Single-molecule Protein Analysis Based on Engineered Nanopores

Human genes can generate millions of proteins due to biological processes such as post-translational modification. Among these, the sequences of low-abundance proteins are difficult to be predicted or measured directly, remaining uncharacterized as “dark matter”. Currently, the gold standard method for de novo protein sequencing is based on mass spectrometry, which is limited by low throughput, short read lengths, and inability to achieve single-molecule resolution. Nanopore technologies have emerged as powerful tools to develop single-molecule protein sequencing. In this study, a nanopore sensor is used for label-free detecting 20 amino acids at the single-molecule resolution. By utilizing exopeptidases, peptides can be cleaved sequentially to amino acids. Then, the released amino acids can be captured and detected by nanopore sensor. Assisted by artificial intelligence algorithms, the identification of amino acid signals and the recovery of sequences can be implemented. Furthermore, by integrating biological nanopore into solid-state nanopore, the stability of the sequencing device can be greatly improved. The research above is expected to establish single-molecule denovo peptide sequencing technique, driving advancements in proteomics, biomarker discovery, and antibody development.