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 (15)N-containing inorganic salts to label whole and mature plants. Employing (15)N salts as the sole nitrogen source for HILEP leads to the production of healthy-looking plants which contain (15)N proteins labeled to nearly 100%. Therefore, HILEP is suitable for quantitative plant proteomic analysis, where plants are grown in either (14)N- or (15)N-hydroponic media and pooled when the biological samples are collected for relative proteome quantitation. The pooled (14)N-/(15)N-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 (15)N salts and how to quantitate the resulting proteomes using a convenient workflow that does not require extensive bioinformatics skills.

The Arabidopsis B-box protein BZS1/BBX20 interacts with HY5 and mediates strigolactone regulation of photomorphogenesis

“…To generate 15 N-labeled seeds, Arabidopsis plants were grown hydroponically (Bindschedler et al, 2012) in diluted Hoagland solution (Hoagland’s No. 2 Basal Salt Mixture, Caisson Laboratories) containing 10 mM K 15 NO 3 (Cambridge Isotope Laboratories,USA).…”

Proteomics in Deciphering the Auxin Commitment in the Arabidopsis thaliana Root Growth

“…These features in fact have offered evident opportunities for global isotope-labeling procedures. Differently from animals, 14 N/ 15 N salts can be used to directly label plant proteomes under controlled growth conditions as in subtle modification of isotope ratio proteomics (SMIRP 67 ), in stable isotope labeling in planta (SILIP 68 ), and in hydroponic labeling of entire plants (HILEP 69 ) methods. A quantitative profiling of the Arabidopsis root proteome has been achieved in plants hydroponically grown and then subjected to heat stress.…”