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

Henry Brinkerhoff, Albert S. W. Kang, Jingqian Liu, Aleksei Aksimentiev, and Cees Dekker
Science 374 1509-1513 (2021)
DOI:10.1021/acs.nanolett.1c04203  BibTex

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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.

Abstract

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.

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Peptide in mixed D/E sequence (E, green; D, light blue) was threaded in a truncated MspA. The system was simulated under electrical bias of 200mV. Waters and ions are not shown.

Peptide with G substitution (E, green; D, light blue; G, blue) was threaded in a truncated MspA. The system was simulated under electrical bias of 200mV. Waters and ions are not shown.

Peptide with W substitution (E, green; D, light blue; W, red) was threaded in a truncated MspA. The system was simulated under electrical bias of 200mV. Waters and ions are not shown.