Isotope Fractionation during Gas Chromatography Can Enhance Mass Spectrometry-Based Measures of ²H-Labeling of Small...

Stable isotope tracers can be used to quantify the activity of metabolic pathways. Specifically, ²H-water is quite versatile, and its incorporation into various products can enable measurements of carbohydrate, lipid, protein and nucleic acid kinetics. However, since there are limits on how much ²H-water can be administered and since some metabolic processes may be slow, it is possible that one may be challenged with measuring small changes in isotopic enrichment. We demonstrate an advantage of the isotope fractionation that occurs during gas chromatography, namely, setting tightly bounded integration regions yields a powerful approach for determining isotope ratios. We determined how the degree of isotope fractionation, chromatographic peak width and mass spectrometer dwell time can increase the apparent isotope labeling. Relatively simple changes in the logic surrounding data acquisition and processing can enhance gas chromatography-mass spectrometry measures of low levels of ²H-labeling, this is especially useful when asymmetrical peaks are recorded at low signal:background. Although we have largely focused attention on alanine (which is of interest in studies of protein synthesis), it should be possible to extend the concepts to other analytes and/or hardware configurations.