Quantification and Mass Isotopomer Profiling of α-Keto Acids in Central Carbon Metabolism

Mass spectrometry has been established as a powerful and versatile technique for studying cellular metabolism. Applications range from profiling of metabolites to accurate quantification and tracing of stable isotopes through the biochemical reaction network. Despite broad coverage of central carbon metabolism, most methods fail to provide accurate assessments of the alpha-keto acids oxaloacetic acid, pyruvate, and glyoxylate because these compounds are highly reactive and degraded during sample processing and mass spectrometric measurement. We present a derivatization procedure to chemically stabilize these compounds readily during quenching of cellular metabolism. Stable derivatives were analyzed by ultrahigh pressure liquid chromatography coupled tandem mass spectrometry to accurately quantify the abundance of alpha-keto acids in biological matrices. Eventually, we demonstrated that the developed protocol is suited to measure mass isotopomers of these alpha-keto acids in tracer studies with stable isotopes. In conclusion, the here described method fills one of the last technical gaps for metabolomics investigations of central carbon metabolism.