Resolving sulfation PTMs on a plant peptide hormone using nanopore sequencing

Peptide phytohormones are decorated with post-translational modifications (PTMs) that are crucial for receptor recognition. Tyrosine sulfation on these hormones is essential for plant growth and development 1 . Measuring the occurrence and position of sulfotyrosine is, however, compromised by major technical challenges during isolation and detection 2 . We recently introduced a nanopore peptide sequencing method that sensitively detects PTMs at the single-molecule level 3 . By translocating PTM variants of the plant pentapeptide hormone phytosulfokine (PSK) through a nanopore, we here demonstrate accurate identification of sulfation and phosphorylation on the two tyrosine residues of PSK. Sulfation can be clearly detected and distinguished (>90%) from phosphorylation on the same residue. Moreover, the presence or absence of PTMs on the two close-by tyrosine residues can be accurately determined (>96% accuracy). Our findings demonstrate the extraordinary sensitivity of nanopore protein measurements, providing a new tool for identifying sulfation on peptide phytohormones and promising wider applications to identify protein PTMs.