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. 2024:2816:25-33.
doi: 10.1007/978-1-0716-3902-3_3.

Optimized Lipidomics Extraction of Sphingosine and Sphinganine from Optic Nerve for Signaling Studies

Alexa Jauregui  1   2 , Emily J Neag  1   2   3 , Amr Almobayed  1   2 , Alexandra Lens  1   4 , Sanjoy K Bhattacharya  5   6
Affiliations

Optimized Lipidomics Extraction of Sphingosine and Sphinganine from Optic Nerve for Signaling Studies

Alexa Jauregui et al. Methods Mol Biol. 2024.

Abstract

Interconvertible sphingolipid metabolites represent germane constituents of eukaryotic membranes and are vital in the regulation of cellular homeostasis, proliferation, survival, and induction of autophagy. This protocol describes a step-by-step method for extractions of sphingosine and sphinganine from mammalian tissue samples, particularly from the murine optic nerve. These lipids are partitioned into a binary mixture of chloroform and methanol in a modified Bligh and Dyer method. This is followed with reverse phase ultrahigh-performance liquid chromatography fractionation with a C18+ column and subsequent tandem mass spectrometry (UHPLC-MS-MS) analysis of the biological abundance. These free sphingoid bases dissociate to form structurally distinctive carbocation product ions that can be confirmed with annotations of lipidomic databases or in-house fragmentation software.

Keywords: Bligh and Dyer; Imaging mass spectrometry; Lipidomics; Mass spectrometry; Optic nerve; Regeneration.

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