Cai Tanxi, Shu Qingbo, Liu Peibin, Niu Lili, Guo Xiaojing, Ding Xiang, Xue Peng, Xie Zhensheng, Wang Jifeng, Zhu Nali, Wu Peng, Niu Lili, Yang Fuquan
Laboratory of Protein and Peptide Pharmaceuticals and Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Laboratory of Protein and Peptide Pharmaceuticals and Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China University of Chinese Academy of Sciences, Beijing 100049, China.
J Lipid Res. 2016 Mar;57(3):388-97. doi: 10.1194/jlr.M063024. Epub 2016 Jan 5.
Phospholipids (PLs), one of the lipid categories, are not only the primary building blocks of cellular membranes, but also can be split to produce products that function as second messengers in signal transduction and play a pivotal role in numerous cellular processes, including cell growth, survival, and motility. Here, we present an integrated novel method that combines a fast and robust TMS-diazomethane-based phosphate derivatization and isotopic labeling strategy, which enables simultaneous profiling and relative quantification of PLs from biological samples. Our results showed that phosphate methylation allows fast and sensitive identification of the six major PL classes, including their lysophospholipid counterparts, under positive ionization mode. The isotopic labeling of endogenous PLs was achieved by deuterated diazomethane, which was generated through acid-catalyzed hydrogen/deuterium (H/D) exchange and methanolysis of TMS-diazomethane during the process of phosphate derivatization. The measured H/D ratios of unlabeled and labeled PLs, which were mixed in known proportions, indicated that the isotopic labeling strategy is capable of providing relative quantitation with adequate accuracy, reproducibility, and a coefficient of variation of 9.1%, on average. This novel method offers unique advantages over existing approaches and presents a powerful tool for research of PL metabolism and signaling.
磷脂（PLs）是脂质类别之一，不仅是细胞膜的主要组成部分，还可分解产生在信号转导中充当第二信使的产物，并在众多细胞过程中发挥关键作用，包括细胞生长、存活和运动。在此，我们提出一种综合的新方法，该方法结合了基于快速且稳健的TMS - 重氮甲烷的磷酸盐衍生化和同位素标记策略，能够同时对生物样品中的磷脂进行谱分析和相对定量。我们的结果表明，在正离子模式下，磷酸盐甲基化能够快速且灵敏地鉴定六种主要的磷脂类别，包括它们的溶血磷脂对应物。内源性磷脂的同位素标记通过氘代重氮甲烷实现，其在磷酸盐衍生化过程中由酸催化的氢/氘（H/D）交换以及TMS - 重氮甲烷的甲醇解反应生成。按已知比例混合的未标记和标记磷脂的测量H/D比值表明，同位素标记策略能够以足够的准确性、可重复性提供相对定量，平均变异系数为9.1%。这种新方法相对于现有方法具有独特优势，为磷脂代谢和信号传导研究提供了强大工具。
