A quasi-digital approach to peptide sequencing using tandem nanopores with endo- and exo-peptidases

Abstract

Abstract.A method of sequencing peptides using tandem cells (RSC Adv., 2015, 5, 167-171; RSC Adv., 2015, 5, 30694-30700) and peptidases is considered. A double tandem cell (two tandem cells in tandem) has three nanopores in series, an amino-acid-specific endopeptidase attached downstream of the first pore, and an exopeptidase attached downstream of the second pore. The endopeptidase cleaves a peptide threaded through the first pore into fragments that are well separated in time. Fragments pass through the second pore and are each cleaved by the exopeptidase into a series of single residues; the latter pass through the third pore and cause separate current blockades that can be counted. This leads to an ordered list of integers corresponding to the number of residues in each fragment. With 20 cells, one per amino acid type, and 20 peptide copies, the resulting 20 lists of integers are used by a simple algorithm to assemble the sequence. This is a quasi-digital process that uses the lengths of subsequences to sequence the peptide, it differs from conventional analog methods which seek to identify monomers in a polymer via differences in blockade levels, residence times, or transverse currents. Several implementation issues are discussed. In particular the problem of fast analyte translocation, widely considered intransigent, may be resolved through the use of a sufficiently long (40-60 nm) third pore. This translates to a required bandwidth of 1-2 MHz, which is within the range of currently available CMOS circuits. IntroductionNanopores have been investigated over several years for their potential use in the analysis of polymers, in particular DNA [1]. There is now a growing interest in other kinds of polymers such as polyethylene glycol [2] and proteins/peptides [3,4]. Here a scheme for peptide sequencing is described that is based on a modified version of a tandem cell with cleaving enzymes that was proposed and studied earlier [5,6]. A tandem cell consists of two electrolytic cells joined in tandem and has the structure [cis1, membrane with upstream nanopore (UNP), trans1/cis2, membrane with downstream nanopore (DNP), trans2]. An enzyme attached to the downstream side of UNP successively cleaves the leading monomer in a polymer that has threaded through UNP; the cleaved monomer then translocates to and through DNP where the resulting ionic current blockade level (and/or other discriminators [6]) is used to identify it. With DNA the enzyme is an exonuclease [5], with peptides it is an amino or carboxy peptidase [6]. The approach does not use labels or immobilization.The present communication describes a method of peptide sequencing using a double tandem cell (two tandem cells connected in tandem), an endopeptidase to break the peptide into fragments, and an exopeptidase to obtain the lengths of the latter. With 20 such cells these length data are used by a simple algorithm to assemble the sequence.