A single protein molecule can carry dozens of chemical modifications that change its function, and this project aims to read those modifications directly from the full-length protein chain. Current protein analysis methods chop proteins into short fragments, losing the combinatorial patterns of modifications—known as proteoforms—that determine how proteins actually behave in cells. This matters because diseases such as cancer and neurodegeneration are driven by specific proteoform changes that current techniques cannot see. The researchers plan to pull individual protein chains through a nanopore without using enzymes, using electric fields and chemical steps instead. They will detect modifications either directly or after they bind to specific ligands, and integrate the system into commercial nanopore arrays for higher throughput. If successful, this fundamental science could eventually allow researchers to catalogue every proteoform in a single cell. That would transform how we understand protein regulation in health and disease, much as DNA sequencing transformed genetics. There is no immediate practical application—the work is about establishing whether enzymeless nanopore proteoform identification is possible at all. But if it works, it would open a new window onto the molecular machinery that runs every living cell.
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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
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