SUMMARY We propose to develop the foundations of a platform for direct sequencing of native, full-length protein strands using unfoldase-coupled nanopore array technology. In principle, this technology could be used to identify protein primary sequence, in addition to certain post-translational modifications (PTMs) found in prokaryotic and eukaryotic cells, with single-molecule resolution. It is a foundational advance over existing and other next-gen proteomic technologies such as Edman degradation, mass spectrometry, fluorescent label approaches, and immunoaffinity-based methods that suffer from limitations in read length, throughput, sensitivity, labeling efficiency, and/or the availability of suitable affinity reagents. Nanopore sequencing of intact protein strands overcomes these limitations because the ~1 nanometer-long sensor directly interacts with the protein strand as it is linearly-driven through the pore by the unfoldase motor protein, manifesting sequence-specific ionic current signals. Thus, complete sequence analysis of native protein molecules can be achieved. This method is a natural technical extension of current nanopore sequencing platforms that use molecular motors to control movement of nucleic acid strands through nanopores in DNA/RNA sequencing. During the grant period, we will pursue three specific aims: 1) Establish baseline methods of controlled protein translocation through nanopore sensor arrays using unfoldase motors; 2) Develop computational and bioinformatic methods to translate raw nanopore signal data into protein sequence information (amino acid calling and PTM detection); and 3) Establish techniques for analysis of native proteins and proteomic samples. Our team of investigators is uniquely qualified to take on this project: i) We pioneered the analysis of full-length protein strands using unfoldase-coupled nanopore sensors and recently demonstrated that the Oxford Nanopore MinION nanopore array device can be used to directly detect peptide strands and resolve single amino acid substitutions (Nivala). ii) Co-investigators on this application have elucidated and exquisitely characterized the enzymatic mechanisms of unfoldase motor activity through in vitro biochemical, single-molecule, and structural studies (Martin), and have led the development of nanopore raw signal analyses for sequencing of nucleic acids, including direct RNA sequencing, genome and transcriptome-wide detection of modified bases, and assembly of a human genome using ultra-long DNA nanopore reads (Jain). iii) Collaborators will provide access to enabling nanopore technology platforms and expertise, including highly-parallel nanopore sensor arrays and customized nanopore proteins, and offer natural routes to technology transfer (Oxford Nanopore), contribute to characterization and comparison of project results to traditional analysis methods such as protein mass spectrometry (Guttman), and advise on compelling technological applications that wi...