Enzymeless nanopore proteoform identification

Means to sequence DNA and RNA quickly and cheaply have revolutionized biology and medicine. The ability to analyse cellular
proteins and their millions of variants, "proteoforms", would be an advance of comparable impact. The state-of-the-art proteomic
technologies largely focus on short peptide fragments; they are unable to recapitulate the dynamic and complex picture of
proteoform populations. Nanopore sensing, which has enabled ultra-long-read DNA and RNA sequencing, is emerging as a promising
solution with the potential ability to characterise full-length polypeptide chains.

This project aims to establish enzymeless means to capture, unfold, and drive the translocation of individual full-length polypeptides
through protein nanopores to map sites of variation (e.g. post-translational modifications, PTMs). A strong body of preliminary data
has been collected for two parallel but complementary strategies: electroosmosis-driven translocation and chemically-controlled
stepping of polypeptides through protein nanopores. Building on this initial work, my group will optimise the enzymeless systems for
rapid PTM detection within full-length polypeptides and establish a general approach to identify and count individual proteoforms.

We propose to engineer new electroosmotically active nanopores and screen their ability to accommodate long molecular tracks
forchemical stepping. By these means, we will detect PTMs during polypeptide translocation, either directly or after they have bound
to specific ligands. Various means to bias protein unfolding will be built into the systems, including the modulation of voltage and
temperature, and the use of a range of denaturants. Finally, the enzymeless systems will be integrated into commercial nanopore
arrays to assay proteoforms of biological significance with a meaningful throughput. Our results will lay the groundwork for
cataloguing proteoforms in single cells and ultimately, the proteome-wide comparison of cells or tissues