Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298.
Department of Biology, Virginia Commonwealth University, Richmond, VA 23284.
J Lipid Res. 2020 Jun;61(6):953-967. doi: 10.1194/jlr.D120000809. Epub 2020 Apr 27.
MS-assisted lipidomic tissue analysis is a valuable tool to assess sphingolipid metabolism dysfunction in disease. These analyses can reveal potential pharmacological targets or direct mechanistic studies to better understand the molecular underpinnings and influence of sphingolipid metabolism alterations on disease etiology. But procuring sufficient human tissues for adequately powered studies can be challenging. Therefore, biorepositories, which hold large collections of cryopreserved human tissues, are an ideal retrospective source of specimens. However, this resource has been vastly underutilized by lipid biologists, as the components of OCT compound used in cryopreservation are incompatible with MS analyses. Here, we report results indicating that OCT compound also interferes with protein quantification assays, and that the presence of OCT compound impacts the quantification of extracted sphingolipids by LC-ESI-MS/MS. We developed and validated a simple and inexpensive method that removes OCT compound from OCT compound-embedded tissues. Our results indicate that removal of OCT compound from cryopreserved tissues does not significantly affect the accuracy of sphingolipid measurements with LC-ESI-MS/MS. We used the validated method to analyze sphingolipid alterations in tumors compared with normal adjacent uninvolved lung tissues from individuals with lung cancer and to determine the long-term stability of sphingolipids in OCT compound-cryopreserved normal lung tissues. We show that lung cancer tumors have significantly altered sphingolipid profiles and that sphingolipids are stable for up to 16 years in OCT compound-cryopreserved normal lung tissues. This validated sphingolipidomic OCT compound-removal protocol should be a valuable addition to the lipid biologist's toolbox.
MS 辅助脂质组学组织分析是评估疾病中鞘脂代谢功能障碍的一种有价值的工具。这些分析可以揭示潜在的药理靶点或直接的机制研究，以更好地理解鞘脂代谢改变对疾病病因的分子基础和影响。但是，为了充分研究，获得足够的人类组织可能具有挑战性。因此，生物库是冷冻保存的人类组织的大型集合，是标本的理想回溯来源。然而，脂质生物学家对这一资源的利用还远远不够，因为冷冻保存中使用的 OCT 化合物的成分与 MS 分析不兼容。在这里，我们报告的结果表明，OCT 化合物还会干扰蛋白质定量分析，并且 OCT 化合物的存在会影响 LC-ESI-MS/MS 提取的鞘脂的定量。我们开发并验证了一种简单且廉价的方法，可以从 OCT 化合物包埋的组织中去除 OCT 化合物。我们的结果表明，从冷冻保存的组织中去除 OCT 化合物不会显著影响 LC-ESI-MS/MS 分析鞘脂的准确性。我们使用经过验证的方法分析了肺癌患者的肿瘤与正常相邻未受累肺组织之间的鞘脂变化，并确定了 OCT 化合物冷冻保存的正常肺组织中鞘脂的长期稳定性。我们表明，肺癌肿瘤的鞘脂谱发生了显著改变，并且 OCT 化合物冷冻保存的正常肺组织中的鞘脂在长达 16 年内保持稳定。这种经过验证的脂质组学 OCT 化合物去除方案应该是脂质生物学家工具包的有价值的补充。
