Obtaining Narrow Distributionsof Single-MoleculePeptide Signals Enables Sensitive Peptide Discrimination with α‑HemolysinNanopores

Biological nanopore technology has emerged as a promising tool for analyzing peptides and post-translational modifications at the single-molecule level. However, a broader application is currently limited by the partial separation of peptides and low-throughput, mainly due to the nonuniform peptide signals detected by nanopores. Narrowing the peptide signal distribution is crucial for improving the nanopore’s sensing ability but remains a bottleneck. Here, we demonstrate that capturing peptides with electrophoretic force against electroosmotic flow can provoke more uniform blockades in α-hemolysin nanopores. By using buffers with 2 M KCl at pH 3.8, we obtain the most uniform peptide signals, which may be correlated to the shape, linearization, and actual dwelling position of peptides. Five peptides with acetylation and phosphorylation, including isomeric peptides, can be readily separated from each other. The citrullination replacement of arginine and the β-hydroxybutyrylation modification in another peptide sequence are also discriminated in a mixture. A series of peptides with different compositions induced uniform peptide blockades when they were analyzed with our method. Our work presents an efficient approach to optimize nanopore signals for peptide analysis using α-hemolysin nanopores.