Peptide sequencing in an electrolytic cell with two nanopores in tandem and exopeptidase

Abstract

A nanopore-based approach to peptide sequencing without labels or immobilization is considered. It is based on a tandem cell (RSC Adv., 2015, 5, 167-171) with the structure [cis1, upstream pore (UNP), trans1/cis2, downstream pore (DNP), trans2]. An amino or carboxyl exopeptidase attached to the downstream side of UNP cleaves successive leading residues in a peptide threading from cis1 through UNP. A cleaved residue translocates to and through DNP where it is identified. A Fokker-Planck model is used to compute translocation statistics for each amino acid type. Multiple discriminators, including a variant of the current blockade level and translocation times through trans1/cis2 and DNP, identify a residue. Calculations show the 20 amino acids to be grouped by charge (+, -, neutral) and ordered within each group (which makes error correction easier). The minimum cleaving interval required of the exopeptidase, the sample size (number of copies of the peptide to sequence or runs with one copy) to identify a residue with a given confidence level, and confidence levels for a given sample size are calculated. The results suggest that if the exopeptidase cleaves each and every residue and does so in a reasonable time, peptide sequencing with acceptable (and correctable) errors may be feasible. If validated experimentally the proposed device could be an alternative to mass spectrometry and gel electrophoresis. Implementation-related issues are discussed.