Bascom Palmer Eye Institute, Miller School of Medicine at University of Miami, Miami, FL, USA.
Miami Integrative Metabolomics Research Center, Miami, FL, USA.
Methods Mol Biol. 2024;2816:25-33. doi: 10.1007/978-1-0716-3902-3_3.
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.
可相互转化的神经酰胺代谢物是真核细胞膜的重要组成部分，对细胞内稳态、增殖、存活和自噬的诱导的调节至关重要。本方案描述了从哺乳动物组织样品（特别是从鼠视神经）中提取神经醇和神经氨酸的分步方法。这些脂质在改良的 Bligh 和 Dyer 法中分配到氯仿和甲醇的二元混合物中。然后进行反相超高效液相色谱（UHPLC）分离，采用 C18+柱，随后对生物丰度进行串联质谱（UHPLC-MS-MS）分析。这些游离的神经鞘碱基解离形成结构独特的碳正离子产物离子，可以通过脂质组学数据库的注释或内部片段化软件进行确认。
