Highlighting the tricarboxylic acid cycle: Liquid and gas chromatography–mass spectrometry analyses of 13C-labeled organic acids

The tricarboxylic acid (TCA) cycle is involved in the complete oxidation of organic acids to carbon dioxide in aerobic cells. It not only uses the acetyl-CoA derived from glycolysis but also uses breakdown products of proteins, fatty acids, and nucleic acids. Therefore, the TCA cycle involves numerous carbon fluxes through central metabolism to produce reductant power and transfer the generated electrons to the aerobic electron transport system where energy is formed by oxidative phosphorylation. Although the TCA cycle plays a crucial role in aerobic organisms and tissues, the lack of direct isotopic labeling information in its intermediates (organic acids) makes the quantification of its metabolic fluxes rather approximate. This is the major technical gap that this study intended to fill. In this work, we established and validated liquid and gas chromatography–mass spectrometry methods to determine 13C labeling in organic acids involved in the TCA cycle using scheduled multiple reaction monitoring and single ion monitoring modes, respectively. Labeled samples were generated using maize embryos cultured with [13C]glucose or [13C]glutamine. Once steady-state labeling was reached, 13C-labeled organic acids were extracted and purified. When applying our mass spectrometric methods to those extracts, mass isotopomer abundances of seven major organic acids were successfully determined.