In vivo uniform ¹⁵N‐isotope labelling of plants: Using the greenhouse for structural proteomics

Abstract

Isotope labelling of proteins is important for progress in the field of structural proteomics. It enables the utilisation of the power of nuclear magnetic resonance spectroscopy (NMR) for the characterisation of the three-dimensional structures and corresponding dynamical features of proteins. The usual approach to obtain isotopically labelled protein molecules is by expressing the corresponding gene in bacterial or yeast host organisms, which grow on isotope-enriched media. This method has several drawbacks. Here, we demonstrate that it is possible to fully label a plant with (15)N-isotopes. The advantage of in vivo labelling of higher organisms is that all constituting proteins are labelled and become available as functional, post-translationally modified, correctly folded proteins. A hydroponics set-up was used to create the first example of a uniformly (15)N-labelled (> 98%) plant species, the potato plant (Solanum tuberosum L., cv. Elkana). Two plants were grown at low costs using potassium-[(15)N]-nitrate as the sole nitrogen source. At harvest time, a total of 3.6 kg of potato tubers and 1.6 kg of foliage, stolons and roots were collected, all of which were fully (15)N-labelled. Gram quantities of soluble (15)N-labelled proteins (composed mainly of the glycoprotein patatin and Kunitz-type protease inhibitors) were isolated from the tubers. NMR results on the complete proteome of potato sap and on an isolated protease inhibitor illustrate the success of the labelling procedure. The presented method of isotope labelling is easily modified to label other plants. Its envisioned impact in the field of structural proteomics of plants is discussed.

Stable Isotope Labeling of Arabidopsis thaliana Cells and Quantitative Proteomics by Mass Spectrometry

“…This method has also been applied to label plants with 15 N for analysis of proteins by NMR (10). However, the mass shift introduced by the incorporated 15 N is different for each tryptic peptide depending on the length and amino acid composition.…”

Discovery and validation of colonic tumor-associated proteins via metabolic labeling and stable isotopic dilution

“…However, an isotopic label may be introduced instead by growth on isotopically enriched food or media. This process, metabolic labeling, often has been used with bacteria, yeast (11), cultured cells (12), and plants (13,14). Less frequently, it also has been applied in mammals (15)(16)(17).…”

Quantitative Proteomics in Plants: Choices in Abundance

“…By contrast with SILAC, metabolic labeling with 15 N has been shown to achieve .98% incorporation in both intact plants (Ippel et al, 2004) and cell cultures (Engelsberger et al, 2006) and is more cost effective than SILAC. The trade-off is that because all amino acids will incorporate the label, the mass difference between labeled and unlabeled peptides will vary depending on their sequence.…”