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

Abstract

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 (NO ) and ammonium (NH ) 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 N, but a second form, N, is present in very low concentrations. N 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 N-labeled nitrate and ammonium and convert them into amino acids and proteins. A protocol for extracting and quantifying amino acids and N enrichment in maize (Zea mays L.) leaves labeled with NH is described. Following amino acid extraction, purification, and separation by GC/MS, a calculation of the N 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.

Isotopic labelling reveals the efficient adaptation of wheat root TCA cycle flux modes to match carbon demand under ammonium nutrition

“…15 N analyses were carried out by GC-MS (436 GC-MS Scion; Bruker) according to the protocol described in Cukier et al . 61 . The quantification of 15 N enrichment (%) and the amount of each amino acid is based on the determination of the areas of characteristic peaks: The peak of mass M, corresponding to the unlabelled ( 14 N) amino acid, and the peaks of mass M + n, corresponding to labelled amino acids with n designing the number of 15 N atoms integrated either naturally or during the labelling experiment.…”

“…13 C analyses were performed by GC-MS (436 GC-MS Scion; Bruker) as described in Cukier et al . 61 for 15 N analyses. Importantly, for an optimal separation of derivatized 13 C compounds, the temperature of the oven was regulated according to the following program: 5 min at 70 °C followed by an increase of the temperature at 5 °C min −1 until reaching 300 °C and finally 5 min at 300 °C.…”

NADH-GOGAT Overexpression Does Not Improve Maize (Zea mays L.) Performance Even When Pyramiding with NAD-IDH, GDH and GS

“…These unexpected findings suggest that 2-Oxoglutarate could be used to assimilate ammonium in order to restore the homeostasis of glutamate and maintain the level of the organic acid to a physiological level comparable to that of the untransformed control plants. It would thus be interesting to verify experimentally this hypothesis by feeding the leaves of the GOGAT/IDH and GDH over-expressors with 15 N-ammonium [40] in order to verify if they are able to synthesize 15 N-glutamate.…”

Analysis of 18 Free Amino Acids in Honeybee and Bumblebee Honey from Eastern and Northern Europe and Central Asia Using HPLC-ESI-TQ-MS/MS Approach Bypassing Derivatization Step

“…Isolation and purification of AA from honey matrix were performed based on the protocol described by Cukier et al [ 21 ]. Briefly, 1.0 g honey with accuracy ± 0.01 g was transferred into 15.0 mL conical centrifuge tubes (Sarstedt AG & Co. KG, Nümbrecht, Germany), and 10.0 mL of H 2 O was added.…”