Infinite re-reading of single proteins at single-amino-acid resolution using nanopore sequencing

Abstract

As identifying proteins is of paramount importance for cell biology and applications, it is of interest to develop a protein sequencer with the ultimate sensitivity of decoding individual proteins. Here, we demonstrate a nanopore-based single-molecule sequencing approach capable of reliably detecting single amino-acid substitutions within individual peptides. A peptide is linked to a DNA molecule that is pulled through the biological nanopore MspA by a DNA helicase in single amino-acid steps. The peptide sequence yields clear stepping ion current signals which allows to discriminate single-amino-acid substitutions in single reads. Molecular dynamics simulations show these signals to result from size exclusion and pore binding. Notably, we demonstrate the capability to 'rewind' peptide reads, obtaining indefinitely many independent reads of the same individual molecule, yielding virtually 100% read accuracy in variant identification, with an error rate less than 10-6. These proof-of-concept experiments constitute a promising basis for developing a single-molecule protein sequencer.

Mining folded proteomes in the era of accurate structure prediction

“…We next decided to perform these foldome-wide searches for several other pore-forming protein families, identifying new members of aerolysins, lysenins, cry1 toxins and more ( S1 Fig , S1 Table and File 1 in https://zenodo.org/record/5893808#.YiE_LOhKhPY ). Members of these toxin families have applications in next-generation sequencing (both DNA/RNA [ 22 , 23 ] and polypeptide [ 24 – 27 ]), as well as agricultural applications in crop protection. We anticipate the new members of these families to be of utility in translational research programs.…”

Challenges and approaches to studying pore-forming proteins

“…There has also been work to extend nanopore-based technology to various proteomic applications, including mass identification [117,123,141,142], peptide, and protein sequencing [143][144][145][146][147] (Figure 4). While nanopore-based proteomics promises extremely high sensitivity, and even the possibility of single-cell proteomics [31], sequencing polypeptides remains a frontier challenge in PFP engineering.…”

“…Another challenge in nanopore proteomics is that protein sequencing requires unfolding and regular ratcheting of the denatured polypeptide chain through the pore. Partnering pores with other enzymes, such as unfoldases, helicases, DNA polymerases, and proteasomes has allowed for polypeptide translocation [ 144 , 146 , 148 , 149 ]. However, there is currently an insufficient level of control over single-pass translocation for accurate residue assignment.…”

“…However, there is currently an insufficient level of control over single-pass translocation for accurate residue assignment. To address this, the use of a DNA-peptide conjugate enabled repeated re-reading of peptides, improving discrimination between a limited set of peptide variants [ 144 ]. While several challenges exist, proteomic nanopores could eventually be used on-bench to detect protein point variants, cleavage events, or post-translational modifications — all at the single-molecule level [ 123 ].…”

In silico assessment of a novel single-molecule protein fingerprinting method employing fragmentation and nanopore detection

“…Furthermore, no single pore aperture size is suitable for proteome-wide analysis due to the wide variety of protein sizes found in nature ( Brocchieri and Karlin, 2005 ). Alternatively, proteins may be unfolded and threaded single file through the pore using a molecular motor ( Brinkerhoff et al., 2021 ; Nivala et al., 2014 ). This approach allows for finer interrogation of the residue sequence and may analyze proteins of any size using a single pore aperture size.…”