NMR-based metabolite studies with 15N amino acids

Abstract

¹⁵N labeled amino acids are routinely used to label proteins or nucleic acids for study by NMR. However, NMR studies of ¹⁵N labeled amino acids in metabolite studies have not been pursued extensively, presumably due to line broadening present under standard experimental conditions. In this work, we show that lowering the temperature to −5 °C allows facile characterization of ¹⁵N-labeled amino acids. Further, we show that this technique can be exploited to measure ¹⁵NH₃ produced in an enzyme catalyzed reaction and the transport and metabolism of individual amino acids in mammalian cell culture. With respect to ¹³C-labeled amino acids, ¹⁵N-labeled amino acids are less costly and enable direct characterization of nitrogen metabolism in complex biological systems by NMR. In summary, the present work significantly expands the metabolite pools and their reactions for study by NMR.