Discriminating single-amino acid differences and post-translational modifications in negatively charged peptides with α-hemolysin nanopores

Effective and precise detection of peptides and their post-translational modifications (PTMs) is crucial for biomedical research but remains technically challenging. Here, we present a nanopore sensing strategy that enables label-free analysis of negatively charged peptides. By introducing arginine residues into the nanopore and using amino-cyclodextrin as a molecular adaptor, the system enables unambiguous discrimination of short negatively charged homopeptides differing by only a single residue. Building on this capability, the platform further demonstrates high sensitivity for detecting representative PTMs, including acetylation, lactylation, and phosphorylation, introduced on a negatively charged peptide scaffold. Notably, the system could also analyze fragments generated by proteolytic digestion of full-length proteins, underscoring its potential for accurate protein identification. This work offers an accessible and reliable strategy to characterize peptide variants with single-amino acid differences and PTMs, which may facilitate future applications in peptide biomarker detection and protein sequence analysis.