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

• Published in: Science (American Association for the Advancement of Science) Vol. 374; no. 6574; pp. 1509 - 1513
• Main Authors: Brinkerhoff, Henry, Kang, Albert S W, Liu, Jingqian, Aksimentiev, Aleksei, Dekker, Cees
• Format: Journal Article
• Language: English
• Published: United States The American Association for the Advancement of Science 17.12.2021
• Subjects: Amino acid sequence; Amino Acid Substitution; Amino acids; Amino Acids - chemistry; Cytology; Deoxyribonucleic acid; DNA; DNA helicase; DNA Helicases; Fingerprinting; Ion currents; Molecular dynamics; Molecular Dynamics Simulation; Nanopores; Nucleotides; Peptide mapping; Peptides; Peptides - chemistry; Proteins; Proteomics; Proteomics - methods; Sequence Analysis, Protein; Technology assessment
• ISSN: 0036-8075; 1095-9203
• DOI: 10.1126/science.abl4381

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 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.