FraC nanopores with adjustable diameter identify the mass of opposite-charge peptides with 44 dalton resolution

A high throughput single-molecule method for identifying peptides and sequencing proteins based on nanopores could reduce costs and increase speeds of sequencing, allow the fabrication of portable home-diagnostic devices, and permit the characterization of low abundance proteins and heterogeneity in post-translational modifications. Here we engineer the size of Fragaceatoxin C (FraC) biological nanopore to allow the analysis of a wide range of peptide lengths. Ionic blockades through engineered nanopores distinguish a variety of peptides, including two peptides differing only by the substitution of alanine with glutamate. We also find that at pH 3.8 the depth of the peptide current blockades scales with the mass of the peptides irrespectively of the chemical composition of the analyte. Hence, this work shows that FraC nanopores allow direct readout of the mass of single peptide in solution, which is a crucial step towards the developing of a real-time and single-molecule protein sequencing device.