Wang Ni, Shi Xiaoxiao, Zhang Chao, Zhou Wenwu, Zhu Zengrong
Institute of Insect Sciences, Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, State Key Laboratory of Rice Biology, Hangzhou 310058, China.
Hainan Research Institute, Zhejiang University, Sanya 572000, China.
Bio Protoc. 2021 Feb 20;11(4):e3923. doi: 10.21769/BioProtoc.3923.
Sphingolipids are major structural components of endomembranes and have also been described as an intracellular second messenger involved in various biological functions in all eukaryotes and a few prokaryotes. Ceramides (Cer), the central molecules of sphingolipids, have been depicted in cell growth arrest, cell differentiation, and apoptosis. With the development of lipidomics, the identification of ceramides has been analyzed in many species, mostly in model insects. However, there is still a lack of research in non-model organisms. Here we describe a relatively simple and sensitive method for the extraction, identification, and quantification of ceramides in Hemiptera Insects (brown planthooper), followed by Ultra-Performance Liquid Chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). C18 is used as the separation column for quantitative detection and analysis on the triple quadruple liquid mass spectrometer. In this protocol, the standard curve method is adopted to confirm the more accurate quantification of ceramides based on the optional detection conditions.
鞘脂是内膜的主要结构成分，也被描述为一种细胞内第二信使，参与所有真核生物和少数原核生物的各种生物学功能。神经酰胺（Cer）作为鞘脂的核心分子，已被证明与细胞生长停滞、细胞分化和细胞凋亡有关。随着脂质组学的发展，许多物种中都对神经酰胺进行了鉴定分析，其中大部分是在模式昆虫中。然而，非模式生物中的相关研究仍然较少。在此，我们描述了一种相对简单且灵敏的方法，用于半翅目昆虫（褐飞虱）中神经酰胺的提取、鉴定和定量分析，该方法随后采用超高效液相色谱-串联质谱联用技术（UPLC-MS/MS）。使用C18作为分离柱，在三重四极杆液相质谱仪上进行定量检测和分析。在本实验方案中，采用标准曲线法，基于可选的检测条件来更准确地定量神经酰胺。
