Electro-osmotic capture and ionic discrimination of peptide and protein biomarkers with FraC nanopores

TLDR

Biological nanopores enable high‑throughput DNA sequencing, but protein sequencing is hindered by folding and charge heterogeneity, motivating the development of methods for amino‑acid analysis of both folded and unfolded polypeptides. The study aims to demonstrate that electro‑osmotic flow through engineered FraC nanopores permits polypeptide entry at a fixed potential independent of their charge composition. The authors engineered FraC nanopores to generate electro‑osmotic flow that drives polypeptides into the pore at a constant potential. The engineered FraC nanopores successfully discriminated peptide and protein biomarkers ranging from 25 kDa to 1.3 kDa, distinguishing polypeptides differing by a single amino acid and recognizing both unfolded and folded proteins.

Abstract

Biological nanopores are nanoscale sensors employed for high-throughput, low-cost, and long read-length DNA sequencing applications. The analysis and sequencing of proteins, however, is complicated by their folded structure and non-uniform charge. Here we show that an electro-osmotic flow through Fragaceatoxin C (FraC) nanopores can be engineered to allow the entry of polypeptides at a fixed potential regardless of the charge composition of the polypeptide. We further use the nanopore currents to discriminate peptide and protein biomarkers from 25 kDa down to 1.3 kDa including polypeptides differing by one amino acid. On the road to nanopore proteomics, our findings represent a rationale for amino-acid analysis of folded and unfolded polypeptides with nanopores.Biological nanopore-based protein sequencing and recognition is challenging due to the folded structure or non-uniform charge of peptides. Here the authors show that engineered FraC nanopores can overcome these problems and recognize biomarkers in the form of oligopeptides, polypeptides and folded proteins.