Analysis of Cell Metabolism Using LC-MS and Isotope Tracers

Abstract

Here we discuss our methods to analyze small polar compounds involved in central carbon metabolism using LC-MS. Methods described include sample extraction procedures for cells and medium, as well as for plasma/serum, urine, CSF, and tissue samples. Different extraction solvents are assessed. Our methods for using (13)C stable isotope tracers to examine the kinetics and distributions of mass isotopologues of many metabolites are discussed. Quantification methods are described for (13)C stable isotope tracer experiments as well as for unlabeled experiments. These methods were applied in a fumarate hydratase deficient cell model to show how isotope tracing can demonstrate shifts in metabolic pathways and, together with metabolite exchange rates, can be used to gain insights into changes in cell metabolism.

Keywords: Exactive and Q-Exactive mass spectrometry; Fumarate hydratase; HILIC chromatography; LC-MS; Metabolite extraction; Metabolite quantification; Stable isotope tracers.