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

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.