Differential Isotope Labeling by Permethylation and Reversed-Phase Liquid Chromatography-Mass Spectrometry for Relative Quantification of Intact Neutral Glycolipids in Mammalian Cells

Probing the role of glycolipids in health and disease warrants development of practical strategies to determine these molecules at the intact structural level, namely to simultaneously characterize and quantify the glycan and lipid moieties without breaking the linkage between them. Herein we present such an approach utilizing differential isotope labeling and reversed phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) for structural characterization and relative quantification of intact neutral glycolipids. In this approach, each individual sample and a pooled aliquot of each sample were permethylated using ¹²CH₃I and ¹³CH₃I, respectively, with the latter one serving as internal reference standard. The individual ¹²C-permethylated samples were spiked with equal amounts of the ¹³C-permethylated pooled sample and analyzed by RPLC-MS/MS. Permethylation not only increased the ionization efficiency of glycolipids but also facilitated structural characterization of both moieties. The ratio of the peak areas between the ¹²C- and ¹³C-labeled glycolipids served as surrogate measure of their relative concentrations. The coefficient of variation of the method was <6% measured across four representative glycolipids in five different ratios and triplicate experiments, after correction of natural isotopic distribution. When analyzing the low abundant glycolipids in total lipid extract, permethylation can dramatically reduce the analytical background by depleting most of the highly abundant ester-linked lipids. Application to conduritol B epoxide-, a β-glucocerebrosidase inhibitor, treated RAW 264.7 cells demonstrated the practical utility of this method in profiling the temporal accumulation of different glycolipids. Overall, this methodology offers a practical LC-MS based identification and quantification strategy to advance intact glycolipids analysis in mammalian cells.