Blobs form during the single-file transport of proteins across nanopores.

The transport of biopolymers across nanopores is an important biological process currently under investigation for the rapid analysis of DNA and proteins. While the transport of DNA is generally understood, methods to induce unfolded protein translocation have only recently been discovered (Yu et al., 2023, Sauciuc et al., 2023). Here, we found that during electroosmotically driven translocation of polypeptides, blob-like structures typically form inside nanopores, often obstructing their transport and preventing addressing individual amino acids. This is in contrast with the electrophoretic transport of DNA, where the formation of such structures has not been reported. Comparisons between different nanopore sizes and shapes and modifications by different surface chemistries allowed formulating a mechanism for blob formation. We also show that single-file transport can be achieved by using 1) nanopores that have an entry and an internal diameter smaller than the persistence length of the polymer, 2) nanopores with a nonsticky (i.e ., nonaromatic) inner surface, and 3) moderate translocation velocities. These experiments provide a basis for understanding polypeptide transport under confinement and for improving the design and engineering of nanopores for protein analysis.