Department of Biology, Virginia Commonwealth University.
VCU Lipidomics/Metabolomics Shared Resource, Virginia Commonwealth University School of Medicine.
J Vis Exp. 2021 Apr 27(170). doi: 10.3791/62552.
Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.
鞘脂类是细胞成分，在人类代谢和疾病中有明确的作用。质谱可以用于确定鞘脂类在疾病中是否发生改变，并研究鞘脂类是否可以作为临床治疗靶点。然而，直接从手术室内获取组织的大规模前瞻性研究可能会耗费大量时间，在技术、物流和行政方面也极具挑战性。相比之下，回顾性研究可以利用组织生物库中已经冷冻保存的大量人类标本，通常标本数量庞大。从生物库获取组织的其他优势包括可以获得与组织标本相关的信息，包括组织学、病理学，在某些情况下还包括临床病理变量，所有这些都可以用于检查与脂质组学数据的相关性。然而，与冷冻保存和质谱分析不兼容的最佳切割温度化合物（OCT）相关的技术限制是分析脂质的技术障碍。然而，我们之前已经表明，通过多次洗涤和离心，可以轻松去除 OCT 中保存的人类生物库标本中的 OCT，而不会改变其鞘脂含量。我们还之前已经建立了 OCT 中冷冻保存的人类组织中的鞘脂在长达 16 年内是稳定的。在本报告中，我们概述了分析 OCT 包埋的人类组织样本中鞘脂的步骤和工作流程，包括洗涤组织、为数据归一化称重组织、提取脂质、为液相色谱电喷雾串联质谱分析（LC-ESI-MS/MS）制备样品、质谱数据集成、数据归一化和数据分析。
