Controlled Protein Translocation in Nanopores for Sequencing Applications

项目摘要 蛋白质组学领域的最终目标是单分子、高精度、从头开始的蛋白质测序。 不幸的是，目前的蛋白质组学技术成本高昂，并且缺乏灵敏度、动态范围、吞吐量、 达到这一目标所需的规模和准确性。在这个项目中，我们将开始研究技术 实现最先进的蛋白质测序和表征所需的组件和方法。更多 具体地说，我们将开发通过纳米孔阅读器可控地转移蛋白质的方法，以便 可以对蛋白质进行测序。基于纳米孔的测序提供了一种方法来表征高和 通过一次对一个分子的蛋白质进行测序来获得低丰度蛋白质，但具有高准确性、灵敏度和 产量需要涵盖蛋白质组中蛋白质丰度的广泛动态范围。在这个项目中， 我们将开发一种全新的基于纳米孔的从头蛋白质测序技术。我们将发展 并构建了一个新颖的蛋白质测序系统原型，充分评估和优化了相关的 用于高度受控和通用的蛋白质/多肽表征的工作流程/方法，并初步演示 对各种蛋白质和多肽进行测序。在这个项目结束时，我们将成功地 论证了一种实用的基于纳米孔的蛋白质测序概念的可行性。

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.