Hydroponic Isotope Labeling of Entire Plants and High-Performance Mass Spectrometry for Quantitative Plant Proteomics

Abstract

Hydroponic isotope labeling of entire plants (HILEP) combines hydroponic plant cultivation and metabolic labeling with stable isotopes using ¹⁵N-containing inorganic salts to label whole and mature plants. Employing ¹⁵N salts as the sole nitrogen source for HILEP leads to the production of healthy-looking plants which contain ¹⁵N proteins labeled to nearly 100%. Therefore, HILEP is suitable for quantitative plant proteomic analysis, where plants are grown in either ¹⁴N- or ¹⁵N-hydroponic media and pooled when the biological samples are collected for relative proteome quantitation. The pooled ¹⁴N-/¹⁵N-protein extracts can be fractionated in any suitable way and digested with a protease for shotgun proteomics, using typically reverse phase liquid chromatography nanoelectrospray ionization tandem mass spectrometry (RPLC-nESI-MS/MS). Best results were obtained with a hybrid ion trap/FT-MS mass spectrometer, combining high mass accuracy and sensitivity for the MS data acquisition with speed and high-throughput MS/MS data acquisition, increasing the number of proteins identified and quantified and improving protein quantitation. Peak processing and picking from raw MS data files, protein identification, and quantitation were performed in a highly automated way using integrated MS data analysis software with minimum manual intervention, thus easing the analytical workflow. In this methodology paper, we describe how to grow Arabidopsis plants hydroponically for isotope labeling using ¹⁵N salts and how to quantitate the resulting proteomes using a convenient workflow that does not require extensive bioinformatics skills.