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 phosphoethanolamine-containing lipids in biological samples after one-step in situ derivatization

This article presents a novel methodology for the analysis of ethanolamine glycerophospholipid (PE) and lysoPE molecular species directly from lipid extracts of biological samples. Through brief treatment of lipid extracts with fluorenylmethoxylcarbonyl (Fmoc) chloride, PE and lysoPE species were selectively derivatized to their corresponding carbamates. The reaction solution was infused directly into the ion source of an electrospray ionization mass spectrometer after appropriate dilution. The facile loss of the Fmoc moiety dramatically enhanced the analytic sensitivity and allowed the identification and quantitation of low-abundance molecular species. A detection limitation of attomoles (amoles) per microliter for PE and lysoPE analysis was readily achieved using this technique (at least a 100-fold improvement from our previous method) with a >15,000-fold dynamic range. Through intrasource separation and multidimensional mass spectrometry array analysis of derivatized species, marked improvements in signal-to-noise ratio, molecular species identification, and quantitation can be realized. The procedure is both simple and effective and can be extended to analyze many other lipid classes or other cellular metabolites by adjustments in specific derivatization conditions. Thus, through judicious derivatization, a new dimension exploiting specific functional reactivities in each lipid class can be used in conjunction with shotgun lipidomics to penetrate farther into the low-abundance regime of cellular lipidomes.     

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.     

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.     

Characterization and direct quantitation of sphingoid base-1-phosphates from lipid extracts: a shotgun lipidomics approach

Here, we have extended shotgun lipidomics for the characterization and quantitation of sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (DHS1P) in crude lipid extracts in the presence of ammonium hydroxide by using precursor ion scanning of m/z 79.0 (corresponding to [PO(3)](-)) in the negative-ion mode. It is demonstrated that a broad linear dynamic range for the quantitation of both S1P and DHS1P and a detection limit at low amol/mul concentration are achieved using this approach. The developed method for the quantitation of sphingoid base-1-phosphates is generally simpler and more efficient than other previously published methods. Multiple factors influencing the quantitation of sphingoid base-1-phosphates, including ion suppression, extraction efficiency, and potential overlapping with other molecular species, were examined extensively and/or are discussed. Mass levels of S1P and DHS1P in multiple biological samples, including human plasma, mouse plasma, and mouse brain tissues (e.g., cortex, cerebellum, spinal cord, and brain stem), were determined by the developed methodology. Accordingly, this technique, as a new addition to shotgun lipidomics technology, will be extremely useful for understanding the pathways of sphingolipid metabolism and for exploring the important roles of sphingoid base-1-phosphates in a wide range of physiological and pathological studies.     

Yeast sphingolipids: recent developments in understanding biosynthesis, regulation, and function

Sphingolipids function as required membrane components of virtually all eukaryotic cells. Data indicate that members of the sphingolipid family of lipids, including sphingoid bases, sphingoid base phosphates, ceramides, and complex sphingolipids, serve vital functions in cell biology by both direct mechanisms (e.g., binding to G-protein coupled receptors to transduce an extracellular signal) and indirect mechanisms (e.g., facilitating correct intracellular protein transport). Because of the diverse roles these lipids play in cell biology, it is important to understand not only their biosynthetic pathways and regulation of sphingolipid synthesis, but also the mechanisms by which some sphingolipid species with specific functions are modified or converted to other sphingolipid species with alternate functions. Due to many factors including ease of culture and genetic modification, and conservation of major sphingolipid metabolic pathways, Saccharomyces cerevisiae has served as an ideal model system with which to identify enzymes of sphingolipid biosynthesis and to dissect sphingolipid function. Recent exciting developments in sphingolipid synthesis, transport, signaling, and overall biology continue to fuel vigorous investigation and inspire investigations in mammalian sphingolipid biology.     

Sphingolipids as modulators of membrane proteins

The diversity of the transmembranome of higher eukaryotes is matched by an enormous diversity of sphingolipid classes and molecular species. The intrinsic properties of sphingolipids are not only suited for orchestrating lateral architectures of biological membranes, but their molecular distinctions also allow for the evolution of protein motifs specifically recognising and interacting with individual lipids. Although various reports suggest a role of sphingolipids in membrane protein function, only a few cases have determined the specificity of these interactions. In this review we discuss examples of specific protein–sphingolipid interactions for which a modulator-like dependency on sphingolipids was assigned to specific proteins. These novel functions of sphingolipids in specific protein–lipid assemblies contribute to the complexity of the sphingolipid classes and other molecular species observed in animal cells. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Molecular characterization and targeted quantitative profiling of the sphingolipidome in rice

Recent advances in comprehensive metabolite profiling techniques, the foundation of metabolomics, is facilitating our understanding of the functions, regulation and complex networks of various metabolites in organisms. Here, we report a quantitative metabolomics technique for complex plant sphingolipids, composed of various polar head groups as well as structural isomers of hydrophobic ceramide moieties. Rice (Oryza sativa L.) was used as an experimental model of monocotyledonous plants and has been demonstrated to possess a highly complex sphingolipidome including hundreds of molecular species with a wide range of abundance. We established a high‐throughput scheme for lipid preparation and mass spectrometry‐based characterization of complex sphingolipid structures, which provided basic information to create a comprehensive theoretical library for targeted quantitative profiling of complex sphingolipids in rice. The established sphingolipidomic approach combined with multivariate analyses of the large dataset obtained clearly showed that different classes of rice sphingolipids, particularly including subclasses of glycosylinositol phosphoceramide with various sugar‐chain head groups, are distributed with distinct quantitative profiles in various rice tissues, indicating tissue‐dependent metabolism and biological functions of the lipid classes and subclasses. The sphingolipidomic analysis also highlighted that disruption of a lipid‐associated gene causes a typical sphingolipidomic change in a gene‐dependent manner. These results clearly support the utility of the sphingolipidomic approach in application to wide screening of sphingolipid‐metabolic phenotypes as well as deeper investigation of metabolism and biological functions of complex sphingolipid species in plants.     

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.     

Novel advances in shotgun lipidomics for biology and medicine

The field of lipidomics, as coined in 2003, has made profound advances and been rapidly expanded. The mass spectrometry-based strategies of this analytical methodology-oriented research discipline for lipid analysis are largely fallen into three categories: direct infusion-based shotgun lipidomics, liquid chromatography-mass spectrometry-based platforms, and matrix-assisted laser desorption/ionization mass spectrometry-based approaches (particularly in imagining lipid distribution in tissues or cells). This review focuses on shotgun lipidomics. After briefly introducing its fundamentals, the major materials of this article cover its recent advances. These include the novel methods of lipid extraction, novel shotgun lipidomics strategies for identification and quantification of previously hardly accessible lipid classes and molecular species including isomers, and novel tools for processing and interpretation of lipidomics data. Representative applications of advanced shotgun lipidomics for biological and biomedical research are also presented in this review. We believe that with these novel advances in shotgun lipidomics, this approach for lipid analysis should become more comprehensive and high throughput, thereby greatly accelerating the lipidomics field to substantiate the aberrant lipid metabolism, signaling, trafficking, and homeostasis under pathological conditions and their underpinning biochemical mechanisms.     

Direct quantitation of psychosine from alkaline-treated lipid extracts with a semi-synthetic internal standard

Psychosine is an important bioactive sphingolipid metabolite and plays an essential role in the pathogenesis of Krabbe's disease. Herein, we extended shotgun lipidomics for the characterization and quantitation of psychosine in alkaline-treated crude lipid extracts by using neutral loss scan of 180 amicro (i.e., galactose) in the positive-ion mode. Specifically, we semi-synthesized N,N-dimethyl psychosine and used it as an internal standard for quantitation of psychosine. After characterization of the fragmentation patterns of psychosine and the selected internal standard and optimization of the experimental conditions, we demonstrated that a broad linear dynamic range for the quantitation of psychosine and a limit of detection at a concentration of low fmol/microl were achieved using this approach. The developed method is generally simpler and more efficient than other previously reported methods. Multiple factors influencing quantitation of psychosine were extensively examined and/or discussed. The levels of psychosine in diabetic mouse nerve tissue samples were determined by the developed methodology. Collectively, the developed approach, as a new addition to the shotgun lipidomics technology, will be extremely useful for understanding the pathways/networks of sphingolipid metabolism and for exploring the important roles of psychosine in a variety of physiological and pathological conditions.     

Molecular lipidomics of exosomes released by PC-3 prostate cancer cells

The molecular lipid composition of exosomes is largely unknown. In this study, sophisticated shotgun and targeted molecular lipidomic assays were performed for in-depth analysis of the lipidomes of the metastatic prostate cancer cell line, PC-3, and their released exosomes. This study, based in the quantification of approximately 280 molecular lipid species, provides the most extensive lipid analysis of cells and exosomes to date. Interestingly, major differences were found in the lipid composition of exosomes compared to parent cells. Exosomes show a remarkable enrichment of distinct lipids, demonstrating an extraordinary discrimination of lipids sorted into these microvesicles. In particular, exosomes are highly enriched in glycosphingolipids, sphingomyelin, cholesterol, and phosphatidylserine (mol% of total lipids). Furthermore, lipid species, even of classes not enriched in exosomes, were selectively included in exosomes. Finally, it was found that there is an 8.4-fold enrichment of lipids per mg of protein in exosomes. The detailed lipid composition provided in this study may be useful to understand the mechanism of exosome formation, release and function. Several of the lipids enriched in exosomes could potentially be used as cancer biomarkers.     

A novel informatics concept for high-throughput shotgun lipidomics based on the molecular fragmentation query language

Shotgun lipidome profiling relies on direct mass spectrometric analysis of total lipid extracts from cells, tissues or organisms and is a powerful tool to elucidate the molecular composition of lipidomes. We present a novel informatics concept of the molecular fragmentation query language implemented within the LipidXplorer open source software kit that supports accurate quantification of individual species of any ionizable lipid class in shotgun spectra acquired on any mass spectrometry platform.     

Mass spectrometric analysis reveals changes in phospholipid, neutral sphingolipid and sulfatide molecular species in progressive epilepsy with mental retardation, EPMR, brain: a case study

Progressive epilepsy with mental retardation, EPMR, belongs to a group of inherited neurodegenerative disorders, the neuronal ceroid lipofuscinoses. The CLN8 gene that underlies EPMR encodes a novel transmembrane protein that localizes to the endoplasmic reticulum (ER) and ER-Golgi intermediate compartment. Recently, CLN8 was linked to a large eukaryotic protein family of TLC (TRAM, Lag1, CLN8) domain homologues with postulated functions in lipid synthesis, transport or sensing. By using liquid chromatography/mass spectrometry we analysed molecular species of major phosholipid and simple sphingolipid classes from cerebral samples of two EPMR patients representing a progressive and advanced state of the disease. The progressive state brain showed reduced levels of ceramide, galactosyl- and lactosylceramide and sulfatide as well as a decrease in long fatty acyl chain containing molecular species within these classes. Among glycerophospholipid classes, an increase in species containing polyunsaturated acyl chains was detected especially in phosphatidylserines and phosphatidylethanolamines. By contrast, saturated and monounsaturated species were overrepresented among phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol classes in the advanced state sample. The observed changes in brain sphingo- and phospholipid molecular profiles may result in altered membrane stability, lipid peroxidation, vesicular trafficking or neurotransmission and thus may contribute to the progression of the molecular pathogenesis of EPMR.     

Proteomic and bioinformatics profile of paired human alveolar macrophages and peripheral blood monocytes

Little is known about proteomic differences between pluripotent human peripheral blood monocytes (MN) and their terminally‐differentiated pulmonary counterparts, alveolar macrophages (AM). To better characterize these cell populations, we performed a label‐free shotgun proteomics assessment of matched AM and MN preparations from eight healthy volunteers. With an FDR of less than 0.45%, we identified 1754 proteins within AM and 1445 from MN. Comparison of the two proteomes revealed that 1239 of the proteins found in AM were shared with MN, whereas 206 proteins were uniquely identified in MN and 515 were unique to AM. Molecular and cellular functions, protein classes, development associations, and membership in physiological systems and canonical pathways were identified among the detected proteins. Analysis of biologic processes represented by these proteomes indicated that MN were most prominently enriched for proteins involved in cellular movement and immune cell trafficking. In contrast, AM were enriched for proteins involved in protein trafficking, molecular transport, and cellular assembly and organization. These findings provide a baseline proteomic resource for further studies aimed at better understanding of the functional differences between MN and AM in both health and disease.     

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.     

Lipidomics: an analysis of cellular lipids by ESI-MS

Recognition of the importance of lipid signaling in cellular function has led to rapid progress in the technology of lipid analysis. Measurements of lipid species changes are central to defining the networks of cell signaling (e.g., receptor activation by hormones or drugs) and lipids are involved in many biochemical and pathological processes. During the last several years our laboratory has focused on developing efficient methods for extraction of glycerophospholipids from biological systems and their detection and identification by mass spectrometry. We analyze phospholipid changes in mammalian cells as a result of a defined ligand stimulation strategy that supports the research questions of the consortium. The improvement of mass spectrometry techniques for phospholipid analysis combined with sophisticated computational methods developed in our group has facilitated simultaneous analysis of hundreds of phospholipid species in mammalian cells. This information is presented as Lipid Arrays (or more precisely as virtual arrays) and allows identification of temporal changes in membrane phospholipid species between two contrasting biological conditions (e.g., unstimulated basal vs. stimulated or as a contrast between normal and disease stages). Using the lipidomics approach, we are able to identify approximately 450 phospholipid species from total membrane extracts and qualitatively measure pattern response changes initiated by cell surface receptors. As such, this approach facilitates the elucidation of the metabolic changes induced by a perturbation in the cell and recognition of patterns of signaling.     

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.     

Shotgun lipidomics identifies a paired rule for the presence of isomeric ether phospholipid molecular species

Background

Ether phospholipids are abundant membrane constituents present in electrically active tissues (e.g., heart and the brain) that play important roles in cellular function. Alterations of ether phospholipid molecular species contents are associated with a number of genetic disorders and human diseases.

Methodology/Principal Findings

Herein, the power of shotgun lipidomics, in combination with high mass accuracy/high resolution mass spectrometry, was explored to identify a paired rule for the presence of isomeric ether phospholipid molecular species in cellular lipidomes. The rule predicts that if an ether phospholipid A′-B is present in a lipidome, its isomeric counterpart B′-A is also present (where the ′ represents an ether linkage). The biochemical basis of this rule results from the fact that the enzymes which participate in either the sequential oxidation of aliphatic alcohols to fatty acids, or the reduction of long chain fatty acids to aliphatic alcohols (metabolic precursors of ether lipid synthesis), are not entirely selective with respect to acyl chain length or degree of unsaturation. Moreover, the enzymatic selectivity for the incorporation of different aliphatic chains into the obligatory precursor of ether lipids (i.e., 1-O-alkyl-glycero-3-phosphate) is also limited.

Conclusions/Significance

This intrinsic amplification of the number of lipid molecular species present in biological membranes predicted by this rule and demonstrated in this study greatly expands the number of ether lipid molecular species present in cellular lipidomes. Application of this rule to mass spectrometric analyses provides predictive clues to the presence of specific molecular species and greatly expands the number of identifiable and quantifiable ether lipid species present in biological samples. Through appropriate alterations in the database, use of the paired rule increases the number of identifiable metabolites in metabolic networks, thereby facilitating identification of biomarkers presaging disease states.