Project Summary As it stands today, traditional/standard protein characterization methods have insufficient limits-of-detection, dynamic range, throughput, cost, accuracy, sensitivity, scale, and/or some combination thereof. Because of these shortcomings, there are no currently available methods capable of meeting the needs within the proteomics field: single-cell, proteome-wide characterization/sequencing. New technology must be developed to advance and revolutionize the field of proteomics, similar to how inventive nanopore-based technology developments have opened and accelerated the fields of genomics and transcriptomics. Nanopore-based technology is a very powerful method for molecular characterization and because of this, it has the potential to also shape the future of protein sequencing. It is one of only a few potential approaches that represent a viable path to direct, high-throughput, high-sensitivity, single-molecule, protein sequencing capable of characterizing both low- and high-abundance proteins, which is an absolute necessity for achieving the accuracy and dynamic range required for comprehensive, enabling protein analyses. During this program, Electronic BioSciences, Inc. (EBS) aims to develop a completely new nanopore-based technology that will enable de novo protein sequencing. During this Phase I project, we will develop and build a novel protein sequencing system prototype, fully assess and optimize the associated workflow/methodology for highly controlled and versatile protein/peptide characterization, and demonstrate initial sequencing for various proteins and peptides. At the conclusion of this project, we will have successfully shown concept feasibility for practical nanopore-based protein sequencing.