A framework for peptide identification on commercial nanopore sequencing platforms

Direct single-molecule peptide analysis could in principle enable rapid and sensitive identification of pathogen-derived or disease-associated biomarkers without reliance on mass spectrometry. However, existing nanopore peptide sensing methods are typically constrained by limited throughput and lack of accessibility beyond specialized setups. Here, we present an integrated experimental-computational framework for DNA-linked peptide translocation on a commercially available, high-throughput nanopore sequencing platform, the MinION. Synthetic peptides were covalently bound to oligonucleotides at both termini. The resulting peptide-DNA constructs were then translocated through the CsgG-CsgF pores using a DNA motor protein. Current traces were segmented using the known DNA sequences to extract peptide-associated signal regions. From these segments, we extracted signal features and trained feature-based and deep-learning classifiers to distinguish peptides, balancing interpretability and classification performance. We establish a framework for peptide identification using standard nanopore sequencing hardware. Across a diverse panel of synthetic peptides, our approach resolves single-amino-acid substitutions, maintains performance across independent sequencing runs, and correctly identifies peptides in blind mixtures. Interpretable model analyses connect classifier decisions and common errors to specific signal motifs. By combining commercially available instrumentation with a reproducible experimental and computational workflow, this framework lowers the barrier to nanopore-based proteomics and enables broader adoption across laboratories. It provides a foundation for future developments in amino acid modification detection and sequence analysis.