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

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

Brinkerhoff, H.D. (TU Delft BN/Cees Dekker Lab; Kavli institute of nanoscience Delft)
Kang, A.S.W. (TU Delft BN/Cees Dekker Lab; Kavli institute of nanoscience Delft)
Liu, Jingqian (University of Illinois at Urbana Champaign)
Aksimentiev, Aleksei (University of Illinois at Urbana Champaign)
Dekker, C. (TU Delft BN/Cees Dekker Lab; Kavli institute of nanoscience Delft)

2021

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 singlemolecule protein fingerprinting and analysis technology.

http://resolver.tudelft.nl/uuid:8d31363e-1576-466b-87ae-b692a6c05ab6

https://doi.org/10.1126/science.abl4381

0036-8075

Science, 374 (6574), 1509-1513

Institutional Repository

journal article

© 2021 H.D. Brinkerhoff, A.S.W. Kang, Jingqian Liu, Aleksei Aksimentiev, C. Dekker