Sphingolipidomics: Methods for the comprehensive analysis of sphingolipids

doi:10.1016/j.jchromb.2008.12.057

Journal of Chromatography B

Volume 877, Issue 26, 15 September 2009, Pages 2696-2708

https://doi.org/10.1016/j.jchromb.2008.12.057 Get rights and content

Abstract

Sphingolipids comprise a highly diverse and complex class of molecules that serve as both structural components of cellular membranes and signaling molecules capable of eliciting apoptosis, differentiation, chemotaxis, and other responses in mammalian cells. Comprehensive or “sphingolipidomic” analyses (structure specific, quantitative analyses of all sphingolipids, or at least all members of a critical subset) are required in order to elucidate the role(s) of sphingolipids in a given biological context because so many of the sphingolipids in a biological system are inter-converted structurally and metabolically. Despite the experimental challenges posed by the diversity of sphingolipid-regulated cellular responses, the detection and quantitation of multiple sphingolipids in a single sample has been made possible by combining classical analytical separation techniques such as high-performance liquid chromatography (HPLC) with state-of-the-art tandem mass spectrometry (MS/MS) techniques. As part of the Lipid MAPS consortium an internal standard cocktail was developed that comprises the signaling metabolites (i.e. sphingoid bases, sphingoid base-1-phosphates, ceramides, and ceramide-1-phosphates) as well as more complex species such as mono- and di-hexosylceramides and sphingomyelin. Additionally, the number of species that can be analyzed is growing rapidly with the addition of fatty acyl Co-As, sulfatides, and other complex sphingolipids as more internal standards are becoming available. The resulting LC–MS/MS analyses are one of the most analytically rigorous technologies that can provide the necessary sensitivity, structural specificity, and quantitative precision with high-throughput for “sphingolipidomic” analyses in small sample quantities. This review summarizes historical and state-of-the-art analytical techniques used for the identification, structure determination, and quantitation of sphingolipids from free sphingoid bases through more complex sphingolipids such as sphingomyelins, lactosylceramides, and sulfatides including those intermediates currently considered sphingolipid “second messengers”. Also discussed are some emerging techniques and other issues remaining to be resolved for the analysis of the full sphingolipidome.

Introduction

Sphingolipids (SLs) are 1,3-hydroxy-2-amino alkanes or alkenes with substantial structural diversity apart from this common core moiety. SLs are structural components of cellular membranes in all eukaryotes and some prokaryotes, but their variety of signal transduction roles is also well established [1], [2]. Selected SLs and a portion of their de novo biosynthetic and turnover pathways are shown in Fig. 1, from which it is clear that some molecular species are free sphingoid bases (non-acylated, long-chain bases), other species are N-acylated sphingoid bases, and still other species are more complex N-acylated sphingoid bases with a polar moiety at the 1-position. While this increasing molecular diversity results from a series of biosynthetic enzymatic reactions, it should not be overlooked that reversal of these reactions (catabolism or turnover) is a commonly encountered aspect of SL metabolism. Cells exert fine-tuned control over SL metabolic flux and opposing outcomes in cellular state may hang in the balance. For example, sphingosine and sphingosine-1-phosphate are interconverted by kinases and phosphatases [3]; the non-phosphorylated sphingoid base is pro-apoptotic [4] while the phosphorylated sphingoid base is anti-apoptotic [5]. Studies of SL biology clearly require a high degree of specificity for quantitation of multiple molecular species in a single sample because of the structural similarity and interconversion of SL species. Thus, the ability to analytically distinguish (resolve) these species is critical for the accurate determination of metabolic flux. The focus of this review is the development of state-of-the-art technologies in SL analysis, including a compilation of SL publications based upon classical analytical approaches. Although the topics are mentioned here, more comprehensive reviews of SL metabolism [6], [7], metabolic inhibition, and signaling [8], [9], [10], [11] are available.

Section snippets

Sphingoid bases

Sphingoid bases (also called long-chain bases) include 3-ketosphinganine, sphinganine, phytosphingosine (4-hydroxysphinganine), and sphingosine (also called sphing-4-enine). The first products of de novo SL biosynthesis are 3-ketosphinganine and sphinganine. In many mammalian cell types, phytosphingosine and sphingosine are the products of phytoceramide and ceramide de-N-acylation, respectively, not direct 4-hydroxylation and 4,5-E-desaturation of sphinganine. However, yeast cells and some

Thin-layer chromatography (TLC)

TLC has been used for the qualitative study of sphingoid bases and their phosphates, ceramides [28], sphingomyelins [29] and more complex GSLs. Advances in high-performance TLC (HPTLC) have allowed improved resolution of some molecular species [29], [30]. However, TLC does not offer sufficient structural specificity to guarantee homogeneity within a single spot of lipid using detection by Rhodamine 6G or iodine vapor. This has lead to its use as a step to check fraction collections in

Electron ionization (EI) mass spectrometry

Electron ionization mass spectrometry was initially used to elucidate the structures of ceramides [48], [49] and neutral GSL species [50], [51]. These early experiments permitted the analysis of SLs as intact molecular species, and yielded diagnostic fragmentations that could distinguish isomeric SL structures [52], [53]. Because these molecules were either relatively large or polar they required derivatization of the SL to trimethylsilyl or permethyl ethers to reduce their polarity and

Emerging technologies for sphingolipidomic analyses

The term “sphingolipidomic” analysis refers to the structure specific and quantitative measurement of all the individual molecular species of sphingolipids so that the role of each in a given biological context (i.e. biosynthesis or turnover) may be determined. To this end, LC–MS/MS is currently the analytical tool of choice for “sphingolipidomic” analyses for several important reasons. First, LC–MS/MS provides three orthogonal points of specificity with regard to differentiation of complex

“Shotgun” lipidomics

Other mass spectrometric techniques have been introduced for the identification, structure determination, and quantitation of sphingolipids. For example, two-dimensional (2D) ESI MS, using a triple quadrupole instrument has been described by Han and Gross [77], [78]. This technique utilizes MS and MS/MS analyses of an infused sample in both positive and negative ESI conditions. The formation of charged molecular adducts in the positive mode and anionic species in the negative mode is promoted

Summary and perspective

Initially described and named in 1884 by Jo-han Ludwig Wilhelm Thudichum [98], sphingolipids are now known to mediate many important cellular processes, such as apoptosis, differentiation, and chemotaxis. The structural diversity of SLs has been established by numerous chromatographic separation techniques, including TLC and HPLC. Mass spectrometry provides many advantages in the detection of SLs, especially sensitivity, and both specificity and structural information may be provided by MS/MS

Acknowledgement

The authors thank Prof. Alfred H. Merrill, Jr. for critical reading of this manuscript and Lipid MAPS (U54 GM069338) for continued support

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The limits of detection and quantification of 11 ceramides and 6 dihydroceramides were 0.01–0.5 ng/mL and 0.02–1 ng/mL, respectively, and all detected ceramides had good linearities (R² > 0.997). The extraction recoveries of ceramides at three levels were within 82.32%–115.24% in the liver and within 83.21%–118.70% in the small intestine. The relative standard deviations of intra- and inter-day precision were all within 15%. The extracting solutions of the liver and small intestine could be stably stored in the autosampler 24 h at 10 °C, the lyophilized liver and small intestine for ceramides quantification could be stably stored at least 1 week at −80 °C. The ceramides and dihydroceramides in normal mice liver and small intestinal tissues analyzed by the developed method indicated that the detected 9 ceramide and 5 dihydroceramides levels were significantly different, in which Cer d18:1/16:0, Cer d18:1/22:0, Cer d18:1/24:1, Cer d18:1/24:0 and dHCer d18:0/24:1 are the main components in the liver, whereas Cer d18:1/16:0 and dHCer d18:0/16:0 accounts for the majority of proportion in the intestinal tissues.
A simple and rapid method for the quantification of 11 ceramides and 6 dihydroceramides in the animal tissues was developed and applied. The compositions of ceramides in two tissues suggested that the compositional features should to be considered when exploring the biomarkers or molecular mechanisms.
Shotgun lipidomics combined targeted MRM reveals sphingolipid signatures of coronary artery disease
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Sphingolipids (SPLs) are bioactive lipids that manifest structural diversity and complexity in eukaryotes. However, the distributions and functions of these molecules in mammalian tissues/cells have not been systematically investigated. Herein, we integrated shotgun lipidomics with targeted LC-MRM/MS approach to comprehensively analyze SPL species in various biological samples with high accuracy. Preliminarily, 1311 SPL molecules were identified in 18 kinds of mammalian samples, including 3 groups of human sera, 10 mouse tissues and 5 cell lines via 26 sphingoid long-chain bases scanning. The sphingolipidome compositions and distributions were systematically characterized and distinct qualitative and quantitative profiles were clearly exhibited in various samples, indicating unique biological functions of the sphingolipidomes. Next, targeted SPLs analysis by LC-MRM/MS with critical criteria monitoring two characteristic fragments of one precursor was applied to human serum samples from 24 coronary artery disease (CAD) patients and 12 healthy controls, which successfully quantified 170 SPL molecules. Ten novel SPL molecules were discovered as a potential diagnostic panel for CAD patients via multivariate exploratory receiver operating characteristic curve-based biomarker analysis. The diagnostic panel with the 10 SPL molecules achieved 97.2% accuracy, with a favorable auxiliary diagnostic value (AUC = 1.000), for the detection of CAD. These results clearly support the sphingolipidomic approach in application to discovering disease biomarker panel as well as deep investigation of biological functions of complex SPLs in mammalian samples.
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Glycolipids encompass a wide variety of compounds that contain both glycans (i.e., carbohydrates) and lipids. The type of lipid moiety and attached carbohydrate(s) determine the categories and subcategories, which include: fatty acyl glycolipids, glycosphingolipids (cerebrosides, globosides, gangliosides, sulfatides and others), glycoglycerolipids, glycophospholipids (e.g., phosphatidylinositols), glycosylated prenols (e.g., dolichol-phospho-glycans), glycosylated sterols, glycosylated polyketides and saccharolipids. This review provides a brief overview of the types of functions these compounds perform, the structural features of the different categories, and a few examples of how structural diversity is achieved. Although the “glycolipidolome” is formidably large, it is becoming more assessable to characterization as the tools of the “omics” revolution expand to include mass spectrometry and other techniques for structural analysis.
Profiling of sphingolipids in Caenorhabditis elegans by two-dimensional multiple heart-cut liquid chromatography – mass spectrometry
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Sphingolipids exert important functions in cells, ranging from stabilising the cell membrane to bioactive signalling in signal transduction pathways. Changed concentrations of sphingolipids are associated with, among others, neurodegenerative and cardiovascular diseases. In this work, we present a novel two-dimensional liquid chromatography method (2D-LC) coupled to tandem mass spectrometry (MS/MS) for the identification of ceramides, hexosylceramides and sphingomyelins in the model organism Caenorhabditis elegans (C. elegans). The method utilises a multiple heart-cut approach with a hydrophilic interaction liquid chromatography (HILIC) separation in the first dimension. The fractions of the sphingolipid classes were cut out and thereby separated from the abundant glycerolipids, which offers a simplified sample preparation and a high degree of automation as it compensates the alkaline depletion step usually conducted prior to the chromatographic analysis. The fractions were stored in a sample loop and transferred onto the second column with the combination of two six port valves. A reversed phase liquid chromatography was performed as the second dimension and allowed for a separation of the species within a sphingolipid class and according to the fatty acid moiety of the sphingolipid. The segregation of the abundant glycerolipids and the reduced matrix effects allowed for better identification of low abundant species, especially dihydro-sphingolipids with a saturated sphingoid base. In addition, the separation of the three fractions was carried out parallel to the separation and equilibration in the first dimension, which leads to no extension of the analysis time for the 2D-LC compared to the one-dimensional HILIC method. In total 45 sphingolipids were detected in the C. elegans lipid extract and identified via accurate mass and MS/MS fragments.
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Sphingolipids (SLs) are endogenously bioactive molecules with diverse structures, and its metabolic disorders are involved in the progression of many diseases. In this study, an ultra-performance liquid chromatography quadrupole exactive mass spectrometry (UPLC-Q-Exactive-MS) method was established to comprehensively profile SLs in plasma. First, the fragment patterns of SL standards of each subclass were investigated. Then, the SL species in plasma were characterized based on the fragmentation rules. Finally, a total of 144 endogenous SL species consisting of 216 regioisomers were identified in plasma of human, golden hamster and C57BL/6 mice, which was the most comprehensive identification for SLs in plasma. In addition to the known species, 19 SL species that have never been reported were also identified. The profile of SLs in plasma of human and two rodent species was compared subsequently. It was worth noting that a total of 9 SL molecular species consisting of 11 regioisomers with low abundance were successfully identified in human plasma through comparison among species. Those findings contribute to a deeper understanding of SLs in human plasma and provide scientific basis for the selection of animal model. The established profile of SLs in plasma could be used for screening of lipid biomarkers of various diseases.

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^☆: This paper is part of the special issue “Lipidomics: Developments and Applications”, X. Han (Guest Editor)”.

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ReviewSphingolipidomics: Methods for the comprehensive analysis of sphingolipids☆

Abstract

Introduction

Section snippets

Sphingoid bases

Thin-layer chromatography (TLC)

Electron ionization (EI) mass spectrometry

Emerging technologies for sphingolipidomic analyses

“Shotgun” lipidomics

Summary and perspective

Acknowledgement

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Review
Sphingolipidomics: Methods for the comprehensive analysis of sphingolipids☆