PT - JOURNAL ARTICLE AU - G. Sampath TI - A digital approach to protein identification and quantification using tandem nanopores and peptidases and search through a proteome database AID - 10.1101/024158 DP - 2015 Jan 01 TA - bioRxiv PG - 024158 4099 - http://biorxiv.org/content/early/2015/08/07/024158.short 4100 - http://biorxiv.org/content/early/2015/08/07/024158.full AB - A single-molecule method of identifying proteins based on electrical measurements and database search without labels or immobilization is considered. It uses electrolytic cells with two or three nanopores in tandem and one or two peptidases covalently attached to the trans side of a pore. An unknown protein is digested into peptides ending in a known amino acid; the peptides enter the cell, pass through the first pore, and are fragmented by a high-specificity endopeptidase. The second enzyme, if present, is an exopeptidase that cleaves the fragments into residues after the second pore. Level transitions in a blockade pulse due to the pore ionic current or transverse current pulse caused by a fragment in the second pore or individual such pulses caused by single residues in the third pore are counted. N residue-specific cells produce N integer lists from which a partial sequence is assembled. Search through the Uniprot database shows that for small N (3 to 5) over 98% of proteins in the human proteome can be identified from such sequences. A Fokker-Planck model is used to derive minimum enzyme turnover intervals required for correct sequencing. With thick (80-100 nm) pores the pulse width is ∼1 μs/residue, which is within the capability of CMOS detector circuits. If digested peptides are assumed to enter a cell in random order then over a long run the quantity of a protein in a mixture of proteins can be estimated from the number of its identifying peptides.