Sphingolipid Analysis by High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS)

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Sphingolipid (SPL) metabolism (Fig. 1) serves a key role in the complex mechanisms regulating cellular stress responses to environment. Several SPL metabolites, especially ceramide (Cer), sphingosine (Sph) and sphingosine1-phosphate (S1P) act as key bioactive molecules governing cell growth and programmed cell death (Fig. 2). Perturbations in sphingolipids of one type may enhance or interfere with the action of another. To monitor changes in SPL composition therefore, reliable analytical methods are necessary.

Here we present the liquid chromatography tandem mass spectrometry (LC-MS/MS) approach for simultaneous qualitative and quantitative monitoring of SPL components (classes and molecular species) in biological material as an effective tool to study sphingolipid signaling events. The LC-MS/MS methodology is the only available technique that provides high specificity and sensitivity, along with a wealth of structural identification information. Figure 1.

Sphingolipid biosynthesis and metabolic pathways; metabolomic profiling of sphingolipids. Abbreviations used in the figure: 3-keto-dhSph, 3-keto-dihydrosphingosine; dhSph, dihydrosphingosine; dhS1P, dihydrosphingosine 1-phosphate; dhCer, dihydroceramide; Sph, sphingosine; S1P, sphingosine 1-phosphate; Cer1P, ceramide-1-phosphate; SM, sphingomyelin; lyso-SM, lyso-sphingomyelin; DAG, diacylglycerol; O-Acyl-Sph. O-acyl-sphingosine, O-Acyl-Cer, O-acyl-ceramide; N-Me-Sph, N-methyl-sphingosine; N.N-DMSph, N,N-dimethyl-sphingosine; PA, palmitic acid; EAP, ethanolamine phosphate; HD, hexadecenal; GL, glycerolipids; de-SM, demethylated sphingomyelin; PGLs, phosphoglycerolipids; GlcCer, glucosylceramide; GalCer, galactosylceramide; GSLs, glycosphingolipids.
Figure 2.
Natural sphingolipids are a highly heterogenous system related to the sphingoid bases and derivatization made on the amino- and hydroxy-functions. Structures shown in this figure represent derivatives of 18C-SB (sphingoid bases containing C18-backbone chain) indicating SPLs containing 2-amino-1,3-dihydroxy-octadecene-4E, 2-amino-1,3-dihydroxy-octadecane and 2-amino-1,3,4-trihydroxy-octadecane. General structures, nomenclature and abbreviations for SPLs are cited and described in this presentation. Cn -indicates the chain length of N-acyl part of SPLs.