A dynamical anthrax toxin nanopore biosensor for high-fidelity single-peptide classification.

Nanopore sensing holds the potential to revolutionize proteomics, yet current methods often rely on ensemble aggregation, where thousands of events must be statistically aggregated or averaged to identify a protein or peptide signature. While effective for pure samples, this aggregation strategy fails in complex, heterogeneous mixtures where the identity of individual molecules must be determined in real-time. Here, we demonstrate high-fidelity classification of peptides from single, individual translocation events, eliminating the need for ensemble averaging. This sensitivity is achieved using the anthrax toxin protective antigen (PA) nanopore. Unlike static pores used more generally, the PA pore's dynamic active-site clamps generate information-rich, multi-state signals. These clamps also enable the utility of high-affinity capture, permitting analysis at low nanomolar concentrations. We developed a machine learning framework that makes inferences on these dynamical multi-state signals and achieves ~91% accuracy on single events. This work establishes a framework for true single-molecule proteomics, enabling the resolution of complex mixtures that bulk aggregation methods cannot decipher.