An engineered electroosmotic flow transports unravelled proteins across nanopores

Abstract

The development of a technology capable of sequencing single proteins holds promise to unravel new biological information hidden in ensemble analysis. However, new techniques must be first developed. In one approach, proteins are unfolded and translocate across a nanopore under an external bias. Unlike DNA, however, proteins do not have a uniform charge, and the electrophoretic force cannot be used to translocate proteins. Here, we show that by introducing sets of charges spaced by ~1 nm an otherwise neutral nanopore an electroosmotic force is created that induces the unidirectional transport of polypeptides, even against relatively strong electrophoretic forces. Unstructured polypeptides and native proteins unfolded with urea produce current signatures as they traversed the nanopore, which could lead to quick protein identifcation. This approach can be used to translocate and stretch proteins in non-enzymatic protein identification and enzymatic protein sequencing approaches.

Resolving sulfation PTMs on a plant peptide hormone using nanopore sequencing

“…This suggests that the early signal peak is not related to the linker, but more likely due to the closer proximity of the 2 nd tyrosine to the negatively charged poly-D tail. Recent nanopore studies also described ionic current alterations from charge interactions near nanopore constriction site 21,22 . By contrast, the delayed tail signals may be explained by hydrophobic interactions between the unmodified 1 st tyrosine with the pore inner surface (similar to observations described in a previous study 16 ) that are disrupted by charged PTMs on this tyrosine.…”