Project Summary The ultimate goal in the field of proteomics is single-molecule, high-accuracy, de novo protein sequencing. Unfortunately, current proteomics technologies are costly, and lack the sensitivity, dynamic range, throughput, scale, and accuracy needed to meet this goal. During this project, we will begin examining the technological components and methods required to enable state-of-the-art protein sequencing and characterization. More specifically, we will develop methods for controllably translocating a protein through a nanopore reader such that the protein can be sequenced. Nanopore-based sequencing provides a path to characterizing both high- and low-abundance proteins by sequencing proteins one molecule at a time, but with high accuracy, sensitivity and throughput needed to cover the wide dynamic range in protein abundance in the proteome. During this project, we will develop a completely new nanopore-based technology for de novo protein sequencing. 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 demonstrated feasibility for a practical nanopore-based protein sequencing concept.

Project Narrative This program is aimed at developing and validating the individual components and the overall methodology for new nanopore-based technology capable of direct, de novo, single-molecule sequencing of full-length proteins. Rapid, reliable, high-throughput, low-cost, protein sequencing and characterization will enable basic biological research; a more comprehensive assessment of gene expression, protein variations and post translational modifications; and faster disease biomarker discovery and validation, as well as enhanced clinical testing and monitoring of diseases.