Alkaline methanolysis of lipid extracts extends shotgun lipidomics analyses to the low-abundance regime of cellular sphingolipids

Abstract

Sphingolipids that contain a sphingoid base are composed of hundreds to thousands of distinct compounds, many of which serve as lipid regulators of biological functions. The global analysis of the large number of low-abundance sphingolipid molecular species has been hampered in many cases by the sphingolipid molecular species being overwhelmed by the quantity of other classes of lipid (e.g., glycerophospholipid) molecular species present, thereby imposing severe restrictions on the dynamic range of their measurement using shotgun lipidomics. Herein, we developed a facile approach in which the sphingolipids of cellular extracts were dramatically enriched by direct alkaline methanolysis of lipid extracts followed by extraction to remove the large majority of other endogenous lipid classes. Through direct infusion of the resultant enriched solution, we identified and quantitated a variety of very-low-abundance sphingolipid classes (e.g., sphingosine, psychosine, and lysosphingomyelin) and molecular species (e.g., sphingomyelin) using electrospray ionization mass spectrometry (i.e., shotgun sphingolipidomics). Accordingly, through utilization of these facile enrichment techniques, direct penetrance into the sphingolipidomes has been greatly extended, facilitating new insights into their metabolism and signaling functions in biological systems. KeywordsElectrospray ionization mass spectrometry; Shotgun lipidomics; Shotgun sphingolipidomics; Sphingolipidome; Lysosphingomyelin; Psychosine; Sphingomyelin; Sphingosine Lipidomics, the large-scale study of the pathways and networks of cellular lipids, is an emerging and rapidly expanding research field [1][2][3]. Currently, lipidomics research has been focused on identifying alterations in lipid metabolic pathways and networks induced by a disease state, a gene mutation (knockout or overexpression), a therapeutic treatment, or other perturbations. In the future, research in lipidomics will expand to include the dynamics of lipidomes, subcellular organizations of lipidomes, and interactions of lipids with lipids, proteins, and other cellular moieties. Although lipidomics has emerged as a distinct field only within the past few years [1][2][3], numerous new discoveries and/or advances have already been made [3][4][5][6][7][8][9][10][11][12]. Its essential roles in identifying the biochemical mechanisms of lipid metabolism, investigating the functions of an individual gene of interest, identifying novel biomarkers, and evaluating drug efficacy, among others, are becoming increasingly recognized.One of the major analytical platforms in current lipidomics practice is multidimensional massspectrometry (MS) 1 -based shotgun lipidomics [13][14][15]. This platform has now evolved into a mature technology that includes a series of simple steps such as multiplexed extractions, *Corresponding author. Fax: +1 314 362 1402. E-mail address: xianlin@wustl.edu (X. Han).. intrasource separation, identification of individual lipid molecular species using multidimensional MS, and quan...

Large-scaled human serum sphingolipid profiling by using reversed-phase liquid chromatography coupled with dynamic multiple reaction monitoring of mass spectrometry: Method development and application in hepatocellular carcinoma

“…Besides, efforts have been made, by applying mild alkaline hydrolysis [16,18,19,23,32], or solid phase extraction (SPE) [20,33], aimed at reduced ion suppression, especially in a shotgun pattern. Nevertheless, these methods were hampered either by limited throughput in using SPE, or by inconstant factors of dripping loss or possible interfering protein in using chloroform/methanol/water.…”

Methods in Comprehensive Mass Spectrometry-Based Measurement of Sphingolipids

“…Direct infusion based shotgun lipidomics enables determination of the levels of many low-abundance sphingolipid metabolites e.g. ceramide-phosphates, sphinganine and sphingosinephosphates in a variety of biological samples [25,26]. In a recent study the determination of sphingosine levels in the lysosome was achieved using this method [25].…”

Direct quantitation of psychosine from alkaline-treated lipid extracts with a semi-synthetic internal standard

“…The solvent of the combined crude extract from each sample was evaporated under a nitrogen stream. The residue was treated with lithium methoxide (1 M) in MeOH (200 ml) as described previously (20). The reaction mixtures were quenched with water (2 ml) and washed with diethyl ether (2 ml) and hexane (2 ml 3 2).…”

“…This analytical platform has very recently been extended to the analyses of low-abundant lipid classes of the sphingolipidome after treatment of lipid extracts with lithium methoxide followed by cleaning and recovering of sphingolipidome through liquid-liquid partition (20). This approach has now been termed shotgun sphingolipidomics.…”

“…The levels of psychosine in multiple biological samples including cortex, cerebellum, spinal cord, and brain stem from diabetic mice and controls at different ages were determined by the developed methodology. With this extension, shotgun lipidomics not only allows us to analyze the major lipid classes, but also to analyze many of the minor lipid classes [e.g., psychosine, sphingoid-based-1-phosphates (21), sphingosine (20,22), and ceramide (23)] all from one lipid extract of a biological sample (e.g., approximately 10 mg tissue sample). Accordingly, this developed method, as a new addition to the shotgun lipidomics technology, will be extremely useful for understanding the pathways/networks of altered sphingolipid metabolism in a disease state (e.g., Alzheimerʼs disease) and for exploring the important roles of psychosine in a wide range of physiological and pathological conditions.…”