Zero-Dalton Resolution in Nanopore Peptide Recognition.

Achieving zero-dalton resolution─the discrimination of biomolecules with identical mass─remains a fundamental challenge in molecular analysis. Nanopore sensing can resolve short peptide isomers, but whether discrimination persists when peptide length approaches the nanopore saturation limit is unclear. Here, we demonstrate robust nanopore-based discrimination of two 13-residue α-synuclein isomers using wild-type aerolysin at lengths approaching full nanopore sensing region occupancy. Isomer separation requires full peptide accommodation within the nanopore and arises from intrinsic structural differences amplified by ion-specific effects under nanoscale confinement. Complementary ion mobility-mass spectrometry and molecular simulations corroborate these intrinsic conformational distinctions under native conditions. The presence of salts strongly modulates peptide conformation; specifically, KCl and NaCl favor extended states, whereas LiCl and CaCl2 promote compact, folded conformations that amplify intrinsic structural differences, enabling near-perfect isomer separation (up to 100%). Remarkably, we demonstrate that isomer discrimination is insensitive to the order of pore entry, the specific conformations adopted (stretched, coiled, or globular), or whether peptides fully translocate or remain trapped. This robustness suggests that exploiting folded or partially folded peptide states─maximizing residue occupancy within the nanopore─may provide a path to extending zero-dalton discrimination to longer isomeric peptides. More broadly, this work outlines a strategy for advancing single-molecule proteomics and next-generation molecular fingerprinting, including applications to structurally complex biomolecules.