Sphingolipidomics: Methods for the comprehensive analysis of sphingolipids

Abstract

Sphingolipids comprise a highly diverse and complex class of molecules that serve as both structural components of cellular membranes and signaling molecules capable of eliciting apoptosis, differentiation, chemotaxis, and other responses in mammalian cells. Comprehensive or "sphingolipidomic" analyses (structure specific, quantitative analyses of all sphingolipids, or at least all members of a critical subset) are required in order to elucidate the role(s) of sphingolipids in a given biological context because so many of the sphingolipids in a biological system are interconverted structurally and metabolically. Despite the experimental challenges posed by the diversity of sphingolipid-regulated cellular responses, the detection and quantitation of multiple sphingolipids in a single sample has been made possible by combining classical analytical separation techniques such as high-performance liquid chromatography (HPLC) with state-of-the-art tandem mass spectrometry (MS/MS) techniques. As part of the Lipid MAPS consortium an internal standard cocktail was developed that comprises the signaling metabolites (i.e. sphingoid bases, sphingoid base-1-phosphates, ceramides, and ceramide-1-phosphates) as well as more complex species such as mono-and di-hexosylceramides and sphingomyelin. Additionally, the number of species that can be analyzed is growing rapidly with the addition of fatty acyl Co-As, sulfatides, and other complex sphingolipids as more internal standards are becoming available. The resulting LC-MS/MS analyses are one of the most analytically rigorous technologies that can provide the necessary sensitivity, structural specificity, and quantitative precision with high-throughput for "sphingolipidomic" analyses in small sample quantities. This review summarizes historical and state-of-the-art analytical techniques used for the for the identification, structure determination, and quantitation of sphingolipids from free sphingoid bases through more complex sphingolipids such as sphingomyelins, lactosylceramides, and sulfatides including those intermediates currently considered sphingolipid "second messengers". Also discussed are some emerging techniques and other issues remaining to be resolved for the analysis of the full sphingolipidome.

A rapid and quantitative LC-MS/MS method to profile sphingolipids

“…The latter is of major importance to compensate for matrix effects and varying ionization effi ciencies, especially during gradient elution. Because reversed phase chromatography shows chain length-dependent separation, coelution of analytes and ISs may not be accomplished ( 13,16,19,27 ). Classical normal phase chromatography offers polar head group-specifi c separation but may be impaired by limited reproducibility and insuffi cient peak shapes.…”

“…Due to the relatively low level of the selected sphingolipids in crude lipid extracts, a direct analysis using "shotgun approaches" may be hampered by signal suppression caused by other matrix components ( 12,19,27 ). Therefore, we decided to establish an HPLC separation of sphin- GluCer 24:0 ( m/z 812.7) with and without isotope correction.…”

Tandem Mass Spectrometry of Sphingolipids: Application in Metabolic Studies and Diagnosis of Inherited Disorders of Sphingolipid Metabolism

“…Tandem mass spectrometry exhibits higher selectivity and sensitivity (with a higher signalto-noise ratio because noise is suppressed) than does simple mass spectrometry. This advantage also makes this technique useful when tandem mass spectrometry is coupled with HPLC separation (Haynes, et al, 2009;Sullards, et al, 2011).…”

“…This variation can be used in intrasource separation in lipid analyses (Han & Gross, 2005;Haynes, et al, 2009). Neutral sphingolipids are usually protonated by ammonium formiate, ammonium acetate, formic or acetic acid.…”

A Comparison of Trabecular Meshwork Sphingolipids and Ceramides of Ocular Normotensive and Hypertensive States of DBA/2J Mice

“…29 Ceramides can be generated through introduction of a double bond at the 4, 5 position of the sphingoid base to yield N-acyl-sphingosines. Other sphingolipids such as sphingosine arise through degradation pathways.…”