Extraction of Apoplastic Peptides for the Structural Elucidation of Mature Peptide Hormones in Arabidopsis

Abstract

Various secreted peptides, including peptide hormones, are present in the apoplast, but their biochemical characterization remains a challenge due to their low abundance, difficulty in extraction, and interference from numerous secondary metabolites. Here, we describe a simple and straightforward protocol for the extraction of apoplastic peptides with a high purity. This protocol takes advantage of the fact that apoplastic peptides diffuse and accumulate in the culture medium when Arabidopsis seedlings are subjected to whole-plant submerged culture. The peptides in the culture medium are efficiently recovered by o-chlorophenol extraction followed by acetone precipitation. The recovered peptides can be subjected to nano-liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS) without any additional clean-up. This procedure enables the structural elucidation of mature peptide hormones in the apoplast with the use of Arabidopsis plants that overexpress peptide hormone genes.