TITLE: “Quantification of Absolute Metabolite Concentrations in T cells by Shotgun Metabolomics.”
ABSTRACT: Quantitative understanding of immunometabolism underlies improving immune functions and developing successful immunotherapies. Kinetic and thermodynamic laws rely on absolute, not relative, metabolite concentrations to map metabolism. However, until now, comprehensive absolute metabolite quantification has been inaccessible due to the need for iterative analytical procedures involving internal standards. Here we developed a simple technique to facilitate absolute metabolite quantitation. Shotgun metabolomics leverages the known absolute metabolite concentrations of model systems and 13C labeling to distinguish metabolites from two different cell types in a single extraction sample. We cultured T cells in unlabeled media and E. coli or epithelial cells in 13C-labeled media and extracted their metabolites simultaneously. Using LC-MS and known concentrations of 13C-labeled metabolites as internal standards, we quantified ~80 metabolites en masse in human T cells.