Optimization of lipid extraction and analytical protocols for UHPLC-ESI-HRMS-based lipidomic analysis of adherent mammalian cancer cells

Lipidomics, which reveals comprehensive characterization of molecular lipids, is a rapidly growing technology used in biomedical research. Lipid extraction is a critical step in lipidomic analysis. However, the effectiveness of different lipid extract solvent systems from cellular samples still remains unclear. In the current study, the protocol of reverse-phase liquid chromatography mass spectrometry (LC/MS)-based lipidomics was optimized for extraction and detection of lipids from human pancreatic cancer cell line PANC-1. Four different extraction methods were compared, including methanol/methyl-tert-butyl ether (MTBE)/H2O, methanol/chloroform, methanol/MTBE/chloroform, and hexane/isopropanol. Data were acquired using high-resolution mass spectrometry in positive and negative ion modes respectively. The number of total detected and identified lipids was assessed with the aid of automated lipid identification software LipidSearch. Results demonstrated that methanol/MTBE/H2O provided a better extraction efficiency for different lipid classes, which was chosen as the optimized extraction solvent system. This validated method enables highly sensitive and reproducible analysis for a variety of cellular lipids, which was further applied to an untargeted lipidomic study on human pancreatic cancer PANC-1 cell lines. Moreover, this optimized extraction solvent system can be further applied to other cancer cell lines with similar chemical and physical properties.