SILAC and alternatives in studying cellular proteomes of plants

Quantitative proteomics by metabolic labeling has a high impact on the growing field of plant systems biology. SILAC has been pioneered and optimized for plant cell culture systems allowing for SILAC-based quantitative experiments in specialized experimental setups. In comparison to other model organisms, the application of SILAC to whole plants is challenging. As autotrophic organisms, plants under their natural growth conditions can hardly be fully labeled with stable isotope-coded amino acids. The metabolic labeling with inorganic nitrogen is therefore the method of choice for most whole-plant physiological questions. Plants can easily metabolize different inorganic nitrogen isotopes. The incorporation of the labeled inorganic nitrogen then results in proteins and metabolites with distinct molecular mass, which can be detected on a mass spectrometer. In comparative quantitative experiments, similarly as in SILAC experiments, treated and untreated samples are differentially labeled by nitrogen isotopes and jointly processed, thereby minimizing sample-to-sample variation. In recent years, heavy nitrogen labeling has become a widely used strategy in quantitative proteomics and novel approaches were developed for metabolite identification. Here we present a typical hydroponics setup, the workflow for processing of samples, mass spectrometry and data analysis for large-scale metabolic labeling experiments of whole plants.