Enzymeless nanopore proteoform identification

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Means to sequence DNA and RNA quickly and cheaply have revolutionized biology and medicine. The ability to analyse cellularproteins and their millions of variants, "proteoforms", would be an advance of comparable impact. The state-of-the-art proteomictechnologies largely focus on short peptide fragments; they are unable to recapitulate the dynamic and complex picture ofproteoform populations. Nanopore sensing, which has enabled ultra-long-read DNA and RNA sequencing, is emerging as a promisingsolution 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 polypeptidesthrough protein nanopores to map sites of variation (e.g. post-translational modifications, PTMs). A strong body of preliminary datahas been collected for two parallel but complementary strategies: electroosmosis-driven translocation and chemically-controlledstepping of polypeptides through protein nanopores. Building on this initial work, my group will optimise the enzymeless systems forrapid 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 tracksforchemical stepping. By these means, we will detect PTMs during polypeptide translocation, either directly or after they have boundto specific ligands. Various means to bias protein unfolding will be built into the systems, including the modulation of voltage andtemperature, and the use of a range of denaturants. Finally, the enzymeless systems will be integrated into commercial nanoporearrays to assay proteoforms of biological significance with a meaningful throughput. Our results will lay the groundwork forcataloguing proteoforms in single cells and ultimately, the proteome-wide comparison of cells or tissues