High-resolution nanopore peptide sensing, profiling and sequence assembly.

Nanopores have been explored as a potential platform for protein analysis following the success of nanopore nucleic acid sequencing. However, protein sequencing remains technically challenging and has not yet been established for proteomics use. Here a nickel-immobilized Mycobacterium smegmatis porin A (MspA-NTA-Ni) nanopore is shown to enable the identification of a range of proteomic analytes, including amino acids and peptides up to 39 amino acids in length. Under identical conditions, signals corresponding to 20 proteinogenic amino acids, 4 post-translationally modified amino acids, 32 peptides, 6 modified peptides, 11 bioactive peptides and 2 neoantigen peptides were recorded. Machine-learning-based analysis enabled classification of these analytes with a validation accuracy of up to 97.4% within the studied dataset. The MspA-NTA-Ni nanopore supports both direct peptide identification and peptide profiling following enzymatic hydrolysis. As a proof of concept, a reference peptide was digested using exo- and endopeptidases to generate overlapping peptide fragments. Nanopore measurements combined with machine learning predictions enabled the identification of fragment compositions and partial sequences, allowing reconstruction of the original peptide sequence. This hydrolysis-based approach shows sensitivity to sequence alterations, including mutations, deletions and post-translational modifications, indicating potential utility for targeted peptide characterization.