Peptide sequencing based on host-guest interaction-assisted nanopore sensing

Direct protein sequencing technologies with improved sensitivity and throughput are still needed. Here, we propose an alternative method for peptide sequencing based on enzymatic cleavage and host-guest interaction-assisted nanopore sensing. We serendipitously discovered that the identity of any proteinogenic amino acid in a particular position of a phenylalanine-containing peptide could be determined via current blockage during translocation of the peptide through alpha-hemolysin nanopores in the presence of cucurbit[7]uril. Building upon this, we further present a proof-of-concept demonstration of peptide sequencing by sequentially cleaving off amino acids from C terminus of a peptide with carboxypeptidases, and then determining their identities and sequence with a peptide probe in nanopore. With future optimization, our results point to a different way of nanopore-based protein sequencing. A phenylalanine-containing peptide probe can be used for discriminating all 20 amino acids via current blockage during translocation through an alpha-hemolysin (alpha HL) nanopore. The paper provides proof-of-concept peptide sequencing demonstrations.