GC-MS profiling of leukemia cells: an optimized preparation protocol for the intracellular metabolome

Abstract

Sample preparation is an important step in the untargeted cell metabolomics workflow. In this study, a gas chromatography-mass spectrometry (GC-MS)-based metabolome preparation protocol was optimized with the aim of achieving global, reliable and reproducible intracellular metabolite profiling of leukemia cells. We have evaluated and compared 8 sample preparation methods: 4 extraction solvents (i.e., pure methanol, aqueous acetonitrile, methanol/methanol/water and aqueous methanol) coupled with 2 common trimethylsilylation reagents (i.e., N,O-bis(trimethylsilyl)trifluoroacetamide and N-methyl-N-(trimethylsilyl)trifluoroacetamide). The extraction solvents and derivatization agents were evaluated for their suitability for leukemia cells using criteria based on the number of total detected metabolites, extraction efficiency and repeatability. Based on the overall performance, methanol/methanol/water was chosen as the optimal extraction solvent for covering the highest number of intracellular metabolites with the best reproducibility. The derivatization agent N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) proved to be more suitable for profiling of the leukemia intracellular metabolome with better repeatability than N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). This optimized method provides a robust, efficient and reproducible way to gather information on the metabolome of a leukemia cell. It also offers a solid basis to further understand the physiology of leukemia cells and unravel the mechanism of leukemia’s multidrug resistance from the perspective of metabolomics.