β-Barrel Nanopores with an Acidic-Aromatic Sensing Region Identify Proteinogenic Peptides at Low pH

Roderick Corstiaan Abraham Versloot, Sabine Angenieta Paulina Straathof, Gemma Stouwie, Matthijs Jonathan Tadema, Giovanni Maglia*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

27 Citations (Scopus)
269 Downloads (Pure)

Abstract

Biological nanopores are emerging as sensitive single-molecule sensors for proteins and peptides. The heterogeneous charge of a polypeptide chain, however, can complicate or prevent the capture and translocation of peptides and unfolded proteins across nanopores. Here, we show that two β-barrel nanopores, aerolysin and cytotoxin K, cannot efficiently detect proteinogenic peptides from a trypsinated protein under a wide range of conditions. However, the introduction of an acidic-aromatic sensing region in the β-barrel dramatically increased the dwell time and the discrimination of peptides in the nanopore at acidic pH. Surprisingly, despite the fact that the two β-barrel nanopores have a similar diameter and an acidic-aromatic construction, their capture mechanisms differ. The electro-osmotic flow played a dominant role for aerolysin, while the electrophoretic force dominated for cytotoxin K. Nonetheless, both β-barrel nanopores allowed the detection of mixtures of trypsinated peptides, with aerolysin nanopores showing a better resolution for larger peptides and cytotoxin K showing a better resolution for shorter peptides. Therefore, this work provides a generic strategy for modifying nanopores for peptide detection that will be most likely be applicable to other nanopore-forming toxins.

Original languageEnglish
Article number1c11455
Pages (from-to)7258-7268
Number of pages11
JournalAcs Nano
Volume16
Issue number5
Early online date18-Mar-2022
DOIs
Publication statusPublished - 24-May-2022

Keywords

  • nanopores
  • protein sequencing
  • nanopore spectrometry
  • nanopore-forming toxins
  • single-molecule

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