Elsevier

Methods in Enzymology

Volume 434, 2007, Pages 233-241
Methods in Enzymology

Combination of C17 Sphingoid Base Homologues and Mass Spectrometry Analysis as a New Approach to Study Sphingolipid Metabolism

https://doi.org/10.1016/S0076-6879(07)34012-3Get rights and content

Abstract

In recent years, sphingolipid metabolites ceramide, sphingosine, and sphingosine‐1‐phosphate have emerged as important second messengers in addition to their role as precursors of biomembrane components. The investigation of these sphingolipid metabolites requires the development of new, more sensitive methods for assaying the enzymes involved in their production. This chapter describes the utilization of mass spectrometry technology in combination with nonnaturally occurring C17 sphingoid bases in the in vitro assays of two of the enzymes of the sphingolipid pathway, ceramide synthase and sphingosine kinase. These new in vitro methods provide high sensitivity and extreme accuracy even when crude extracts are used as enzyme sources.

Introduction

Several sphingolipid metabolites, particularly ceramide, sphingosine, and sphingosine‐1‐phosphate, in addition to their role as precursors or breakdown products of key structural components of biological membranes, have been identified as important bioactive molecules that regulate cell growth and death (Hannun 2002, Merrill 2002, Spiegel 2002). This discovery emphasizes the need for developing more efficient and sensitive methods for characterizing the enzymes of sphingolipid metabolic pathways. Until now, the majority of the methods used to measure the activities of most enzymes in the sphingolipid pathway have relied on the use of radioactive substrates. While using radioactivity leads to sensitive methodology, it does not escape the hazards of routinely handling radioactivity, which can lengthen procedures as a consequence of regulations and special care required for such handling. In addition, the substrates and products of sphingolipid enzymes are, for the most part, highly hydrophobic and detection requires separation via thin‐layer chromatography, which has limitations when it comes to separation of species with similar mobilities.

Mass spectrometry (MS) methodology, as discussed in this chapter, is an efficient and sensitive means for measuring even small changes in sphingolipid species. The electrospray ionization (ESI) MS methodology, coupled with high‐performance liquid chromatography, allows generation of intact molecular ions of molecules directly from solutions. In addition to the sensitivity and selectivity of MS, our new approach utilizes synthetic sphingolipids containing a sphingoid‐base backbone that is 17 carbons in length in lieu of the naturally occurring mammalian sphingoid bases, sphingosine (2‐amino‐1,3‐dihydroxyoctadecene) and dihydrosphingosine (2‐amino‐1,3‐dihydroxyoctadecane), which are 18 carbons in length (Fig. 12.1). This elegant approach allows for extremely accurate measurement of the products and/or the substrates of the enzymes of the sphingolipid pathway even in crude cell homogenates. In addition to the in vitro enzyme activity methods, the MS approach, when used in conjunction with sphingoid bases with C17 backbones, is a powerful tool for cellular analysis of sphingolipids. This method analyzes simultaneously C17 and C18 sphingolipid species, which is not available with traditional methods. It also provides detailed information about their composition so that the particular sphingolipid metabolic pathway can be analyzed without being obscured by endogenous sphingolipids. This chapter focuses on the application of these approaches to a novel method to study the in vitro and cellular activities of two sphingolipid pathway enzymes, namely ceramide synthase and sphingosine kinase (SK).

Section snippets

Mass Spectrometry Analysis

The described ESI/MS/MS analyses of sphingolipids, containing the C17 backbone sphingoid base, are developed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer, operating in a multiple reaction monitoring positive ionization mode (Bielawski et al., 2006). The analyzed samples are fortified with 50 pmol of an internal standard, N‐palmitoyl‐d‐erythro‐C13 sphingosine, and extracted with ethyl acetate/isopropanol/water (60/30/10, v/v) solvent system. After evaporation and

Ceramide Synthase

Ceramide synthases catalyze the biosynthesis of dihydroceramide or ceramide from a sphingoid base and fatty acid CoA. Depending on the source of the sphingoid base, members of this family of enzymes can be involved in the de novo synthesis or recycling of ceramide. Ceramide synthases can utilize all four isomers of the sphingoid bases dihydrosphingosine and sphingosine, that is, d‐threo‐, d‐erythro‐, l‐threo, and l‐erythro, but with different specificity. The preferred substrate is the d

In Vitro Ceramide Synthase Method

The pH optimum of ceramide synthase is pH 7.5 (Sribney, 1966). In vitro reactions performed in phosphate buffer show higher activities as compared to in vitro reactions performed in Tris buffer, and ceramide synthase activity is not dependent on metal ions (Morell 1970, Sribney 1966). The reaction mix, usually 100 μl, contains 25 mM potassium phosphate buffer (pH 7.4). The C17 backbone sphingoid base substrates (dihydrosphingosine or sphingosine) are hydrophobic, and therefore special care has to

Sphingosine Kinase

Sphingosine kinases are highly conserved enzymes that catalyze the biosynthesis of sphingosine to sphingosine‐1‐phosphate and the biosynthesis of dihydrosphingosine to dihydrosphingosine‐1‐phosphate. The enzyme has been purified or cloned from a variety of sources, including Saccaromyces cerevisiae (Nagiec et al., 1998), Arabidopsis thaliana (Imai and Nishiura, 2005), Drosophila (Herr et al., 2004), rat (Imamura et al., 2001), mouse (Kohama et al., 1998), and human (Liu 2000, Pitson 2000). SKs

In Vitro Sphingosine Kinase Method

Cells are usually grown to 70 to 80% confluency, washed twice with an appropriate volume of cold phosphate‐buffered saline, and collected by centrifugation at 3000g for 5 min. The pellets are then resuspended in two different buffers, known to selectively favor SK1 or SK2. The buffer for SK1 contains 20 mM Tris‐HCl, pH 7.4, 1 mM EDTA, 0.5 mM deoxypyridoxine, 15 mM NaF, 1 mM β‐mercaptoethanol, 1 mM sodium orthovanadate, 40 mM β‐glycerophosphate, 10% glycerol, 0.5% Triton X‐100, 10 mM KCl, and 1.5 mM

In Cells Labeling with C17 Sphingoid Base

Sphingoid bases with C17 backbone are efficient and easy to handle molecular probes. They are suitable for pulse or long‐term metabolic labeling of sphingolipids in the cells. Aliquots of concentrated ethanol solutions of C17 sphingoid bases can be added directly to the growth media alone or in combination with other treatments. The final concentration of the C17 sphingoid base probe in media depends on the cell type used in the experiment. However, it is important to note that micromolar

Acknowledgments

We thank Drs. L. Siskind, C. Clarke, and Y. Hannun for their helpful comments. We also acknowledge the Lipidomics Core Facility at MUSC (COBRE P20RR17677). This work was supported by the following grants: R01 AG016583, R01 GM062887, P01 CA097132, and a VA Merit award to L. M. O.

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