Author contributions A.O. conceived the project and supervised all ionic current measurements. A.A. conceived and supervised the modeling part of the project and contributed to design of experiments. H.O. carried out experiments and performed data analysis. K.S. performed all MD simulations and SEM calculations and developed the theoretical model. F.P. developed data analysis methods and applied it to experiment results. J.P. contributed to design of the project, suggested the experiment to split suspected cysteine dimers using dithiothreitol, participated to data interpretation and in the general discussion. P.M. participated to the project discussion, suggested an interpretation for the two peaks found for proline and participated in the general discussion and data interpretation, write a draft to answer the referee questions. T.E. with H.O. performed experiments on the high-resolution set-up and analyzed data. J.C.B. conceived and supervised high-resolution recordings, contributed software for data analysis of complex resistive pulses, analyzed data, prepared Supplementary Figs. 12-15 and wrote Supplementary Note 3 and the pertinent part of the Online Methods. A.A. and A.O. wrote the first draft of the manuscript.

There are still unmet needs in finding new technologies for biomedical diagnostic and industrial applications. A technology allowing the analysis of size and sequence of short peptide molecules of only few molecular copies is still challenging. The fast, low-cost and label-free single-molecule nanopore technology could be an alternative for addressing these critical issues. Here, we demonstrate that the wild-type aerolysin nanopore enables the size-discrimination of several short uniformly charged homopeptides, mixed in solution, with a single amino acid resolution. Our system is very sensitive, allowing detecting and characterizing a few dozens of peptide impurities in a high purity commercial peptide sample, while conventional analysis techniques fail to do so.