Translocation of linearized full-length proteins through an engineered nanopore under opposing electrophoretic force

Nanopores have recently been used to identify and fingerprint proteins. However, because proteins, unlike DNA, do not have a uniform charge, the electrophoretic force cannot in general be used to translocate or linearize them. Here we show that the introduction of sets of charges in the lumen of the CytK nanopore spaced by ~1 nm creates an electroosmotic flow that induces the unidirectional transport of unstructured natural polypeptides against a strong electrophoretic force. Molecular dynamics simulations indicate that this electroosmotic-dominated force has a strength of ~20 pN at -100 mV, which is similar to the electric force on single-stranded DNA. Unfolded polypeptides produce current signatures as they traverse the nanopore, which may be used to identify proteins. This approach can be used to translocate and stretch proteins for enzymatic and non-enzymatic protein identification and sequencing.