Peptide sequencing with single amino acid resolution using a sub-nanometer diameter pore

Amino acid (AA) residues of protein primary structures can now be sequenced using a sub-nanometer diameter pore in a few-nanometer-thick amorphous silicon (a-Si) membrane. When denatured peptides are driven by an electric field through the pore, the ionic current is partially blocked, which depends on AA properties such as volume, charge, and solvation, and the sequence is read from blockade fluctuations. Sub-nanopores (0.3–0.4 nm) with biconical geometry were sputtered atom-by-atom using a tightly focused 300 keV electron beam in an aberration-corrected scanning transmission electron microscopy (STEM). Blockade currents were amplified over 1.8 MHz bandwidth and sampled at 3.6 MS/s. To enhance signal-to-noise, similar blockades were clustered using an affinity propagation (AP) algorithm to generate consensus. Molecular dynamics (MD) simulations were performed to predict blockade fluctuation patterns for sequence identification. Finally, a target database was constructed with MD and searched for a match with empirical consensus. Sequencing was demonstrated for amyloid-β (Aβ_1–42), its variants, and post-translationally modified forms, highlighting a path toward label-free single-AA resolution protein sequencing.