Towards single-molecule protein sequencing with nanopores - title: towards single-molecule protein sequencing with nanopores abstract: this proposal concerns the development of a nascent…

Towards single-molecule protein sequencing with nanopores - title: towards single-molecule protein sequencing with nanopores abstract: this proposal concerns the development of a nascent single-molecule proteomics technology based on nanopore sequencing. In recent work by brinkerhoff ET AL., we demonstrated a first-of- kind method for sequentially reading single peptide molecules with a biological nanopore, detecting single-amino acid substitutions in individual molecules and showing that extremely high fidelity may be reached through indefinitely many independent RE-reads of individual proteins. This method does not require chemical labeling of amino acids, can be carried out on commercially available nanopore sequencing hardware, and retains the properties that enabled the success of nanopore DNA sequencing: low overhead cost, physical rather than chemical sensitivity to small changes in single molecules, and the flexibility to be RE-engineered to target specific sequencing applications. In this proposal to fund a collaboration of the gundlach and dekker laboratories, both world leaders in nanopore technology development, we aim to develop this promising new technology into a fully fledged single-molecule proteomics tool. This will involve characterizing reads of a broad array of protein sequences, optimising core elements of the sequencing methodology for protein analysis, preparing natural peptides for reading, and determining the method's applicability to pressing proteomics problems such as post-translational modification and splice variant detection. Achieving these aims will position the nanopore protein reader for immediate application to proteomics problems in fundamental biology and clinical medicine.