Characterization of oligopeptides in Solanum lycopersicum xylem exudates

We collected 120 ml of xylem exudate from the cut surface of the first internodes from the hypocotyls of the tomato plants at 45 DAG for five hours. The exudates were purified using the gel-free method that we developed previously (Okamoto et al., 2015, Plant J.). Using gel filtration chromatography (Superdex Peptide 10/300 GL gel filtration column (10 mm  300 mm; GE Healthcare)), we separated small-protein-containing fractions (small-protein fractions) and oligopeptide-containing fractions (peptide fractions). The small-protein fractions were treated with trypsin, and the peptide fractions were further purified with multimode chromatography (Asahipak GS-320 column (7.5 mm  300 mm; Shodex)). Then, data-dependent MS/MS (ddMS/MS) was performed using an EASY-nLC 1000 (Thermo Fisher Scientific) connected to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific).