Sequential reading of a stepwise-shortened peptide immobilized on nanopore.

Nanopore-based peptide sequencing achieves single-amino-acid resolution with 1-residue stepwise shortening using engineered MspA nanopores and peptide looping.

• Why it matters: Decoding peptide sequences accurately is essential for proteomics but remains challenging due to peptide complexity and structural variability, limiting current methods.
• What they did: Researchers developed the tPAL strategy with dual-modified MspA nanopores and cholesterolized aminopeptidase to sequentially shorten immobilized peptides by single amino acids, generating consistent, sequence-dependent signals.
• The result: This approach enables precise peptide sequence decoding, including detection of mutations, modifications, and unnatural amino acids, advancing nanopore proteomics and chiral peptide analysis.