Shotgun lipidomics: multidimensional MS analysis of cellular lipidomes

Shotgun lipidomics, comprised of intrasource separation, multidimensional mass spectrometry and computer-assisted array analysis, is an emerging powerful technique in lipidomics. Through effective intrasource separation of predetermined groups of lipid classes based on their intrinsic electrical propensities, analyses of lipids from crude extracts of biologic samples can be directly and routinely performed. Appropriate multidimensional array analysis of lipid pseudomolecular ions and fragments can be performed leading to the identification and quantitation of targeted lipid molecular species. Since most biologic lipids are linear combinations of aliphatic chains, backbones and head groups, a rich repertoire of multiple lipid building blocks present in discrete combinations represent experimental observables that can be computer reconstructed in conjunction with their pseudomolecular ions to directly determine the lipid molecular structures from a lipid extract. Through this approach, dramatic increases in the accessible dynamic range for ratiometric quantitation and discrimination of isobaric molecular species can be achieved without any prior column chromatography or operator-dependent supervision. At its current state of development, shotgun lipidomics can analyze over 20 lipid classes, hundreds of lipid molecular species and more than 95% of the mass content of a cellular lipidome. Thus, understanding the biochemical mechanisms underlying lipid-mediated disease states will be greatly facilitated by the power of shotgun lipidomics.     

Shotgun lipidomics: multidimensional MS analysis of cellular lipidomes

Shotgun lipidomics, comprised of intrasource separation, multidimensional mass spectrometry and computer-assisted array analysis, is an emerging powerful technique in lipidomics. Through effective intrasource separation of predetermined groups of lipid classes based on their intrinsic electrical propensities, analyses of lipids from crude extracts of biologic samples can be directly and routinely performed. Appropriate multidimensional array analysis of lipid pseudomolecular ions and fragments can be performed leading to the identification and quantitation of targeted lipid molecular species. Since most biologic lipids are linear combinations of aliphatic chains, backbones and head groups, a rich repertoire of multiple lipid building blocks present in discrete combinations represent experimental observables that can be computer reconstructed in conjunction with their pseudomolecular ions to directly determine the lipid molecular structures from a lipid extract. Through this approach, dramatic increases in the accessible dynamic range for ratiometric quantitation and discrimination of isobaric molecular species can be achieved without any prior column chromatography or operator-dependent supervision. At its current state of development, shotgun lipidomics can analyze over 20 lipid classes, hundreds of lipid molecular species and more than 95% of the mass content of a cellular lipidome. Thus, understanding the biochemical mechanisms underlying lipid-mediated disease states will be greatly facilitated by the power of shotgun lipidomics.     

Characterization and direct quantitation of cerebroside molecular species from lipid extracts by shotgun lipidomics

By using shotgun lipidomics based on the separation of lipid classes in the electrospray ion source (intrasource separation) and two-dimensional (2D) MS techniques (Han, X., and R. W. Gross. 2004. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of the cellular lipidomes directly from crude extracts of biological samples. Mass Spectrom. Rev. First published on June 18, 2004; doi: 10.1002/mas.20023, In press), individual molecular species of most major and many minor lipid classes can be quantitated directly from biological lipid extracts. Herein, we extended shotgun lipidomics to the characterization and quantitation of cerebroside molecular species in biological samples. By exploiting the differential fragmentation patterns of chlorine adducts using electrospray ionization (ESI) tandem mass spectrometry, hydroxy and nonhydroxy cerebroside species are readily identified. The hexose (either galactose or glucose) moiety of a cerebroside species can be distinguished by examination of the peak intensity ratio of its product ions at m/z 179 and 89 (i.e., 0.74 +/- 0.10 and 4.8 +/- 0.7 for galactose- and glucose-containing cerebroside species, respectively). Quantitation of cerebroside molecular species (as little as 10 fmol) from chloroform extracts of brain tissue samples was directly conducted by 2D ESI/MS after correction for differences in (13)C-isotopomer intensities. This method was demonstrated to have a greater than 1,000-fold linear dynamic range in the low concentration region; therefore, it should have a wide range of applications in studies of the cellular sphingolipid lipidome.     

Shotgun lipidomics of cardiolipin molecular species in lipid extracts of biological samples

Cardiolipin is a prominent component of the mitochondrial inner membranes contributing to the regulation of multiple discrete mitochondrial functions. Here, we extend shotgun lipidomics to identify and quantitate cardiolipin molecular species directly from lipid extracts of biological samples. Three shotgun lipidomics approaches for analyses of cardiolipin molecular species were developed using either a continuous ion-transmission instrument (i.e., triple-quadrupole type) with either low or high mass resolution settings or a high mass resolution hybrid pulsed instrument [i.e., quadrupole time-of-flight (QqTOF) type]. Three chemical principles were used for the development of these approaches. These include the marked enrichment of linoleate in cardiolipin to maximize the signal-to-noise ratio, the specific neutral loss of ketenes from doubly charged cardiolipin molecular ions to yield doubly charged triacyl monolysocardiolipins, and the doubly charged character of two phosphates in each cardiolipin molecular species. Through these techniques, we identified and quantified the specific molecular species profiles of cardiolipin directly from lipid extracts of mouse heart, liver, and skeletal muscle. The accuracy ( approximately 5%) and the low end of the linear dynamic range (10 fmol/microl) for quantitation make these approaches useful for studying alterations in cardiolipin metabolism in multiple disease states using either type of mass spectrometer.     

Alkaline methanolysis of lipid extracts extends shotgun lipidomics analyses to the low-abundance regime of cellular sphingolipids

Sphingolipids that contain a sphingoid base are composed of hundreds to thousands of distinct compounds, many of which serve as lipid regulators of biological functions. The global analysis of the large number of low-abundance sphingolipid molecular species has been hampered in many cases by the sphingolipid molecular species being overwhelmed by the quantity of other classes of lipid (e.g., glycerophospholipid) molecular species present, thereby imposing severe restrictions on the dynamic range of their measurement using shotgun lipidomics. Herein, we developed a facile approach in which the sphingolipids of cellular extracts were dramatically enriched by direct alkaline methanolysis of lipid extracts followed by extraction to remove the large majority of other endogenous lipid classes. Through direct infusion of the resultant enriched solution, we identified and quantitated a variety of very-low-abundance sphingolipid classes (e.g., sphingosine, psychosine, and lysosphingomyelin) and molecular species (e.g., sphingomyelin) using electrospray ionization mass spectrometry (i.e., shotgun sphingolipidomics). Accordingly, through utilization of these facile enrichment techniques, direct penetrance into the sphingolipidomes has been greatly extended, facilitating new insights into their metabolism and signaling functions in biological systems.     

Quantitative analysis and molecular species fingerprinting of triacylglyceride molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry

Herein we describe a rapid, simple, and reliable method for the quantitative analysis and molecular species fingerprinting of triacylglycerides (TAG) directly from chloroform extracts of biological samples. Previous attempts at direct TAG quantitation by positive-ion electrospray ionization mass spectrometry (ESI/MS) were confounded by the presence of overlapping peaks from choline glycerophospholipids requiring chromatographic separation of lipid extracts prior to ESI/MS analyses. By exploiting the rapid loss of phosphocholine from choline glycerophospholipids, in conjunction with neutral-loss scanning for individual fatty acids, overlapping peaks in the ESI mass spectrum were deconvoluted generating a detailed molecular species fingerprint of individual TAG molecular species directly from chloroform extracts of biological samples. This method readily detects as little as 0.1 pmol of each TAG molecular species from chloroform extracts and is linear over a 1000-fold dynamic range. The sensitivity of individual TAG molecular species to ESI/MS/MS analyses correlated with the unsaturation index and inversely correlated with total aliphatic chain length of TAG. An algorithm was developed which identifies sensitivity factors, thereby allowing the rapid quantitation and molecular species fingerprinting of TAG molecular species directly from chloroform extracts of biological samples.     

Non-enzymatically derived minor lipids found in Escherichia coli lipid extracts

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.     

Quantitative profiling of PE, MMPE, DMPE, and PC lipid species by multiple precursor ion scanning: a tool for monitoring PE metabolism

We report a method for the simultaneous identification and quantification of phosphatidylethanolamine (PE), monomethyl-phosphatidylethanolamine (MMPE), dimethyl-phosphatidylethanolamine (DMPE), and phosphatidylcholine (PC) species in lipid extracts. The method employs a specific "mass-tag" strategy where DMPE, MMPE, and PE species are chemically methylated with deuterated methyliodide (CD(3)I) to produce PC molecules having class-specific mass offsets of 3, 6 and 9Da, respectively. The derivatized aminoglycerophospholipids release characteristic phosphorylcholine-like fragment ions having specific mass offsets that powers sensitive and quantitative analysis by multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer. Using the mass-tag strategy, we could for the first time determine the stoichiometric relationship between the biosynthetic intermediates MMPE and DMPE, and abundant PE and PC species in a single mass spectrometric analysis. We demonstrated the efficacy of the methodology by conducting a series of biochemical experiments using stable isotope labeled ethanolamine to survey the activities and substrate specificities of enzymes involved in PE metabolism in Saccharomyces cerevisiae. Finally, we benchmarked the mass-tag strategy by specific and sensitive profiling of intermediate MMPE and DMPE species in liver.     

Identification of oxidized phospholipids in bronchoalveolar lavage exposed to low ozone levels using multivariate analysis

Chemical reactions with unsaturated phospholipids in the respiratory tract lining fluid have been identified as one of the first important steps in the mechanisms mediating environmental ozone toxicity. As a consequence of these reactions, complex mixtures of oxidized lipids are generated in the presence of mixtures of non-oxidized naturally occurring phospholipid molecular species, which challenge methods of analysis. Untargeted mass spectrometry and statistical methods were employed to approach these complex spectra. Human bronchoalveolar lavage (BAL) was exposed to low levels of ozone, and samples with and without derivatization of aldehydes were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry. Data processing was carried out using principal component analysis (PCA). Resulting PCA scores plots indicated an ozone dose-dependent increase, with apparent separation between BAL samples exposed to 60 ppb ozone and non-exposed BAL samples as well as a clear separation between ozonized samples before and after derivatization. Corresponding loadings plots revealed that more than 30 phosphatidylcholine (PC) species decreased due to ozonation. A total of 13 PC and 6 phosphatidylglycerol oxidation products were identified, with the majority being structurally characterized as chain-shortened aldehyde products. This method exemplifies an approach for comprehensive detection of low-abundance, yet important, components in complex lipid samples.     

Strategies to Improve/Eliminate the Limitations in Shotgun Lipidomics

Direct infusion‐based shotgun lipidomics is one of the most powerful and useful tools in comprehensive analysis of lipid species from lipid extracts of various biological samples with high accuracy/precision. However, despite many advantages, the classical shotgun lipidomics suffers some general dogmas of limitations, such as ion suppression, ambiguous identification of isobaric/isomeric lipid species, and ion source–generated artifacts, restraining the applications in analysis of low‐abundance lipid species, particularly those less ionizable or isomers that yield almost identical fragmentation patterns. This article reviews the strategies (such as modifier addition, prefractionation, chemical derivatization, charge feature utilization) that have been employed to improve/eliminate these limitations in modern shotgun lipidomics approaches (e.g., high mass resolution mass spectrometry–based and multidimensional mass spectrometry–based shotgun lipidomics). Therefore, with the enhancement of these strategies for shotgun lipidomics, comprehensive analysis of lipid species including isomeric/isobaric species is achieved in a more accurate and effective manner, greatly substantiating the aberrant lipid metabolism, signaling trafficking, and homeostasis under pathological conditions.     

A comprehensive method for lipid profiling by liquid chromatography-ion cyclotron resonance mass spectrometry

This work aims to combine chromatographic retention, high mass resolution and accuracy, MS/MS spectra, and a package for automated identification and quantitation of lipid species in one platform for lipidomic analysis. The instrumental setup elaborated comprises reversed-phase HPLC coupled to a Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FT), and Lipid Data Analyzer (LDA) software. Data analysis for lipid species quantification in this platform is based on retention time, mass resolution of 200,000, and mass accuracy below 2 ppm. In addition, automatically generated MS/MS spectra provide structural information at molecular level. This LC/MS technology allows analyzing complex biological samples in a quantitative manner as shown here paradigmatically for murine lipid droplets having a huge surplus of triacylglycerol species. Chromatographic preseparation of the bulk lipid class alleviates the problem of ion suppression of lipid species from other classes. Extension of 1D to 2D chromatography is possible, yet time consuming. The platform affords unambiguous detection of lipid species as low as 0.1‰ within major lipid classes. Taken together, a novel lipidomic LC/MS platform based on chromatographic retention, high mass resolution and accuracy, MS/MS analysis, and quantitation software enables analysis of complex samples as demonstrated for lipid droplets.     

A targeted mass spectrometric analysis of phosphatidylinositol phosphate species

The development of a new mass spectrometric lipid profiling methodology permits the identification of cellular phosphatidylinositol monophosphate/phosphatidylinositol bisphosphate/phosphatidylinositol trisphosphate (PIP/PIP2/PIP3) species that includes the fatty acyl composition. Using electrospray ionization mass spectrometry, we were able to resolve and identify 28 PIP and PIP2 compounds as well as 8 PIP3 compounds from RAW 264.7 or primary murine macrophage cell extracts. Analysis of PIP profiles after agonist stimulation of cells revealed the generation of differential PIP3 species and permitted us to propose a novel means for regulation and specificity in signaling through PIP3. This is the first reported identification of intact, cellular PIP3 by mass spectral analysis. The ability to analyze the fatty acyl chain composition of signaling lipids initiates new venues for investigation of the processes by which specific polyphosphoinositide species mediate.     

Characterization and direct quantitation of ceramide molecular species from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry

A rapid, simple, and reliable method has been developed for the characterization and quantitation of ceramide molecular species directly from chloroform extracts of biological samples by electrospray ionization tandem mass spectrometry (ESI/MS/MS). By exploiting the differential fragmentation patterns of deprotonated ceramide ions, individual 2-hydroxy and nonhydroxy ceramide molecular species were readily identified by ESI/MS/MS with the neutral loss of fragments of mass 256.2 and 327.3 which correspond to sphingosine derivatives. The ions generated from the neutral loss of 256.2 (i.e., [M - H - 256.2](-)) are unique for ceramides with N-acyl sphingosine with the 18-carbon homolog. However, the sensitivity for nonhydroxy ceramides in ESI/MS/MS with the neutral loss of 256.2 is approximately threefold higher than that for 2-hydroxy ceramides. The ions resulting from the neutral loss of 327.3 (i.e., [M - H - 327.3](-)) are specific for 2-hydroxy ceramides. Additionally, all ceramides including both 2-hydroxy and nonhydroxy forms can be confirmed and accurately quantitated by ESI/MS/MS with the neutral loss of 240.2 after correction for (13)C isotope factors. This methodology demonstrated a 1000-fold linear dynamic range and a detection limit at the subfemtomole range and was applied to directly quantitate ceramide molecular species in chloroform extracts of biological samples including brain tissues and cell cultures.     

A method for profiling gangliosides in animal tissues using electrospray ionization-tandem mass spectrometry

Gangliosides are critical in many functions of mammalian cells but present as a minor lipid component with many molecular species of subtle differences. Conventional strategies for profiling gangliosides suffer from poor reproducibility, low sensitivity, and low-throughput capacity. Prior separation of gangliosides by thin-layer chromatography and/or high-performance liquid chromatography not only was laborious and tedious but also could introduce uneven losses of molecular species. We developed a new strategy of using electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to profile gangliosides with high-throughput potential. This strategy involves three new findings: (i) collision-induced fragmentation of gangliosides gave rise to a common ion of m/z 290, a derivative of N-acetylneuraminic acid; (ii) phospholipids exert a profound suppression of ganglioside detection in ESI-MS/MS to prevent a direct detection in total cellular lipid extracts; and (iii) enrichment of gangliosides in the aqueous phase from total cellular lipid extracts eliminates the damping effect of phospholipids and permits direct precursor scan.     

Direct ESI-MS analysis of O-acyl glycosylated cardiolipins from the thermophilic bacterium Alicyclobacillus acidoterrestris

We used direct ESI-MS analysis to identify derivatives of cardiolipin molecular species (i.e. O-acyl glycosylated cardiolipins) from the thermophilic bacterium Alicyclobacillus acidoterrestris. We used triple-quadrupole type mass spectrometer for analysis of this complex lipid and enzymatic hydrolysis and ¹H and ¹³C NMR for the identification of these cardiolipin derivatives. These techniques enabled us to identify and quantify the specific molecular species profiles of derivatives of cardiolipin directly from lipid extracts of the bacterium including the identification of the sugar moiety as α-d-mannose and all five acyls including their positional isomers.     

LIPID arrays: new tools in the understanding of membrane dynamics and lipid signaling

Phospholipids are the structural building blocks of the membrane bilayer, which retains and regulates intra-cellular content. In addition to creating a protective barrier around the cell, lipids modulate membrane trafficking and are themselves precursors of important intracellular signaling molecules. Identification and quantification of these molecular species is essential for a more complete understanding of cell signaling pathways, and more reliable and sensitive methods are needed for determining membrane phospholipid content. Recent improvements in electrospray ionization mass spectrometry have made possible the direct identification of more than 400 phospholipid species from biological extracts of a single cell type. Changes in the cellular concentration of diverse lipids can be determined by analysis of the mass spectra by statistical algorithms. In the future, lipid arrays will be integrated with other high-throughput profiling technologies, and computational lipidomics will expand our understanding of the molecular basis of cellular processes and diseases.     

Lipidomics reveals that adiposomes store ether lipids and mediate phospholipid traffic

Lipid droplets are accumulations of neutral lipids surrounded by a monolayer of phospholipids and associated proteins. Recent proteomic analysis of isolated droplets suggests that they are part of a dynamic organelle system that is involved in membrane traffic as well as packaging and distributing lipids in the cell. To gain a better insight into the function of droplets, we used a combination of mass spectrometry and NMR spectroscopy to characterize the lipid composition of this compartment. In addition to cholesteryl esters and triacylglycerols with mixed fatty acid composition, we found that approximately 10-20% of the neutral lipids were the ether lipid monoalk(en)yl diacylglycerol. Although lipid droplets contain only 1-2% phospholipids by weight, >160 molecular species were identified and quantified. Phosphatidylcholine (PC) was the most abundant class, followed by phosphatidylethanolamine (PE), phosphatidylinositol, and ether-linked phosphatidylcholine (ePC). Relative to total membrane, droplet phospholipids were enriched in lysoPE, lysoPC, and PC but deficient in sphingomyelin, phosphatidylserine, and phosphatidic acid. These results suggest that droplets play a central role in ether lipid metabolism and intracellular lipid traffic.     

Spatial mapping of lipids at cellular resolution in embryos of cotton

Advances in mass spectrometry (MS) have made comprehensive lipidomics analysis of complex tissues relatively commonplace. These compositional analyses, although able to resolve hundreds of molecular species of lipids in single extracts, lose the original cellular context from which these lipids are derived. Recently, high-resolution MS of individual lipid droplets from seed tissues indicated organelle-to-organelle variation in lipid composition, suggesting that heterogeneity of lipid distributions at the cellular level may be prevalent. Here, we employed matrix-assisted laser desorption/ionization-MS imaging (MALDI-MSI) approaches to visualize lipid species directly in seed tissues of upland cotton (Gossypium hirsutum). MS imaging of cryosections of mature cotton embryos revealed a distinct, heterogeneous distribution of molecular species of triacylglycerols and phosphatidylcholines, the major storage and membrane lipid classes in cotton embryos. Other lipids were imaged, including phosphatidylethanolamines, phosphatidic acids, sterols, and gossypol, indicating the broad range of metabolites and applications for this chemical visualization approach. We conclude that comprehensive lipidomics images generated by MALDI-MSI report accurate, relative amounts of lipid species in plant tissues and reveal previously unseen differences in spatial distributions providing for a new level of understanding in cellular biochemistry.     

Comparative Lipid Profiling of the Cnidarian Aiptasia pallida and Its Dinoflagellate Symbiont

Corals and other cnidarians house photosynthetic dinoflagellate symbionts within membrane-bound compartments inside gastrodermal cells. Nutritional interchanges between the partners produce carbohydrates and lipids for metabolism, growth, energy stores, and cellular structures. Although lipids play a central role in the both the energetics and the structural/morphological features of the symbiosis, previous research has primarily focused on the fatty acid and neutral lipid composition of the host and symbiont. In this study we conducted a mass spectrometry-based survey of the lipidomic changes associated with symbiosis in the sea anemone Aiptasia pallida, an important model system for coral symbiosis. Lipid extracts from A. pallida in and out of symbiosis with its symbiont Symbiodinium were prepared and analyzed using negative-ion electrospray ionization quadrupole time-of-flight mass spectrometry. Through this analysis we have identified, by exact mass and collision-induced dissociation mass spectrometry (MS/MS), several classes of glycerophospholipids in A. pallida. Several molecular species of di-acyl phosphatidylinositol and phosphatidylserine as well as 1-alkyl, 2-acyl phosphatidylethanolamine (PE) and phosphatidycholine were identified. The 1-alkyl, 2-acyl PEs are acid sensitive suggestive that they are plasmalogen PEs possessing a double bond at the 1-position of the alkyl linked chain. In addition, we identified several molecular species of phosphonosphingolipids called ceramide aminoethylphosphonates in anemone lipid extracts by the release of a characteristic negative product ion at m/z 124.014 during MS/MS analysis. Sulfoquinovosyldiacylglycerol (SQDG), an anionic lipid often found in photosynthetic organisms, was identified as a prominent component of Symbiodinium lipid extracts. A comparison of anemone lipid profiles revealed a subset of lipids that show dramatic differences in abundance when anemones are in the symbiotic state as compared to the non-symbiotic state. The data generated in this analysis will serve as a resource to further investigate the role of lipids in symbiosis between Symbiodinium and A. pallida.