This article was originally published in the October 2013 edition of Matreya’s Newsletter for Glyco/Sphingolipid Research (PDF).

Over the past decade sphingolipids have gained enormous recognition as vital and complex components of biological systems. For many years, lipids in general, and sphingolipids in particular, have received far less attention than their critical functions deserve. Many reasons contributed to this oversight including the difficulty of extraction and analysis as well as their tremendous diversity in structure and function.^1,2 Another historical problem was the very limited availability of appropriate natural and synthetic sphingolipid standards. Fortunately, there has been a welcome advance in making standards available, which has greatly accelerated the analytical field known as sphingolipidomics, leading to our enhanced understanding and classification of sphingolipids.

Sphingolipidomics is the study of the individual sphingolipid species and its metabolism within a given biological system using mass spectrometry. It began to appear as a distinct subfield of the greater lipidomics discipline around 2005.^3,4 There are tens of thousands of possible sphingolipids that vary in their polar head groups, acyl chains, and sphingoid bases. The metabolic pathway of these sphingolipids has been extensively studied to understand and treat diseases related to sphingolipids. Many of these sphingolipids are present in only picomole to nanomole amounts, making detection difficult. However, with the incorporation of soft ionization techniques in mass spectrometry, the detection of very small amounts of sphingolipids is possible. Most sphingolipidomics studies use either liquid chromatography (LC)- or shotgun-based mass spectrometry approaches.¹ With both approaches, internal standards are used to correct for sample extraction efficiency and instrumentation variability. While adding an internal standard for each individual sphingolipid detected with either of these methods would be ideal, this is impractical due to the vast number of possible sphingolipids in a sample. Thus, internal standards for each class of sphingolipid expected to be found in a sample are used. Appropriate sphingolipidomics internal standards include sphingolipids that are modified on either the oligosaccharide head, ceramide acyl chain, or the sphingosine backbone. These standards are usually stable isotope labeled, unusual in chain length, or fluorescently tagged.

Stable Isotope-Labeled Standards

The most preferred internal standards for lipidomics studies are stable isotope-labeled standards. These standards can be detected by mass spectrometry, while demonstrating nearly identical physical properties compared to natural sphingolipids. This is very important to ensure similar extraction properties between the analytes and the internal standards.¹ Most commonly, deuterium or ¹³C atoms are introduced in the N-acyl chain of the ceramide. The label can also be introduced into the sphingosine tail or oligosaccharide head group, where it is typically more stable.

Stable Isotope-Labeled Sphingolipids

New Deuterium-Labeled Glycosphingolipids

Stable Isotope-Labeled Sphingolipids as Highly Specific Mass Spectrometry Standards

Novel Adamantane-Labeled Sphingolipids

Glycinated Lyso-Glycosphingolipids as New Mass Spectrometry Internal Standards

References

1. Han, X. and Jiang, X. A review of lipidomic technologies applicable to sphingolipidomics and their relevant applications. Eur. J. Lipid Sci. Technol. 111(1), 39-52 (2009).

2. Futerman, A.H. and Hannun, Y.A. The complex life of simple sphingolipids. EMBO Rep. 5(8), 777-782 (2004).

3. Maceyka, M., Milstien, S., and Spiegel, S. Sphingosine kinases, sphingosine-1-phosphate and sphingolipidomics. Prostaglandins Other Lipid Mediat. 77(1-4), 15-22 (2005).

4. Merrill, A.H., Jr., Sullards, M.C., Allegood, J.C., et al. Sphingolipidomics: High-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry. Methods 36(2), 207-224 (2005).

5. Merrill, A.H., Jr. Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chem. Rev. 111(10), 6387-6422 (2011).

6. Lipsky, N.G. and Pagano, R.E. Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: Endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus en route to the plasma membrane. J. Cell Biol. 100(1), 27-34 (1985).