Institute of Clinical Chemistry, University Hospital Zurich, University of Zurich, Zurich, Switzerland.
Methods Mol Biol. 2025;2855:209-223. doi: 10.1007/978-1-0716-4116-3_14.
Sphingolipids (SLs) are essential lipids with important functions in membrane formation and cell signaling. The presence of a long chain base (LCB) structure is common to all SLs. De novo SL synthesis is initiated by the enzyme serine-palmitoyltransferase (SPT), which forms an LCB by the conjugation from serine and fatty acyl-CoAs. SPT can metabolize a variety of acyl-CoA substrates, which form diverse LCB structures within and across species. The LCB then undergoes further metabolic modifications resulting in an extraordinarily diverse spectrum of sphingolipids formed. SL analysis, using liquid chromatography-mass spectrometry (LC-MS)-based methods, poses challenges due to the diverse range of frequently isobaric species. This complexity complicates the identification of underlying LCB structures using standard lipidomics approaches. Here, we describe a simplified method to analyze the LCB profile in cells, tissue, and blood. The procedure involves chemical hydrolysis to remove the conjugated headgroups and N-acyl chains, allowing to specifically resolve the underlying LCB structures by LC-MS. This method can also be combined with an isotope labeling approach to determine in vivo SPT activity and total SL de novo synthesis over time.
鞘脂（SLs）是一类具有重要生物学功能的必需脂质，它们在膜形成和细胞信号转导中发挥作用。所有 SLs 都具有长链碱基（LCB）结构。从头合成 SL 的过程由丝氨酸棕榈酰转移酶（SPT）酶起始，该酶通过丝氨酸和脂肪酸辅酶 A 的缩合形成 LCB。SPT 可以代谢多种酰基辅酶 A 底物，从而在种内和种间形成不同的 LCB 结构。然后，LCB 进一步经历代谢修饰，从而形成具有极高多样性的鞘脂谱。使用基于液相色谱-质谱（LC-MS）的方法进行 SL 分析，由于经常存在等质异位的多种物质，因此具有挑战性。这种复杂性使得使用标准脂质组学方法来鉴定潜在的 LCB 结构变得复杂。在这里，我们描述了一种简化的方法来分析细胞、组织和血液中的 LCB 图谱。该方法涉及化学水解以去除共轭的头部基团和 N-酰基链，从而通过 LC-MS 特异性地解析潜在的 LCB 结构。该方法还可以与同位素标记方法结合使用，以确定体内 SPT 活性和总 SL 从头合成随时间的变化。
