Spike Signals and MD Simulations Reveal the Significance of Peptide Stretching in Nanopore Protein Sequencing.

Current nanopore protein sequencing methods lack detailed insights into the translocation dynamics of peptides within nanopores, impeding further advancements and optimizations. In this study, we have identified a unique set of spike signals that characterize the translocation process of a single-stranded DNA (ssDNA)-peptide conjugate through nanopores. We propose a spring oscillation model (which only exhibits contraction and stretch, not classic harmonic oscillation or vibration) to explain the generation of these spike signals and validate it through experiments involving base loss, the introduction of the C6 spacer in ssDNA, and molecular dynamics (MD) simulations. Additionally, the introduction of negatively charged amino acids to induce a stretching force stabilizes the peptide conformation, halting spring oscillations and restoring distinct step-like signals for amino acid identification. Our findings revealed the central difficulty to achieve single amino acid resolution in universal peptide sequencing, which can be fixed by stretching the peptides during translocation using additional field force, paving the way for enhancing high-resolution nanopore-based protein sequencing technologies.