Digestion and Orthogonal Click Conjugation Enables Nanopore Reading of Proteins

Accurate characterization of bioactive peptides and proteins is needed to fully understand the origin of their activity. Nanopore reading enables polypeptide characterization at the single-molecule level. Here we describe a digestion-ligation method based on readily available reagents and benchtop equipment to convert peptides and proteins into DNA-peptide-DNA conjugates that can be analyzed by nanopores. Chymotrypsin digestion yields peptide fragments with C-terminal tyrosine residues suitable for oxidation-induced click chemistry. Subsequent selective installation of an azide on the N terminus enables installation of a template DNA strand needed for nanopore reading. The findings provide a facile approach to prepare DNA-peptide-DNA conjugates that has great potential for applicability to nanopore reading of many proteins.