Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.     

An improved assay for platelet-activating factor using HPLC-tandem mass spectrometry

We describe an improved assay for platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) using HPLC-tandem mass spectrometry (LC-MS/MS). The present method can readily detect as little as 1 pg (1.9 fmol) of PAF, a significant improvement over previously described LC-MS/MS methods, and gives a linear response up to 1,000 pg of PAF. Our method also overcomes the artifacts from isobaric lipids that have limited the usefulness of certain existing LC-MS/MS assays for PAF. In the course of these studies, we detected three novel lipid species in human neutrophils. One of the novel lipids appears to be a new molecular species of PAF, and the other two have chromatographic and mass spectrometric properties consistent with stearoyl-formyl-glycerophosphocholine and oleoyl-formyl-glycerophosphocholine. These observations identify previously unknown potential interferences in the measurement of PAF by LC-MS/MS. Moreover, our data suggest that the previously described palmitoyl-formyl-glycerophosphocholine is not unique but rather is a member of a new and poorly understood family of formylated lipids.     

Lipidomic analysis of bacterial plasmalogens

Plasmalogens are a group of lipids with potentially important, and not yet fully known, functions in organisms from bacteria to protozoans, invertebrates, and mammals. They can protect cells against the damaging effects of reactive oxygen species, protect other phospholipids or lipoprotein particles against oxidative stress, and have been implicated as signaling molecules and modulators of membrane dynamics. They have been found in many anaerobic bacterial species, and their biosynthetic pathways differ in aerobic and anaerobic organisms. The use of advanced techniques permits the identification of not only plasmalogen classes but also their positional isomers and often also individual molecular species. This paper describes direct analyses of plasmalogens from natural sources, frequently very unusual, using electrospray ionization mass spectrometry in combination with high-performance liquid chromatography and/or shotgun lipidomics.     

Photo-crosslinking analysis of preferential interactions between a transmembrane peptide and matching lipids

In this study, a novel method is presented by which the molecular environment of a transmembrane peptide can be investigated directly. This was achieved by incorporating a photoactivatable crosslinking probe in the hydrophobic segment of a model transmembrane peptide. When this peptide was incorporated into lipid bilayers and irradiated with UV light, a covalent bond was formed between the crosslinking probe and a lipid. This crosslinking reaction could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the resulting product could be characterized by mass spectrometry. By use of phospholipases, it was demonstrated that the peptide crosslinks to both acyl chains of the lipids. The peptide showed a clear preference to partition into fluid lipids and was excluded from lipids in the gel phase. However, when the peptide was incorporated into bilayers containing two lipid species with different acyl chain lengths, molecular sorting of the lipids around the peptide based on hydrophobic matching was not observed. It is proposed that the size of the transmembrane part plays an important role in the dynamic interactions of membrane proteins with the surrounding lipids and hence in determining whether molecular sorting can occur.     

New applications of mass spectrometry in lipid analysis

Mass spectrometry has emerged as a powerful tool for the analysis of all lipids. Lipidomic analysis of biological systems using various approaches is now possible with a quantitative measurement of hundreds of lipid molecular species. Although availability of reference and internal standards lags behind the field, approaches using stable isotope-labeled derivative tagging permit precise determination of specific phospholipids in an experimental series. The use of reactivity of ozone has enabled assessment of double bond positions in fatty acyl groups even when species remain in complex lipid mixtures. Rapid scanning tandem mass spectrometers are capable of quantitative analysis of hundreds of targeted lipids at high sensitivity in a single on-line chromatographic separation. Imaging mass spectrometry of lipids in tissues has opened new insights into the distribution of lipid molecular species with promising application to study pathophysiological events and diseases.     

Lipidomics: practical aspects and applications

Lipidomics is the characterization of the molecular species of lipids in biological samples. The polar lipids that comprise the bilayer matrix of the constituent cell membranes of living tissues are highly complex and number many hundreds of distinct lipid species. These differ in the nature of the polar group representing the different classes of lipid. Each class consists of a range of molecular species depending on the length, position of attachment and number of unsaturated double bonds in the associated fatty acids. The origin of this complexity is described and the biochemical processes responsible for homeostasis of the lipid composition of each morphologically-distinct membrane is considered. The practical steps that have been developed for the isolation of membranes and the lipids there from, their storage, separation, detection and identification by liquid chromatography coupled to mass spectrometry are described. Application of lipidomic analyses and examples where clinical screening for lipidoses in collaboration with mass spectrometry facilities are considered from the user point of view.     

Glycerolipid and cholesterol ester analyses in biological samples by mass spectrometry

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. .     

Binding of verocytotoxin 1 to its receptor is influenced by differences in receptor fatty acid content.

Globotriaosylceramide [(Gal alpha 1-4Gal beta 1-4Glc-ceramide (Gb3)] was separated from human kidney, and the fatty acid composition was determined. Semisynthetic Gb3 molecular species of corresponding fatty acid chain length were prepared and compared for verotoxin (VT) binding affinity by TLC overlay, and a quantitative binding assay was performed in the presence of auxiliary lipids. Our results indicate that, within the natural range, fatty acid chain length has little effect on verotoxin binding but that Gb3 molecular species containing different fatty acids can interact to provide a higher affinity toxin receptor than any of the individual component receptor species. Receptor function as assayed by TLC overlay was not always found to correlate with binding in a lipid environment. Short-chain fatty acid Gb3 molecular species could not function as VT receptors under these conditions. Evidence is presented to suggest that fatty acid chain length can have a stereoselective effect on carbohydrate conformation.     

Acidic glycerol lipids of Trichomonas vaginalis and Tritrichomonas foetus

The isolation and characterization of acidic lipids from both Trichomonas vaginalis and Tritrichomonas foetus have been carried out using radiolabeling, a combination of high performance liquid and thin layer chromatographic techniques, and mass spectrometry. Unique among the eukaryotes, these organisms produce phosphatidylglycerols and O-acyl phosphatidylglycerol-like compounds. In this study, the molecular weight distributions of the phosphatidylglycerols and acyl phosphatidylglycerols were determined by negative-ion liquid secondary ionization mass spectrometry (LSIMS) and the fatty acyl groups within each molecular species were assessed by collision-induced decomposition tandem mass spectrometry (CID MS/MS). Both species were found to contain primarily oleic acid in the sn-2 position. The lipids of T. vaginalis had approximately equal amounts of C16 and C18 in the sn-1 position, with varying degrees of unsaturation, especially in the C18 species. The T. foetus lipids had C18 almost exclusively, but also varied in the unsaturation. Other acidic lipids included inositol phosphosphingolipids and inositol diphosphosphingolipids.     

Determining seabird body condition using nonlethal measures

Energy stores are critical for successful breeding, and longitudinal studies require nonlethal methods to measure energy stores ("body condition"). Nonlethal techniques for measuring energy reserves are seldom verified independently. We compare body mass, size-corrected mass (SCM), plasma lipids, and isotopic dilution with extracted total body lipid content in three seabird species (thick-billed murres Uria lomvia, all four measures; northern fulmars Fulmarus glacialis, three measures; and black-legged kittiwakes Rissa tridactyla, two measures). SCM and body mass were better predictors of total body lipids for the species with high percent lipids (fulmars; R2 = 0.5-0.6) than for the species with low percent lipids (murres and kittiwakes; R2 = 0.2-0.4). The relationship between SCM and percent body lipids, which we argue is often a better measure of condition, was also poor (R2 < 0.2) for species with low lipids. In a literature comparison of 17 bird species, percent lipids was the only predictor of the strength of the relationship between mass and total body lipids; we suggest that SCM be used as an index of energy stores only when lipids exceed 15% of body mass. Across all three species we measured, SCM based on the ordinary least squares regression of mass on the first principal component outperformed other measures. Isotopic dilution was a better predictor of both total body lipids and percent body lipids than were mass, SCM, or plasma lipids in murres. Total body lipids decreased through the breeding season at both sites, while total and neutral plasma lipid concentrations increased at one site but not another, suggesting mobilization of lipid stores for breeding. A literature review showed substantial variation in the reliability of plasma markers, and we recommend isotopic dilution (oxygen-18, plateau) for determination of energy reserves in birds where lipid content is below 15%.     

A novel lipoprotein from Oomycete fungi

This work reports on the presence of an endogenous lipoprotein inLagenidium giganteum andPhytophthora cinnamomi as well as from the related non-sterol-deficient Oomycete speciesLagenidium callinectes, andZoophagus insidians. The lipoproteins comprise 0.2-1.0% of the total cell protein and are easily extracted with 0.2% Tween-80. The lipoprotein is homogeneous following heparin-agarose chromatography and nondenaturing gel electrophoresis. This holoprotein from all species had a molecular mass of 320,000 Da and contained four identical protein subunits of molecular mass 40,600. The lipoprotein exhibited similar physical characteristics to human low-density lipoprotein (LDL) as it comigrated with LDL on agarose gels, coeluted with LDL from heparin-agarose columns, and had a similar buoyant density of 1.006-1.063 g/cm³. The associated lipids varied quantitatively by species and included dolichol, diglycerides, and phospholipids. The lipoproteins from sterol-deficient species bound exogenous cholesterol without displacement of any of the native lipids of the lipoprotein.     

Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation

The molecular composition of phosphatidylcholines (PCs) in total lipid extracts was characterized by a combination of multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer and MS3 fragmentation on an ion trap mass spectrometer. Precursor ion spectra for 50 acyl anion fragments of fatty acids (fatty acid scanning) acquired in parallel increased the specificity and the dynamic range of the detection of PCs and identified the fatty acid moieties in individual PC species. Subsequent analysis of detected PC peaks by MS3 fragmentation on an ion trap mass spectrometer quantified the relative amount of their positional isomers, thus providing the most detailed and comprehensive characterization of the molecular composition of the pool of PCs at the low-picomole level. The method is vastly simplified, compared with conventional approaches, and does not require preliminary separation of lipid classes or of individual molecular species, enzymatic digestion, or chemical derivatization. The approach was validated by the comparative analysis of the molecular composition of PCs from human red blood cells. In the total lipid extract of Madin-Darby canine kidney II cells, we detected 46 PC species with unique fatty acid composition and demonstrated that the presence of positional isomers almost doubled the total number of individual molecular species.     

Quantitation of individual molecular species of phosphatidylcholines by reversed-phase high-performance liquid chromatography with fluorometric detection

The multiplicity of phosphatidylcholines is caused by the presence of different pairs of fatty acids in their individual molecular species and at least 27 miscellaneous fatty acids were identified in phosphatidylcholines in the serum of healthy individuals by combined gas–liquid chromatography and mass spectrometry in our present experiments. A method is described for the separation and quantitation of molecular species of phosphatidylcholine in human serum. Total phosphatidylcholine is isolated from lipids extracted from the serum with chloroform–methanol (2:1) by reversed-phase liquid–liquid extraction and subjected to reversed-phase high-performance liquid chromatography with a discontinuous descending gradient of water. Separation is monitored by fluorometry (340/460 nm) and absorption at 205 nm, if required. Up to 25 different molecular species of phosphatidylcholine may be quantified with a satisfactory reproducibility (±5–8%). Data on the distribution of individual molecular species in phosphatidylcholine of 53 normal serums are presented. The method may be used for quantitation of these phospholipids also in other biological materials (cell lines, leukemic cells from patients), and on a micropreparative scale to isolate individual compounds. The speed of separation as well as a satisfactory reproducibility are its principal advantages.     

Determination of molecular species of enantiomeric diacylglycerols by chiral phase high performance liquid chromatography and polar capillary gas-liquid chromatography

A simple method is described for the determination of molecular species of enantiomeric sn-1,2- and sn-2,3-diacylglycerols derived from natural triacylglycerols by Grignard degradation. The method is based on a preparative separation of the enantiomeric diacylglycerols as 3,5-dinitrophenylurethane (DNPU) derivatives by high performance liquid chromatography (HPLC) on a chiral column (25 cm x 4.6 mm ID) containing R-(+)-1-(1-naphthyl)ethylamine as a stationary phase. This is followed by polar capillary gas-liquid chromatography (GLC) of the trimethylsilyl (TMS) ether derivatives of the enantiomeric diacylglycerols derived from the DNPU derivatives using trichlorosilane, which does not cause acyl migration and racemization during the reaction. The cleavage is better than 94% complete. The method was standardized with synthetic sn-1,2- and sn-2,3-dipalmitoyl- and rac-1,2-dioleoylglycerols and was applied to the identification and quantitation of individual molecular species of enantiomeric diacylglycerols generated by Grignard degradation of the triacylglycerols from corn oil, cocoa butter, and lard.     

Molecular speciation and dynamics of oxidized triacylglycerols in lipid droplets: Mass spectrometry and coarse-grained simulations

Lipid droplets (LDs) are ubiquitous and physiologically active organelles regulating storage and mobilization of lipids in response to metabolic demands. Among the constituent LD neutral lipids, such as triacylglycerols, cholesterol esters, and free fatty acids, oxidizable polyunsaturated molecular species may be quite abundant, yet the structural and functional roles of their oxidation products have not been studied. Our previous work documented the presence of these peroxidized species in LDs. Assuming that hydrophilic oxygen-containing functionalities may markedly change the hydrophobic/hydrophilic molecular balance, here we utilized computational modeling to test the hypothesis that lipid peroxidation causes redistribution of lipids between the highly hydrophobic core and the polar surface (phospho)lipid monolayer—the area enriched with integrated enzymatic machinery. Using quantitative liquid chromatography/mass spectrometry, we characterized molecular speciation of oxTAGs in LDs of dendritic cells in cancer and hypoxic trophoblasts cells as two cellular models associated with dyslipidemia. Among the many types of oxidized lipids identified, we found that oxidatively truncated forms and hydroxyl derivatives of TAGs were the prevailing oxidized lipid species in LDs in both cell types. Using coarse-grained molecular dynamics (CG-MD) simulations we established that lipid oxidation changed their partitioning whereby oxidized lipids migrated into the outer monolayer of the LD, where they can affect essential metabolic pathways and undergo conversions, possibly leading to the formation of oxygenated lipid mediators.     

The uses and limitations of the analysis of cellular phosphoinositides by lipidomic and imaging methodologies

The advent of mass spectrometric methods has facilitated the determination of multiple molecular species of cellular lipid classes including the polyphosphoinositides, though to date methods to analyse and quantify each of the individual three PtdInsP and three PtdInsP₂ species are lacking. The use of imaging methods has allowed intracellular localization of the phosphoinositide classes but this methodology does not determine the acyl structures. The range of molecular species suggests a greater complexity in polyphosphoinositide signaling than yet defined but elucidating this will require further method development to be achieved. This article is part of a Special Issue entitled Tools to study lipid functions.     

alpha-methylene ordering of acyl chains differs in glucolipids and phosphatidylglycerol from Acholeplasma laidlawii membranes: (2)H-NMR quadrupole splittings from individual lipids in mixed bilayers

A Acholeplasma laidlawii strain A-EF22 was grown in a medium supplemented with alpha-deuterated oleic acid. Phosphatidylglycerol (PG), the glucolipids monoglucosyldiacylglycerol (MGlcDAG), diglucosyldiacylglycerol (DGlcDAG) and monoacyldiglucosyldiacylglycerol, and the phosphoglucolipid glycerophosphoryldiglucosyldiacylglycerol (GPDGlcDAG) were purified, and the phase behaviour and molecular ordering for the individual lipids, as well as for mixtures of the lipids, were studied by (2)H-, (31)P-NMR and X-ray scattering methods. The chemical structure of all the A. laidlawii lipids, except PG, has been determined and verified previously; here also the chemical structure of PG was verified, utilising mass spectrometry and (1)H and (13)C high resolution NMR spectroscopy. For the first time, lipid dimers were found in the mass spectrometry measurements. The major findings in this work are: (1) addition of 50 mol% of PG to the non-lamellar-forming lipid MGlcDAG does not significantly alter the transition temperature between lamellar and non-lamellar phases; (2) the (2)H-NMR quadrupole splitting patterns obtained from the lamellar liquid crystalline phase are markedly different for PG on one hand, and DGlcDAG and GPDGlcDAG on the other hand; and (3) mixtures of PG and DGlcDAG or MGlcDAG give rise to (2)H-NMR spectra consisting of a superposition of splitting patterns of the individual lipids. These remarkable features show that the local ordering of the alpha-carbon of the acyl chains is different for PG than for MGlcDAG and DGlcDAG, and that this difference is preserved when PG is mixed with the glucolipids. The results obtained are interpreted in terms of differences in molecular shape and hydrophilicity of the different polar headgroups.     

Detection of the abundance of diacylglycerol and triacylglycerol molecular species in cells using neutral loss mass spectrometry

Triacylglycerols (TAGs) are neutral lipids present in all mammalian cells as energy reserves, and diacylglycerols (DAGs) are present as intermediates in phospholipid biosynthesis and as signaling molecules. The molecular species of TAGs and DAGs present in mammalian cells are quite complex, and previous investigations revealed multiple isobaric species having molecular weights at virtually every even mass between 600 and 900 Da, making it difficult to assess changes of individual molecular species after cell activation. A method has been developed, using tandem MS and neutral loss scanning, to quantitatively analyze changes in those glyceryl ester molecular species containing identical fatty acyl groups. This was carried out by neutral loss scanning of 18 common fatty acyl groups where the neutral loss corresponded to the free carboxylic acid plus NH(3). Deuterium-labeled internal standards were used to normalize the signal for each nominal [M+NH(4)](+) ion undergoing this neutral loss reaction. This method was applied in studies of TAGs in RAW 264.7 cells treated with the toll-like receptor 4 ligand Kdo(2)-lipid A. A 50:1-TAG containing 18:1 was found to increase significantly over a 24-h time course after Kdo(2)-lipid A exposure, whereas an isobaric 50:1-TAG containing 16:1 did not change relative to controls.     

Application of the accurate mass and time tag approach in studies of the human blood lipidome

We report a preliminary demonstration of the accurate mass and time (AMT) tag approach for lipidomics. Initial data-dependent LC-MS/MS analyses of human plasma, erythrocyte, and lymphocyte lipids were performed in order to identify lipid molecular species in conjunction with complementary accurate mass and isotopic distribution information. Identified lipids were used to populate initial lipid AMT tag databases containing 250 and 45 entries for those species detected in positive and negative electrospray ionization (ESI) modes, respectively. The positive ESI database was then utilized to identify human plasma, erythrocyte, and lymphocyte lipids in high-throughput LC-MS analyses based on the AMT tag approach. We were able to define the lipid profiles of human plasma, erythrocytes, and lymphocytes based on qualitative and quantitative differences in lipid abundance.     

Characterisation of sphingolipids in the human lens by thin layer chromatography–desorption electrospray ionisation mass spectrometry

The lipidome of the human lens is unique in that cholesterol and dihydrosphingomyelin are the dominant classes. Moreover, the lens lipidome is not static with dramatic changes in several sphingolipid classes associated with both aging and cataract. Accordingly, there is a clear need to expand knowledge of the molecular species that constitute the human lens sphingolipidome. In this study, human lens lipids have been extracted and separated by thin-layer chromatography (TLC). Direct analysis of the TLC plates by desorption electrospray ionisation–mass spectrometry (DESI–MS) allowed the detection over 30 species from 11 classes of sphingolipids. Significantly, novel classes of lens lipids including sulfatides, dihydrosulfatides, lactosylceramide sulfates and dihydrolactosylceramide sulfates were identified.