Elsevier

Methods

Volume 39, Issue 2, June 2006, Pages 82-91
Methods

Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry

https://doi.org/10.1016/j.ymeth.2006.05.004Get rights and content

Abstract

There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), sphingosine (Sph) and sphingosine 1-phosphate (Sph-1P) that necessitates development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring changes in SPL levels and composition from biological materials. Simultaneous ESI/MS/MS analysis of sphingoid bases (SBs), sphingoid base 1-phosphates (SB-1Ps), Cers and sphingomyelins (SMs) is performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer operating in a multiple reaction monitoring (MRM) positive ionization mode. Biological materials (cells, tissues or physiological fluids) are fortified with internal standards (ISs), extracted into a one-phase neutral organic solvent system, and analyzed by a Surveyor/TSQ 7000 LC/MS system. Qualitative analysis of SPLs is performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of SPLs. Quantitative analysis is based on calibration curves generated by spiking an artificial matrix with known amounts of target synthetic standards and an equal amount of IS. The calibration curves are constructed by plotting the peak area ratios of analyte to the respective IS against concentration using a linear regression model. This robust analytical procedure can determine the composition of endogenous sphingolipids (ESPLs) in varied biological materials and achieve a detection limit at 1 pmol or lower level. This and related methodology are already defining unexpected specialization and specificity in the metabolism and function of distinct subspecies of individual bioactive SPLs.

Introduction

Prevalent complex sphingolipids (SPLs): phosphosphingolipids (PSPLs) and glycosphingolipids (GSPLs) are found in all eukaryotes, some prokaryotes and viruses, mainly as components of the plasma membrane and related organelles. SPLs constitute about 30% of the total lipid of plasma membranes, but these percentages can be considerably higher because SPLs are asymmetrically distributed, and can spontaneously aggregate to form liquid ordered micro domains termed “rafts”. SPLs form specialized structures, mediate cell–cell and cell-substratum interactions, modulate the behavior of cellular proteins and receptors, and participate in signal transduction. Several SPL metabolites, especially ceramide (Cer), sphingosine (Sph) and sphingosine-1-phosphate (Sph-1P), have been identified as bioactive key molecules that control cell growth and death [1], [2], [3], [4], [5], [6], [7], [8], [9]. This discovery emphasizes the need for examination of SPL metabolic pathways [1], [2], [3], [4], [5], [6], [7], [8], [9]. To understand how SPL biosynthesis and turnover regulates cell behavior under normal and abnormal conditions, how perturbations in SPL of one type may enhance or interfere with the action of another, and where and how all these SPLs are made and removed, we must be able to establish the metabolomic profile of SPLs. Mass spectrometry (MS) methodology offers an efficient tool to monitor changes in the composition of all these bioactive species under different environments, and may provide a missing link in the search for novel and effective health therapy.

A variety of sample preparation, ionization modes and instrumental design have been developed so far to analyze particular SPL classes by MS technology [10]. Design for this methodology has been based on the fact that different SPL subclasses dissociate into structurally distinctive patterns corresponding to their sphingoid bases, N-acyl chain and polar head group [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21]. Recent advances in electrospray ionization (ESI) have provided a new approach to successfully examine total SPL components in crude lipid extracts [15], [18], [19], [21]. ESI methodology allows generation of intact molecular ions of molecules directly from solution by coupling a high-performance liquid chromatography (HPLC) column directly to the mass spectrometer. Further improvements in instrumentation, such as the triple quadrupole with robust ion sources, fast scanning mass analyzers and reduced chemical noise (possible in MS/MS technique), allow the identification and quantitation of SPLs with great sensitivity (sub-picomole detection limit) in a highly reproducible manner. SPL identification is accomplished by tandem mass spectrometry (MS/MS) with precursor ion scans to distinguish various molecular species in crude lipid extract by taking advantage of the unique molecular decomposition pattern [15], [21] for each SPL class. SPL quantitation is performed by using positive ionization and multiple reaction monitoring (MRM) in conjunction with HPLC separation [21].

In this paper, we describe method for simultaneous analysis of the following SPLs: sphingoid bases (SB): Sph, dhSph; sphingoid base phosphates (SB-1P): Sph-1P, dhSph-1P, ceramides (Cers), dihydroceramides (dhCers), and sphingomyelins (SMs) using state-of-the-art HPLC-MS/MS techniques. The developed protocol describes a new and efficient one-phase neutral organic solvent system used for the preparation of lipid extract containing the above SPLs, and their identification and quantitation.

In general, the correct, sufficient, simple, and safe method for lipid extract preparation is one of the major considerations in lipid analysis, because analytical methods are applied specifically to the SPL components present in the particular lipid extract. In this protocol lipid extracts are prepared under a safe and neutral condition to avoid destruction of the parent “soft” SPLs (e.g. SPLs containing O-acyl group) and to efficiently and quantitatively extract the SB-1Ps from biological material since the latter are notoriously difficult to recover quantitatively [22], [23].

Our ultimate goal is to provide a total metabolomic profile of SPLs. SPL components missing in this protocol are ceramide 1-phosphate (Cer-1P), psychosine (GlcSph), glucosylceramide (GlcCer), lactosylceramide (LacCer) and inositolceramide 1-phosphate (IPC). The missing SPL components are under development and a full protocol will be presented after completion of the synthetic project for the unavailable analytical standards necessary for reliable quantitation of these SPLs. A semiquantitative LC/MS method for analysis of GlcCer, LacCer, and other glycosphingolipids has been described by Sullards et al. [21].

General structures and nomenclature for SPLs cited and described in this paper are shown in Fig. 1. SPLs constitute one of the most structurally diversified classes of amphipathic lipids abundant in all living organisms. Variations in the nature of the head group attached to the primary hydroxyl group (carbohydrates, phosphorylcholine, phosphate or phosphoinositol), N-acyl group, and sphingoid-base backbone result in a great number of chemically distinct SPLs, where Sph, dhSph or phytoSph are the core structural moieties. Some 3-O-alkyl, 3-O-alkenyl (vinyl ether linkage) and 1- or 3-O-acyl-analogs of natural components have also been identified. Thousands of natural, complex SPLs have been isolated based on almost 60 distinct species of SBs, although most of them are very minor components. SBs, the backbone of all SPLs, encompass a wide array of (2S, 3R, 4E)-2-amino-1,3-dihydroxyalkenes (Sphs), (2S, 3R)-2-amino-1,3-dihydroxyalkanes (dhSphs), and (2S, 3S, 4R)-2-amino-1,3,4-trihydroxyalkanes (phytoSphs) with alkyl chain lengths from 14 to 22 carbon atoms and variations in the number and position of the double bonds, hydroxyl groups and branching methyl groups. Mammal SPLs are predominantly composed of 2-amino-1,3-dihydroxy-octadecene (18Sph, abbreviated here as Sph) and 2-amino-1,3-dihydroxy-octadecane (18dhSph, abbreviated here as dhSph), yeast and plant SBs are mainly composed of 2-amino-1,3,4-trihydroxy-octadecane (18phytoSph), 18dhSph and their eicosa-homologs (20phytoSph and 20dhSph). Additionally, some SPLs may contain a double bond in position 8 or have double bonds in positions 4 and 8 (which can be found in plant SPLs). Ceramides are N-acyl-derivatives of SBs. Combinations of different SBs with different fatty acids (including their hydroxy-analogs) generates a huge variety of Cer, dhCer, and phytoCer. These basic SPLs are modified at the 1-hydroxyl group to: (i) phosphates (e.g. Sph-1P and Cer-1P), (ii) phosphocholine-analogs (e.g. SM and lysosphingomyelin, lyso-SM), and (iii) glucosyl- and galactosyl-analogs (e.g. glucosylceramide and galactosylceramide, known as cerebrosides, and their lyso-form: psychosine). The latter group also serve as precursors to hundreds of different species of complex GSPLs, with lactosylceramide (containing only two sugar residues) being the simplest. Additionally, some Cers and GSPLs can be modified on their hydroxyl groups (e.g. O-acyl-Cers), and some SBs can by N-methylated.

The structural diversity of SPLs dictates that every step in analysis of these natural products must be carefully evaluated.

Section snippets

Biological material

Cells in culture: (1 × 106–10 × 106 cells) and biological samples: tissues (mass equivalent to 0.5–1.0 mg of protein), serum (50–100 μl), culture media (2.0–5.0 ml).

Internal standards (ISs)

Internal standards (ISs) are prepared by the Lipidomics Core, MUSC or are purchased from a commercial source, if available (Metreya Inc., AVANTI Polar Lipids Inc.). Additional SPL standards are under development by the Lipidomics Core, MUSC.

  • 1 μM IS “Solution A” containing: 17Sph, 17Sph-1P, 17C16-Cer, and 18C17-Cer in methanol.

  • 5 μM IS

Recipes

Note: All the following solutions should be prepared in volumetric flasks

  • Recipe 1: 1 M Ammonium formate. Prepare 1 M ammonium formate in water.

  • Recipe 2: 1 M NaOH. Prepare 1 M NaOH in methanol.

  • Recipe 3: Tissue homogenation buffer. Prepare buffer containing: 0.25 M sucrose, 25 mM KCl, 50 mM Tris, and 0.5 mM EDTA, pH 7.4.

  • Recipe 4: Cell or tissue extraction mixture. Prepare a solution of iso-propanol:water:ethyl acetate (30:10:60; v:v:v).

  • Recipe 5: Media extraction mixture. Prepare a solution of

Cells in culture

Cells grown in suspension: Transfer cell suspension to 15 ml polyethylene vials kept on ice, separate cell pellet from media by centrifugation at 1000 rpm for 5 min at 5–10 °C, aspirate media, wash cell pellet twice with ice-cold phosphate-buffered saline (1× PBS), separate pellet from PBS wash, use cell pellet for lipid extraction.

Adherent cells: Remove media, wash cells twice with ice-cold PBS, scrape cells with ice-cold PBS and transfer to 15 ml polyethylene vial placed on ice (repeat this

Concluding remarks and hints for troubleshooting

Very low sub-picomole detection limits have been achieved by applying rigorous sample preparation procedures, excellent HPLC separation and MRM experiments, which greatly reduce chemical noise in the final LC/MS chromatograms. We have selected ISs for particular SPL classes based on C17-SB as the closest ‘unnatural’ sphingoid base to the natural C18-Sph counterpart. This selection gives us a confidence that physicochemical properties such as the elution order and mass fragmentation pattern

Related techniques

A variety of different techniques (mostly radio-labeling, HPLC analysis of fluorescent analogs, and enzymatic methods) in addition to MS methodology are used for SPLs measurement. Up to now the enzymatic method employing diacylglycerol kinase and [32P] ATP has been the most commonly used procedure for total Cer quantitation in the range of 25 pmol to 2 nmol [34], [36]. Cellular SBs are most often analyzed by the HPLC technique developed for their fluorescent derivatives [37]. Cellular SB-1Ps are

Acknowledgments

This work was supported by NIH Grant No. RR17677-03 from the COBRE Program of the National Center for Research Resources: “COBRE in Lipidomics and Pathobiology” and by NCI Grant Number IPO1CA097132-01A1 from Program Project Grant Application “Sphingolipids in Cancer Therapy and Angiogenesis”.

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