles in regulating key events in a wide range of experimental inflammatory diseases. These pro-resolving mediators also provide a molecular link between omega-3 essential fatty acids (e.g. EPA, DHA) and the resolution process of inflammation and tissue homeostasis. Here, we review recent evidence obtained using chiral LC-MS-MS-based lipidomics to identify a novel 18S-series of resolvins derived from EPA. Resolvin E1 possesses potent actions in vivo and in vitro demonstrated now in many laboratories, and herein we review comparisons in E-series resolvin biosynthesis and action of 18S-resolvin E1 and 18S-resolvin E2. The biosynthesis and formation of both 18S and 18R-series are enhanced with aspirin treatment and involve the utilization of dietary EPA as well as recombinant human 5-lipoxygenase and LTA4 hydrolase in their stereospecific biosynthesis. Herein we also demonstrate the utility of LC-MS-MS-based lipidomics in identifying resolvins, protectins and related products in marine organisms such as Engraulis (Peruvian anchovy). These new findings emphasize the utility of chiral LC-MS-MS lipidomics and the potential for identifying new resolution circuits with chiral LC-MS-MS-based lipidomics and metabolomics. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► Herein we review recent evidence obtained using chiral LC-MS-MS-based lipidomics to identify a novel 18S-series of resolvins derived from EPA. ► We review comparisons in E-series resolvin biosynthesis and action of 18S-resolvin E1 and 18S-resolvin E2. ► The biosynthesis and formation of both 18S and 18R-series are enhanced with aspirin treatment and involve the utilization of dietary EPA as well as recombinant human 5-lipoxygenase and LTA4 hydrolase in their stereospecific biosynthesis.
Keywords: Resolution; Inflammation; Omega-3 fatty acid; Leukocyte; Nutrition;

Quantitative analysis of glycerophospholipids by LC–MS: Acquisition, data handling, and interpretation by David S. Myers; Pavlina T. Ivanova; Stephen B. Milne; H. Alex Brown (748-757).
As technology expands what it is possible to accurately measure, so too the challenges faced by modern mass spectrometry applications expand. A high level of accuracy in lipid quantitation across thousands of chemical species simultaneously is demanded. While relative changes in lipid amounts with varying conditions may provide initial insights or point to novel targets, there are many questions that require determination of lipid analyte absolute quantitation. Glycerophospholipids present a significant challenge in this regard, given the headgroup diversity, large number of possible acyl chain combinations, and vast range of ionization efficiency of species. Lipidomic output is being used more often not just for profiling of the masses of species, but also for highly-targeted flux-based measurements which put additional burdens on the quantitation pipeline. These first two challenges bring into sharp focus the need for a robust lipidomics workflow including deisotoping, differentiation from background noise, use of multiple internal standards per lipid class, and the use of a scriptable environment in order to create maximum user flexibility and maintain metadata on the parameters of the data analysis as it occurs. As lipidomics technology develops and delivers more output on a larger number of analytes, so must the sophistication of statistical post-processing also continue to advance. High-dimensional data analysis methods involving clustering, lipid pathway analysis, and false discovery rate limitation are becoming standard practices in a maturing field. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.►We review specifics of glycerophospholipid (GPL) analysis by MS and data handling. ►GPL quantitation is challenging given species diversity and range of ionizability. ►Lipidomics is moving beyond profiling to include flux-based measurements.
Keywords: Mass spectrometry; Glycerophospholipids; LC–MS; Quantitation; Data handling; Lipidomics;

Methods for analyzing phosphoinositides using mass spectrometry by Michael J.O. Wakelam; Jonathan Clark (758-762).
The polyphosphoinositides are key signaling lipids whose levels are tightly regulated within cells. As with other cellular lipids multiple species exist with distinct acyl chain makeups. There are methods which analyze the phosphoinositides as their deacylated derivatives which cannot address these distinct forms. Lipidomic analysis of the polyphosphoinositides has been hampered by difficulties with extraction and problems associated with binding of the lipids to surfaces. This review outlines the available MS methodologies, highlighting the difficulties associated with each. However, at present, no single methodology is available that can successfully and reproducibly quantitate each inositol phospholipid. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► In contrast to other lipids methods for phosphoinositide analysis are limited. ► Analysis and quantification of these signaling lipids is important. ► Methods available to analyze polyphosphoinositide are reviewed. ► Derivatization of PtdIns3,4,5P3 allows characterization and quantitation.
Keywords: Mass spectrometry; Phosphoinositide; HPLC; Acyl chain; PtdIns3,4,5P3;

This review deals with the LC-MS analysis of phospholipids. The advantages of including liquid chromatography in phospholipids are highlighted. Special attention is paid to the most-used ionization methods and the role of solvents in chromatography and ionization. Difficulties associated with different quantification strategies are discussed. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► Advantages of liquid chromatography in lipidomic analysis. ► Added value of other ionization techniques than ESI. ► Normal- and reverse phase separations. ► How to deal with ion suppression in quantification.
Keywords: Liquid chromatography; Mass spectrometry; Ionization; Quantification;

Glycerolipid and cholesterol ester analyses in biological samples by mass spectrometry by Robert C. Murphy; Thomas J. Leiker; Robert M. Barkley (776-783).
Neutral lipids are a diverse family of hydrophobic biomolecules that have important roles in cellular biochemistry of all living species but have in common the property of charge neutrality. A large component of neutral lipids is the glycerolipids composed of triacylglycerols, diacylglycerols, and monoacylglycerols that can serve as cellular energy stores as well as signaling molecules. Another abundant lipid class in many cells is the cholesterol esters that are on one hand sterols and the other fatty acyl lipids, but in either case are neutral lipids involved in cholesterol homeostasis and transport in the blood. The analysis of these molecules in the context of lipidomics remains challenging because of their charge neutrality and the complex mixtures of molecular species present in cells. Various techniques have been used to ionize these neutral lipids prior to mass spectrometric analysis including electron ionization, atmospheric chemical ionization, electrospray ionization and matrix assisted laser desorption/ionization. Various approaches to deal with the complex mixture of molecular species have been developed including shotgun lipidomics and chromatographic-based separations such as gas chromatography, reversed phase liquid chromatography, and normal phase liquid chromatography. Several applications of these approaches are discussed. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► APCI and APCI/LC/MS analysis of neutral lipids. ► Electrospray ionization of cholesterol esters and glycerolipids.
Keywords: Glycerolipid; Electrospray ionization; Mass spectrometry; Triacylglycerol; Diacylglycerol; Cholesterol ester;

Analysis of oxysterol metabolomes by William J. Griffiths; Yuqin Wang (784-799).
Oxysterols are oxygenated forms of cholesterol. This definition can, however, be expanded to include oxygenated derivatives of plant sterols and also of cholesterol precursors. Oxysterols are formed in the first steps of cholesterol metabolism and also from cholesterol by reactive oxygen species. Oxysterols were once thought of as simple intermediates, or side-products, in the conversion of cholesterol to hormonal steroids and bile acids, however, they have subsequently been shown to be biologically active molecules in their own right. In this article we will discuss methods of oxysterol analysis including “classical” gas chromatography–mass spectrometry (GC–MS) methods and more recent liquid chromatography (LC)–MS methods. Our main focus, however, will be on analytical methods based on “charge-tagging” and LC–tandem mass spectrometry (MS/MS or MSn) which we have developed over the last decade in our laboratory. Examples will be given of oxysterol analysis in brain, cerebrospinal fluid (CSF) and blood. The advantages and disadvantages of the various methods of oxysterol analysis will be discussed. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► Review of existing GC–MS and LC–MS methods for oxysterol analysis ► Description of sample preparation protocols ► Discussion of newly introduced “charge-tagging” LC–MS/MS and LC–MSn methods ► Evaluation of the “pros and cons” of new analytical methods
Keywords: Oxysterol; Cholesterol; Sterol; Bile acid; Nuclear receptor; Liquid chromatography–mass spectrometry;

Across evolution, dolichols and polyprenols serve as sugar carriers in biosynthetic processes that include protein glycosylation and lipopolysaccharide biogenesis. Liquid chromatography coupled with electrospray ionization mass spectrometry offers a powerful tool for studying dolichols and polyprenols in their alcohol or glycan-modified forms in members of all three domains of life. In the following, recent examples of the how different versions of this analytical approach, namely reverse phase liquid chromatography-multiple reaction monitoring, normal phase liquid chromatography/tandem mass spectrometry and normal phase liquid chromatography-precursor ion scan detection have respectively served to address novel aspects of dolichol or polyprenol biology in Eukarya, Archaea and Bacteria. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► RP LC-MRM allows sensitive detection of polyprenols and dolichols in animal tissues. ► NP LC-MS/MS allows structural characterization of DolP-linked glycans in Archaea. ► NP LC-precursor ion scan allows rapid, global profiling of C55-P-derived sugars in Bacteria.
Keywords: Liquid chromatography/tandem mass spectrometry; Multiple reaction monitoring; Precursor ion scan; Dolichol; Polyprenol;

Analysis of unsaturated lipids by ozone-induced dissociation by Simon H.J. Brown; Todd W. Mitchell; Stephen J. Blanksby (807-817).
Recent developments in analytical technologies have driven significant advances in lipid science. The sensitivity and selectivity of modern mass spectrometers can now provide for the detection and even quantification of many hundreds of lipids in a single analysis. In parallel, increasing evidence from structural biology suggests that a detailed knowledge of lipid molecular structure including carbon–carbon double bond position, stereochemistry and acyl chain regiochemistry is required to fully appreciate the biochemical role(s) of individual lipids. Here we review the capabilities and limitations of tandem mass spectrometry to provide this level of structural specificity in the analysis of lipids present in complex biological extracts. In particular, we focus on the capabilities of a novel technology termed ozone-induced dissociation to identify the position(s) of double bonds in unsaturated lipids and discuss its possible role in efforts to develop workflows that provide for complete structure elucidation of lipids by mass spectrometry alone: so-called top-down lipidomics. This article is part of a Special Issue entitled: Lipodomics and Imaging Mass Spectrom.► The importance of molecular structure in understanding lipid biochemistry ► The strengths and limitations of contemporary mass spectrometry ► The capabilities of ozone induced dissociation
Keywords: Lipid; Lipidomics; Mass spectrometry; Ozonolysis; Ozone-induced dissociation; Collision-induced dissociation;

Oxidized phospholipids (OxPLs) are rapidly becoming recognized as important mediators of cellular and immune signaling. They are generated either enzymatically or non-enzymatically and 100s of structures exist of which only a small fraction have been analyzed to date. Pleiotropic activities, including regulation of adhesion molecule expression, pro-coagulant activity and inhibition of Toll-like receptor signaling have been observed and some are detected in models of human and animal disease, including atherosclerosis and infection. More recently, the acute generation of specific oxidized phospholipids by cellular enzymes in immune cells was reported. Assays for analysis and quantification of OxPLs were first developed approx 15 years ago, primarily for hydro(pero)xy-species. Many were based on monitoring a single precursor ion with/without LC separation, based on the PL headgroup. Others combined LC with monitoring precursor to product transitions, but were unable to provide information regarding position of oxidation on unsaturated sn-2 fatty acid due to sensitivity issues. More recently, LC/MS/MS methods for specific OxPLs have been reported that enable high sensitivity quantitation in biological samples. In this review, widely used methods for detecting and quantifying various classes of OxPL will be summarized, along with practical advice for their use. In particular, the focus will be on LC/MS/MS, which today is almost universally the method of choice. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► Methods for analysis of oxidized phospholipids are highlighted. ► Oxidized phospholipids are important signalling mediators in inflammation. ► LC/MS/MS is rapidly becoming the method of choice for analysis of oxidized phospholipids.
Keywords: Phospholipid; Phosphatidyethanolamine; Phosphatidylcholine; Lipoxygenase; Oxidation; Mass spectrometry;

Non-enzymatically derived minor lipids found in Escherichia coli lipid extracts by Teresa A. Garrett; Christian R.H. Raetz; Jennifer D. Son; Travis D. Richardson; Craig Bartling; Ziqiang Guan (827-837).
Electrospray ionization mass spectrometry is a powerful technique to analyze lipid extracts especially for the identification of new lipid metabolites. A hurdle to lipid identification is the presence of solvent contaminants that hinder the identification of low abundance species or covalently modify abundant lipid species. We have identified several non-enzymatically derived minor lipid species in lipid extracts of Escherichia coli; phosphatidylmethanol, ethyl and methyl carbamates of PE and N-succinyl PE were identified in lipid extracts of E. coli. Phosphatidylmethanol (PM) was identified by exact mass measurement and collision induced dissociation tandem mass spectrometry (MS/MS). Extraction in the presence of deuterated methanol leads to a 3 atomic mass unit shift in the [M–H] ions of PM indicating its formation during extraction. Ethyl and methyl carbamates of PE, also identified by exact mass measurement and MS/MS, are likely to be formed by phosgene, a breakdown product of chloroform. Addition of phosgene to extractions containing synthetic PE significantly increases the levels of PE-MC detected in the lipid extracts by ESI-MS. Extraction in the presence of methylene chloride significantly reduced the levels of these lipid species. N-succinyl PE is formed from reaction of succinyl-CoA with PE during extraction. Interestingly N-succinyl PE can be formed in an aqueous reaction mixture in the absence of added E. coli proteins. This work highlights the reactivity of the amine of PE and emphasizes that careful extraction controls are required to ensure that new minor lipid species identified using mass spectrometry are indeed endogenous lipid metabolites. This article is part of a Special Issue entitled: Lipodomics and Imaging Mass Spectrometry.► Extraction of lipids using solvents can generate artifacts. ► Mass spectrometry identifies lipids formed non-enzymatically during extraction. ► The amine of phosphatidylethanolamine is particularly reactive during chlorofom extraction.
Keywords: Mass spectrometry; E. coli; Lipid; Chloroform; Phosgene; Artifact;

Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids give rise to their biological activity, there is a need for comprehensive or “sphingolipidomic” methods for identification and quantitation of as many individual subspecies as possible. This review defines sphingolipids as a class, briefly discusses classical methods for their analysis, and focuses primarily on liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Recently, a set of evolving and expanding methods have been developed and rigorously validated for the extraction, identification, separation, and quantitation of sphingolipids by LC-MS/MS. Quantitation of these biomolecules is made possible via the use of an internal standard cocktail. The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, sulfatides, and novel compounds such as the 1-deoxy- and 1-(deoxymethyl)-sphingoid bases and their N-acyl-derivatives. These methods can be altered slightly to separate and quantitate isomeric species such as glucosyl/galactosylceramide. Because these techniques require the extraction of sphingolipids from their native environment, any information regarding their localization in histological slices is lost. Therefore, this review also describes methods for TIMS. This technique has been shown to be a powerful tool to determine the localization of individual molecular species of sphingolipids directly from tissue slices. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► Classical methods for the analysis of sphingolipids are reviewed. ► LC-MS/MS methods for identification and quantitation of sphingolipids are presented. ► Tissue Imaging Mass Spectrometry of sphingolipids is presented. ► Applications of new technologies to the analysis of sphingolipids are discussed.
Keywords: Sphingolipid; LC-MS/MS; Tissue imaging mass spectrometry; Tandem mass spectrometry; Quantitation; Internal standard;

Mass spectrometric analysis of neutral sphingolipids: Methods, applications, and limitations by Hany Farwanah; Thomas Kolter; Konrad Sandhoff (854-860).
Sphingolipids represent an important class among lipids, especially when considering their vital roles in lipid metabolism. Thus, a variety of methods have been created to accomplish their analysis and the term “sphingolipidomics” has recently been coined to underline the motivation to enable a comprehensive analysis of all sphingolipid species including the acidic and the neutral ones. In this review, we summarize selected mainly biomedical based mass spectrometric approaches for the analysis of neutral sphingolipids regarding their advantages, applications and limitations. To underline some practical aspects of method development, we focus on a new method recently developed in our laboratory, which enables separation, detection, and mass spectrometric profiling of ceramide, hexosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, sphingomyelin species, and cholesterol in one run. This method can be applied to investigate impairments of neutral sphingolipid metabolism in a variety of disorders such as sphingolipidoses and be employed to screen for sphingolipid profile changes as induced by knockout experiments or related studies. This article is part of a Special Issue entitled: Lipodomics and Imaging Mass Spectrometry.► Mass spectrometric methods for the analysis of neutral (glyco)sphingolipids. ► Focus is on ESI-MS-, APCI-MS-, and MALDI-MS- methods used in biomedical research. ► Shotgun methods in comparison to methods with pre-separations. ► Scope and limitations of glycosphingolipid and sphingolipid analysis by LC/APCI-MS.
Keywords: Lipidomics; Sphingolipidoses; Cholesterol; LC/MS; APCI-MS; Q-TOF;

High-performance thin-layer chromatography/mass spectrometry for the analysis of neutral glycosphingolipids by Akemi Suzuki; Masao Miyazaki; Junko Matsuda; Azusa Yoneshige (861-874).
This mini-review summarizes the protocol we have developed for the analysis of neutral glycosphingolipids (GSLs) by high-performance thin layer chromatography (HPTLC)–mass spectrometry (MS). We also present results obtained using this glycolipidomic approach to study neutral GSLs from mouse kidney, spleen, and small intestine. Finally, we discuss what is required for further development of this method, as well as what is expected for the future of glycolipid biology. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► The protocol for the analysis of neutral glycosphingolipids (GSLs) by high-performance thin layer chromatography (HPTLC)-mass spectrometry (MS). ► Mass spectra of neutral GSLs prepared from mouse kidney, spleen, and small intestine. ► Coupling of the bird eye overview of GSLs by HPTLC with precise structural information by MS. ► Discussion on other MS methods for GSLs and further development of HPTLC-MS.
Keywords: HPTLC–MS; Glycosphingolipid; Ceramide; Kidney; Spleen; Small intestine;

Much effort is currently invested in the development of mass spectrometry-based strategies for investigating the entirety of glycosphingolipids (GSLs) of a certain cell type, tissue, organ or body encompassing the respective glycosphingolipidome. As part of the investigation of the vertebrate glycosphingolipidome, GSL analysis is undergoing rapid expansion owing to the application of novel mass spectrometry techniques acting as the linchpin in the network of collaborations challenged to unravel structural and functional aspects of GSLs. Difficulties may arise in the determination of the exact structures of GSLs due to the heterogeneity of the sugar moiety varying in the number and sequence of monosaccharides, and their anomeric configuration and linkage type, which make up the principal items of the glyco code of biologically active carbohydrate chains. The ceramide variability caused by the diversity of the long-chain amino alcohol and the fatty acid, which both may vary in chain length, degree of unsaturation, and type and number of substituents, further contributes to the increasing number of possible GSL species. In view of this heterogeneity, a single-method analytical mass spectrometry (MS) technique without auxiliary tools yields limited data, providing only partial structural information of individual GSLs in complex mixtures. Approaching this challenge, current advances on a triad system matching three complementary methods are described in this review: (i) silica gel based TLC separation of GSLs, (ii) their overlay detection on the TLC plate (mostly based on antibody-mediated recognition), and (iii) direct and indirect MS based structural characterization, i.e. directly on the TLC plate or in lipid extracts from silica gel. We will focus on recent improvements by employing antibodies, AB5 toxins and bacteria for direct IR-MALDI-o-TOF MS and indirect ESI-QTOF MS analysis of GSLs. We believe that the combinatorial approach using conventional TLC and modern mass spectrometry provides a developmental advance in exploring the glycosphingolipidome of biological material.Display Omitted► Combining TLC, overlay assay, and MS allows firm structural characterization of GSLs. ► TLC-MS eases GSL receptor identification of antibodies, toxins, viruses, and bacteria. ► Coupling of TLC with MS provides advances in animal and human glycosphingolipidomics.
Keywords: Glycolipid; Ganglioside; IR-MALDI-o-TOF MS; ESI-QTOF MS; TLC; Overlay assay;

Reprint of: Chip-based nanoelectrospray mass spectrometry of brain gangliosides by Corina Flangea; Alina Serb; Eugen Sisu; Alina D. Zamfir (897-917).
In the past few years, a considerable effort was invested in interfacing mass spectrometry (MS) to microfluidics-based systems for electrospray ionization (ESI). Since its first introduction in biological mass spectrometry, chip-based ESI demonstrated a high potential to discover novel structures of biomarker value. Therefore, recently, microfluidics for electrospray in conjunction with advanced MS instruments able to perform multistage fragmentation were introduced also in glycolipid research. This review is focused on the strategies, which allowed a successful application of chip technology for ganglioside mapping and sequencing by ESI MS and tandem MS (MS/MS). The first part of the review is dedicated to the progress of MS methods in brain ganglioside research, which culminated with the introduction of two types of microfluidic devices: the NanoMate robot and a polymer microchip for electrospray. In the second part a systematic description of most relevant results obtained by using MS in combination with the two chip systems is presented. Chip-based ESI accomplishments for determination of ganglioside expression and structure in normal brain regions and brain pathologies such as neurodegenerative diseases and primary brain tumors are described together with some considerations upon the perspectives of microfluidics-MS to be routinely introduced in biomedical investigation.► Two types of microfluidics for electrospray were introduced in ganglioside field. ► We describe their principles and advantages over the capillary-based electrospray. ► We also review their application to brain gangliosides in health and disease.
Keywords: Brain gangliosides; Mass spectrometry; Chip-based electrospray; Microfluidics; Brain diseases;

Lipid profiling of lipoproteins by electrospray ionization tandem mass spectrometry by Max Scherer; Alfred Böttcher; Gerhard Liebisch (918-924).
Lipoproteins are of fundamental importance for the lipid transport and cardiovascular disease. The function and metabolism of lipoproteins is intimately linked to the biophysical properties of their surface lipids. Although a number of disease associations were found for lipid species in plasma, only a few studies reported lipid profiles of lipoproteins. Here, we provide an overview of techniques for lipoprotein separation, methods for lipid species analysis based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) as well as data from recent lipidomic studies on lipoprotein fractions. We also discuss the different analytical strategies and how lipid profiling can expand our understanding of the biology and structures of lipoproteins. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► Overview of lipoprotein separation techniques. ► Methods for lipid species analysis based on ESI-MS/MS. ► Overview of lipid species data of lipoprotein fractions. ► Discussion of analytical strategies.
Keywords: Lipoprotein separation; Lipidomics; Phospholipids; Sphingolipids; Lipid species;

High throughput quantitative molecular lipidomics by Hye R. Jung; Tuulia Sylvänne; Kaisa M. Koistinen; Kirill Tarasov; Dimple Kauhanen; Kim Ekroos (925-934).
Applications in biomedical research increasingly demand detailed lipid molecule information acquired at high throughput. Although the recent advances in lipidomics offer to delineate the lipidomes in detail, the challenge remains in performing such analyses at the requested quality and to maintain the quality also in a high throughput setting. In this review we describe a high throughput molecular lipidomic solution based on robotic assisted sample preparation and lipid extraction and multiple lipidomic platforms integrated with a sophisticated bioinformatics system. As demonstrated, the virtue of this lipidomic toolkit lies in its high throughput delivery of comprehensive quantitative lipidomic outputs at the molecular lipid level, its ease of scalability and its capability to serve in a regulatory setting. We anticipate that this toolkit will contribute to basic research, nutritional research and promote the discovery of new disease biomarkers, disease related mechanisms of actions and drug targets. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► Molecular lipidomics. ► Robotic assisted sample preparation and lipid extraction. ► Shotgun and LC-MRM lipidomics. ► Lipidomics driven bioinformatics. ► High throughput quantitative molecular lipidomics.
Keywords: Robotic-assisted; Shotgun lipidomics; LC–MRM; High throughput; Bioinformatics; Molecular lipidomics;

Lipid analysis and lipidomics by structurally selective ion mobility-mass spectrometry by Michal Kliman; Jody C. May; John A. McLean (935-945).
Recent advances in mass spectrometry approaches to the analysis of lipids include the ability to incorporate both lipid class identification with lipid structural information for increased characterization capabilities. The detailed examination of lipids and their biosynthetic and biochemical pathways made possible by novel instrumental and bioinformatics approaches is advancing research in fundamental cellular and medical studies. Recently, high-throughput structural analysis has been demonstrated through the use of rapid gas-phase separation on the basis of the ion mobility (IM) analytical technique combined with mass spectrometry (IM-MS). While IM-MS has been extensively utilized in biochemical research for peptide, protein and small molecule analysis, the role of IM-MS in lipid research is still an active area of development. In this review of lipid-based IM-MS research, we begin with an overview of three contemporary IM techniques which show great promise in being applied towards the analysis of lipids. Fundamental concepts regarding the integration of IM-MS are reviewed with emphasis on the applications of IM-MS towards simplifying and enhancing complex biological sample analysis. Finally, several recent IM-MS lipid studies are highlighted and the future prospects of IM-MS for integrated omics studies and enhanced spatial profiling through imaging IM-MS are briefly described. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► A review of ion mobility-mass spectrometry (IM-MS) for the study of lipids. ► IM-MS data dimensionality and reduces spectral complexity. ► IM-MS is used as an enhanced separation technique and as a structural analysis tool. ► Imaging IM-MS has potential high impact for analyzing complex biological samples. ► Contemporary examples of IM-MS lipid research are limited, but are rapidly expanding.
Keywords: Ion mobility-mass spectrometry; Phospholipid; Gas-phase biomolecular separation; Structural selectivity; Imaging IM-MS;

Desorption electrospray ionization mass spectrometry for lipid characterization and biological tissue imaging by Livia S. Eberlin; Christina R. Ferreira; Allison L. Dill; Demian R. Ifa; R. Graham Cooks (946-960).
Desorption electrospray ionization mass spectrometry (DESI-MS) imaging of biological samples allows untargeted analysis and structural characterization of lipids ionized from the near-surface region of a sample under ambient conditions. DESI is a powerful and sensitive MS ionization method for 2D and 3D imaging of lipids from direct and unmodified complex biological samples. This review describes the strengths and limitations of DESI-MS for lipid characterization and imaging together with the technical workflow and a survey of applications. Included are discussions of lipid mapping and biomarker discovery as well as a perspective on the future of DESI imaging.Display Omitted► DESI-MS is a powerful and sensitive ambient MS ionization method. ► 2D and 3D imaging of lipids from untreated biological samples is achieved by DESI-MS under ambient conditions. ► DESI-MS has strengths and limitations for lipid characterization and imaging. ► DESI-MS technical workflow and a survey of applications are described. ► Discussion on lipid mapping and biomarker discovery and a perspective on the future of DESI imaging are included.
Keywords: Ambient mass spectrometry; Imaging; Lipidomics; Disease diagnosis; Desorption electrospray ionization; Lipid characterization;

Imaging mass spectrometry for lipidomics by Naoko Goto-Inoue; Takahiro Hayasaka; Nobuhiro Zaima; Mitsutoshi Setou (961-969).
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool that enables the simultaneous detection and identification of biomolecules in analytes. MALDI-imaging mass spectrometry (MALDI-IMS) is a two-dimensional MALDI-MS technique used to visualize the spatial distribution of biomolecules without extraction, purification, separation, or labeling of biological samples. This technique can reveal the distribution of hundreds of ion signals in a single measurement and also helps in understanding the cellular profile of the biological system. MALDI-IMS has already revealed the characteristic distribution of several kinds of lipids in various tissues. The versatility of MALDI-IMS has opened a new frontier in several fields, especially in lipidomics. In this review, we describe the methodology and applications of MALDI-IMS to biological samples. This article is part of a Special Issue entitled: Lipodomics and Imaging Mass Spectrometry.► MALDI-IMS is becoming an essential tool for molecular imaging of biological samples. ► It can facilitate the discovery of characteristic molecules in regions of interest. ► Here we showed that many great advances have already been made with MALDI-IMS.
Keywords: Lipidomics; Imaging mass spectrometry; Matrix-assisted laser desorption/ionization;

MALDI imaging of lipids after matrix sublimation/deposition by Robert C. Murphy; Joseph A. Hankin; Robert M. Barkley; Karin A. Zemski Berry (970-975).
Mass spectrometric techniques have been developed to record mass spectra of biomolecules including lipids as they naturally exist within tissues and thereby permit the generation of images displaying the distribution of specific lipids in tissues, organs, and intact animals. These techniques are based on matrix-assisted laser desorption/ionization (MALDI) that requires matrix application onto the tissue surface prior to analysis. One technique of application that has recently shown significant advantages for lipid analysis is sublimation of matrix followed by vapor deposition directly onto the tissue. Explanations for enhanced sensitivity realized by sublimation/deposition related to sample temperature after a laser pulse and matrix crystal size are presented. Specific examples of sublimation/deposition in lipid imaging of various organs including brain, ocular tissue, and kidney are presented. This article is part of a Special Issue entitled: Lipodomics and Imaging Mass Spectrom.► Sublimation/deposition purifies matrix and minimizes lateral diffusion of lipids. ► Sublimation results in small matrix crystals, which increases lipid signal strength. ► Examples of MALDI IMS using sublimation include brain, lens, retina, kidney tissues.
Keywords: MALDI Imaging mass spectrometry; Sublimation/deposition; Sphingolipid; Glycerophospholipid;

Lipid imaging with time-of-flight secondary ion mass spectrometry (ToF-SIMS) by Melissa K. Passarelli; Nicholas Winograd (976-990).
Fundamental advances in secondary ion mass spectrometry (SIMS) now allow for the examination and characterization of lipids directly from biological materials. The successful application of SIMS-based imaging in the investigation of lipids directly from tissue and cells are demonstrated. Common complications and technical pitfalls are discussed. In this review, we examine the use of cluster ion sources and cryogenically compatible sample handling for improved ion yields and to expand the application potential of SIMS. Methodological improvements, including pre-treating the sample to improve ion yields and protocol development for 3-dimensional analyses (i.e. molecular depth profiling), are also included in this discussion. New high performance SIMS instruments showcasing the most advanced instrumental developments, including tandem MS capabilities and continuous ion beam compatibility, are described and the future direction for SIMS in lipid imaging is evaluated.► Recent instrumental and sample preparation methodologicaladvances achieved in ToF- SIMS, as well as the successful applications of these advances in the detection of lipid from biological material are described in this review. ► The performance of ToF-SIMS in the analysis of lipids is compared to similar imaging techniques, including MALDI, DESI and dynamic SIMS. ► Various lipid species detected and identified using ToF-SIMS are compiled in tables and organized using the lipid classification system established by the Lipid MAPS consortium. ► State-of-the-art ToF-SIMS instruments, the J105 3D Chemical Imager and C60 +-QSTAR, are described and their potential application in the field of lipidomics is discussed.
Keywords: ToF-SIMS; Lipid; Cluster source; Sample preparation; C60 +QSTAR; J105 3D Chemical Imager Imaging mass spectrometry (IMS);

The ability to translate vast amounts of information, as obtained from lipidomic analysis, into the knowledge and understanding of biological phenomena is an important challenge faced by the lipidomics community. While many of the informatics and computational tools from other domains such as bioinformatics and metabolomics are also applicable to lipidomics data processing and analysis, new solutions and strategies are needed for the studies of lipidomes at the systems level. This is due to enormous functional and structural diversity of lipids as well as because of their complex regulation at multiple spatial and temporal scales. In order to better understand the lipidomes at the physiological level, lipids need to be modeled not only at the level of biological pathways but also at the level of the biophysical systems they are part of, such as cellular membranes or lipoprotein particles. Herein the current state, recent advances and new opportunities in the field of lipid bioinformatics are reviewed. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.► Many bioinformatics tools used in metabolomics are applicable to lipidomic data analysis. ► Regulation of lipid metabolism occurs at multiple spatial and temporal scales. ► New methods are needed for modeling of lipidomics data.
Keywords: Allostasis; Lipidomics; Metabolomics; Molecular dynamics; System biology;