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

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.

Fig. 1:. Reading peptides with a nanopore.

(A) The DNA-peptide conjugate consists of a peptide (pink) attached via a click linker (green) to an ssDNA strand (black). This DNA-peptide conjugate is extended with a typical nanopore adaptor comprised of an extender that acts as a site for helicase loading (blue) and a complementary oligo with a 3’ cholesterol modification (gold). (B) The cholesterol associates with the bilayer as shown in (a), increasing the concentration of analyte near the pore. The complementary oligo blocks the helicase, until it is pulled into the pore (b), causing the complementary strand to be sheared off (c), whereupon the helicase starts to step along DNA. (C) As the helicase walks along the DNA, it pulls it up through the pore, resulting in (a) a read of the DNA portion followed by (b) a read of the attached peptide. (D) Typical nanopore read of a DNA-peptide conjugate (black), displaying step-like ion currents (identified in red). The asterisks * indicate a spurious level not observed in most reads and therefore omitted from further analysis. The dagger † indicates a helicase backstep. (E) Consensus sequence of ion current steps (red), which for the DNA section is closely matched by the predicted DNA sequence (blue). The linker and peptide sections are identified by counting half-nucleotide steps over the known structural length of the linker. Error bars in the measured ion current levels are errors in the mean value, often too small to see. Error bars in the prediction are standard deviations of the ion current levels that were used to build the predictive map in previous work(19).

Fig. 2:. Detection of single amino acid substitutions in single peptides.

(A) Consensus ion current sequences for each of the three measured variants (D, gold; W, red; G, blue), which differ significantly at the site of the amino acid substitution. (B) Difference in ion current between the W (red) and G (blue) variants and the D variant. Error bars are standard deviations. (C) Confusion matrix showing error modes of a blind classifier in identifying variants of reads, demonstrating an 87% single-read accuracy. (D) All-atom model where a reduced-length MspA pore (grey) confines a polypeptide chain (Glu: green, Asp: light blue; Cys: beige). The top end of the peptide is anchored using a harmonic spring potential, representing the action of the helicase at the rim of a full-length MspA. Water and ions are shown as semitransparent surface and spheres, respectively. (E) Top: Ionic current in MspA constriction versus z coordinate of the mutated residue backbone from MD simulations. Bottom: Fraction of nanopore construction volume available for ion transport. Vertical and horizontal error bars denote standard errors and standard deviations, respectively. (G,H) Representative molecular configurations observed in MD simulations of peptide variants. Glycine and tryptophane residues are shown in dark blue and red, respectively. Significant peptide/pore surface interactions are observed.

Fig. 3:. Re-reading of a single peptide.

(A) Highly repetitive ion current signal corresponding to numerous re-reads of the same section of an individual peptide (in this case, the G-substituted variant). The expanded plot below shows a region that contains four rewinding events (red dashed lines), where the trace jumps back to level 52 ± 2 of the consensus displayed in Fig. 2A. (B) Re-reading is facilitated by helicase queueing, where (a) a second helicase binds behind the primary helicase that controls the DNA-peptide conjugate, re-reading starts when (b) the primary helicase dissociates, and (c) the secondary one becomes the primary helicase that drives a new round of reading. (C) By using information from multiple re-reads of the same peptide, the identification accuracy can be raised to very high levels of fidelity. These results indicate that with sufficient numbers of re-reads, random error can be eliminated and single-molecule error rate can be pushed lower than 1 in 10⁶ even with poor single-pass accuracy. Inset is a logarithmic plot of the error rate = 1 - accuracy.