Labeling Maize (Zea mays L.) Leaves with 15NH4+ and Monitoring Nitrogen Incorporation into Amino Acids by GC/MS Analysis

The human body contains approximately 3.2% nitrogen (N), mainly present as protein and amino acids. Although N exists at a high concentration (78%) in the air, it is not readily available to animals and most plants. Plants are however able to take up both nitrate (NO₃⁻) and ammonium (NH₄⁺) ions from the soil and convert them to amino acids and proteins, which are excellent sources for all animals. Most N is available as the stable isotope ¹⁴N, but a second form, ¹⁵N, is present in very low concentrations. ¹⁵N can be detected in extracts of plants by gas chromatography followed by mass spectrometry (GC/MS). In this protocol, the methods are described for tracing the pathway by which plants are able to take up ¹⁵N-labeled nitrate and ammonium and convert them into amino acids and proteins. A protocol for extracting and quantifying amino acids and ¹⁵N enrichment in maize (Zea mays L.) leaves labeled with ¹⁵NH₄⁺ is described. Following amino acid extraction, purification, and separation by GC/MS, a calculation of the ¹⁵N enrichment of each amino acid is carried out on a relative basis to identify any differences in the dynamics of amino acid accumulation. This will allow a study of the impact of genetic modifications or mutations on key reactions involved in primary nitrogen and carbon metabolism. © 2018 by John Wiley & Sons, Inc.