Abstract
A precise, robust, and specific liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) method was developed for profiling and quantifying glucosyl sphingosine (GS), glucosylceramide (GC), ceramide (Cer), lactosylceramide (LacCer) and sulfatide lipid species in a variety of mouse tissues. The linear response ranges of these species were 0.05-25 ng. The major GC species identified in visceral tissues of mice were GCs with N-acyl chains of C24-1, C24, C22, C16 lengths, but the qualitative and quantitative profiles differed among tissues. GC levels in spleen were approximately 3-5 times higher than in liver and lung. Brain differed from visceral tissues in that galactosylceramides (GalCer) were the predominant monohexosylceramide species identified. A silica column used in hydrophobic interaction liquid chromatography (HILIC) mode was capable of differentiating GC and GalCer. The analysis of mouse brain samples revealed that GC accounted for only 0.3% of the total monohexosylceramides. Cer and LacCer were also profiled and quantified in mouse brain, lung, liver and spleen. Application of these methods greatly facilitated a range of targeted sphingolipidomic investigations and will permit a better understanding of the function and mechanism of these diverse molecular species in various disease animal models, including Gaucher disease.
Highlights
Sphingolipids (SLs) and glycosphingolipids (GSLs) are essential structural components of mammalian cell membranes
Ultra performance liquid chromatography (UPLC) columns were used since they have smaller particle sizes than conventional high performance liquid chromatography (HPLC) columns and facilitate superior chromatographic resolution
Our initial studies with a Waters Acquity ultra performance liquid chromatography (UPLC) column (BEH C18-1.7 μm, 50×2.1 mm i.d.) yielded excellent separation of glucosylceramide standards based upon their N-acyl chain length
Summary
Sphingolipids (SLs) and glycosphingolipids (GSLs) are essential structural components of mammalian cell membranes. Several methods [12,13,15] permit a global analysis of sphingolipid species ranging from very polar species, i.e., sphingosine-1-phosphate, to more lipophilic species in the sphingolipid biosynthesis pathway In many of these methods, sphingolipids with varying chain lengths often coelute under the chromatographic conditions used, making differentiation of closely related species reliant upon measurement of their specific masses. We report an LC-MS/MS method for profiling individual sphingolipids with adequate chromatographic resolution to overcome such potential isotopic effects This highly specific and sensitive LC-MS/MS analytical approach affords the characterization of sphingolipids that vary in N-acyl chain lengths. The potential of this methodology is illustrated from the ability to map and compare quantitatively changes in sphingolipid distribution from wild type mice with that of a genetic mouse model of the lysosomal storage disease, Gaucher disease [27,28]
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