Multiple rereads of single proteins at single-amino acid resolution using nanopores

A proteomics tool capable of identifying single proteins would be important for cell biology research and applications. Here, we demonstrate a nanopore-based single-molecule peptide reader sensitive to single-amino acid substitutions within individual peptides. A DNA-peptide conjugate was pulled through the biological nanopore MspA by the DNA helicase Hel308. Reading the ion current signal through the nanopore enabled discrimination of single-amino acid substitutions in single reads. Molecular dynamics simulations showed these signals to result from size exclusion and pore binding. We also demonstrate the capability to rewind peptide reads, obtaining numerous independent reads of the same molecule, yielding an error rate of <10(-6) in single amino acid variant identification. These proof-of-concept experiments constitute a promising basis for the development of a single-molecule protein fingerprinting and analysis technology.

Automated summary

This study presents a nanopore-based single-molecule peptide reader capable of identifying single amino acid substitutions within individual peptides with high accuracy. By pulling a DNA-peptide conjugate through a biological nanopore and reading the ion current signal, discrimination of single amino acid substitutions in single reads was achieved. The ability to obtain numerous independent reads of the same molecule and achieve a low error rate in amino acid variant identification signifies a promising advancement towards single-molecule protein fingerprinting and analysis technology.