Fingerprinting full-length proteins through single-file translocations.

Biological nanopore is a revolutionary approach to single molecule detection of biological polymers including proteins. However, for full-length protein fingerprinting/sequencing, the nonuniform charged backbone, complex secondary structures, and intra-protein interactions, present multiple challenges in protein capture, threading, and transport with velocity control. Here we introduce an approach for realizing unidirectional transport of full-length proteins through nanopores. To achieve this, we combined a high chemical resistant biological nanopore platform with high concentration of denaturant, guanidinium chloride to unfold, capture and translocate full-length proteins through a biological nanopore by an electroosmotic effect. The uniform and slow (∼10 µs/amino acid) translocation of proteins, combined with supervised machine learning, allows us to use their electrical current blockade signatures to discern their threading orientation and identity. When combined with a method for tail-modification of native proteins, our study can pave the way to direct single-molecule protein fingerprinting without the requirement of a motor enzyme. (Yu et al., BioRxiv 2021, https://doi.org/10.1101/2021.09.28.4621550)