Tracing 15N with chemical reaction interface mass spectrometry: a demonstration using 15N-labeled glutamine and asparagine substrates in cell culture.
This research demonstrates how the chemical reaction interface mass spectrometry (CRIMS) approach works for a study of amino acid metabolism in cell culture. 15N-selective chromatograms from both the culture medium and the cytosol of human hepatoma Hep G2 cells that were incubated in the presence of either 12 mM (alpha-15N)glutamine or (alpha-15N)asparagine have been produced. The time course of the distribution of 15N among different amino acids, as well as the enrichment for each amino acid, were observed over a 144 h period. Labeled glutamine was quickly converted into glutamate. After 144 h of incubation, the total amount of 15N was distributed primarily among alanine (50%), proline (28%) and glutamate (21%). The 15N enrichment of alanine and proline reached 44% and 41% respectively. Asparagine was only slowly metabolized by the cells. In addition to the 82% that was retained in asparagine, the remaining 15N in the media at 144 h was found primarily in alanine (8%), glutamate (6.8%) and proline (2.2%). Their enrichments were 20%, 36% and 19% respectively. The minimum detectable amount was 17 pg of 15N entering the CRI. CRIMS appears to be a powerful, facile approach for 15N-tracer experiments.
Kusmierz
J J
JJ
Department of Pharmacology, George Washington University, Washington, DC 20037.
Abramson
F P
FP
D001216:Asparagine / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D003470:Culture Media; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism; D009587:Nitrogen Isotopes; D014407:Tumor Cells, Cultured
D003470:Culture Media; D009587:Nitrogen Isotopes; D005973:Glutamine; D001216:Asparagine