Detection and Quantification of Methionine Turnover with a Deuterium Labeling Scheme
Authors: Liu Haiying 
Published: June 2021

BackgroundBesides being a key building block of proteins in the body, Methionine is involved in many important functions. As an essential amino acid in human and animals, it has to be supplemented via diet. Interestingly, unlike some other amino acids, its blood concentration is only about 30uM between meals. However, its basal turnover may not be as inert as it seems.ObjectivesTo establish a stable isotope labeling platform to evaluate body amino acid homeostasis, which can be effectively used to assess turnover of amino acids in vivo; To probe and characterize the methionine kinetics in mice with the platform.MethodsIn order to practically monitor methionine kinetics non‐invasively, a stable isotope labeling scheme of amino‐acids was developed and evaluated. Two groups of adult C57B6 mice were used and compared: a deuterium labeled (n=3) group and a control group (N=9). In the labeled group, mice were given 5% deuterated drinking water. Blood was collected from mice, from which plasma was obtained. After deleting proteins, plasma samples were analyzed with a LC‐MS/MS based method using a high‐resolution mass spectrometer (MS) (Thermo Scientific, Q Exactive). LC‐MS analysis was performed using a C18 column in reverse phase and positive mode optimized for quantifying blood methionine level as well as the fraction of deuterium labeling. The LC‐MS based method was developed to exam the deuterium enrichment levels in the end methyl hydrogens and methylene hydrogens. Two fragment ions of methionine at m/z of 56 and 104 was evaluated. Their respective M+1 peak intensity, normalized to its naturally abundant peak, was used as an indirect measure of deuterium incorporation (M=56 or 104). Furthermore, with high m/z resolution of the Q Exactive mass spectrometer(~140,000), we can directly quantify the relative contributions of 13C and deuterium to the M+1 peak.ResultsAfter the deuterium labeling period, deuterium enrichment in blood methionine hydrogens was detected and its fractional enrichment was found to be significantly higher in the labeling group than unlabeled group. Normalized intensities of M+1 peak for fragment ion 56 were found to be: 0.064 ± 0.037 and 0.040 ± 0.013 for labeled and unlabeled group respectively (p=0.00072). Relative to that of carbon‐13, deuterium enrichments in methionine hydrogens of fragment ion 104 were quantified to be: 1.136 ± 0.1528 and 0.006963 ± 0.001583 for labeled and unlabeled group respectively (p<0.018). Furthermore, when the end‐methyl hydrogens were evaluated, we noticed its deuterium enrichment exceeded 100x enrichment of the rest hydrogens in the methionine molecule.ConclusionsUsing the new platform and method described above, we were able to detect and evaluate deuterium enrichment in hydrogens of the methionine molecule in mice following a short period of deuterium labeling. The enrichment results reflected its involvement in methionine cycles, by which it was dynamically converted to SAMe (S−Adenosyl Methionine) and regenerated from homocysteine. Considering the platform can allow a quantitative assessment of the labeling kinetics in different parts of the molecule, the method can be used for estimating fluxes of these cycles. In future studies, we will utilize the same approach in our pharmacology studies to assess the kinetics of methionine with various interventions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.