Nanopore Direct Single-Molecule Protein Sequencing

项目摘要/摘要： 蛋白质是细胞和生物体的主力。目前，还没有任何技术可用于 对这类生理上重要的分子进行常规蛋白质组规模测序和量化。在……里面 在这个项目中，我们建议开发一种直接从头生成单分子蛋白质的纳米孔技术。 测序。类似于纳米孔DNA测序，蛋白质分子的序列由 用电子方法测量线状多肽上的残基对离子流的影响 而完全变性的分子通过纳米孔一次一个地进行电泳性转移。 为了实现这项技术，我们需要克服三个主要挑战：(1)利用 区分20个氨基酸残基所需的尺寸(直径~1 nm，厚度~0.5 nm)(~0.38？ 间距)；(2)未折叠的受控单向移位的方法 通过纳米孔的多肽；(3)从当前的堵塞轮廓中解码序列的算法。 通过几年的概念、理论和实验工作，我们已经找到了潜在的解决方案 这些挑战。首先，我们发明了一种新的固态/蛋白质/环肽纳米孔结构 这将使纳米孔的工程能够区分20种不同的氨基酸残基 从头开始蛋白质测序。其次，我们还演示了一种提供均匀电荷密度的策略 沿着多肽链，使蛋白质能够通过纳米孔进行单向转移。第三，我们有 开发了一种策略，使我们能够对所有207个(=1.28x109)的当前阻塞进行建模和计算 20个氨基酸的七聚体组合，因此目前对任何蛋白质的阻断图谱。我们有 还开发了从计算的当前阻塞分布中解码氨基酸序列的算法。 令人兴奋的是，随着最近的这些突破，我们已经能够证明De Novo纳米孔蛋白测序。我们已经证明，12个氨基酸残基可以用&gt；90%进行测序。 共识准确率，2个残基具有85%的准确率，另外6个残基被解码为3对，准确率为90% 精确度。在这个项目中，我们建议实施这些创新的方法，旨在奠定基础 为实验实现纳米孔蛋白质测序奠定基础。排序和枚举的能力 蛋白质将使常规的蛋白质组规模鉴定和数字量化成为可能，最终 单分子和单细胞灵敏度。在单分子水平上对蛋白质进行排序的能力将 使常规蛋白质组规模的鉴定和数字定量的蛋白质最终单一- 分子敏感性。如果成功，这种颠覆性技术将从基础应用到广泛的应用 人类疾病的研究、药物靶标识别和精确临床诊断将有可能 改变生物医学研究、个性化医疗保健和医疗实践的多个方面。

PROJECT SUMMARY/ABSTRACT: Proteins are the workhorses of cells and organisms. At present, no technologies are available for the routine proteome-scale sequencing and quantification of this physiologically important class of molecules. In this project, we propose to develop a nanopore technology for direct de novo single-molecule protein sequencing. Analogous to nanopore DNA sequencing, the sequences of protein molecules are determined by electronically measuring the alteration of the ionic current flow by the residues along the linear polypeptides while the fully denatured molecules are electrophoretically translocated one at a time through the nanopores. To realize the technology, we need to overcome three major challenges: (1) engineering of nanopores with dimensions (~1 nm diameter and ~0.5 nm thickness) required to distinguish 20 amino acid residues (~0.38 Å spacing) along the linear polypeptide chain; (2) a method for controlled unidirectional translocation of unfolded polypeptides through the nanopores; (3) algorithms for decoding sequences from current blockage profiles. Through several years of conceptual, theoretical and experimental work, we have found potential solutions to these challenges. First, we have invented a new hybrid solid-state/protein/cyclopeptide nanopore architecture that will enable the engineering of nanopores capable of distinguishing the 20 different amino acid residues for de novo protein sequencing. Second, we have also demonstrated a strategy to impart uniform charge density along the polypeptide chain to enable unidirectional translocation of protein through nanopores. Third, we have developed a strategy that has enabled us to model and compute the current blockages of all 207 (=1.28x109) heptamer combinations of 20 amino acids, and thus the current blockage profiles of any proteins. We have also developed the algorithms to decode amino acid sequences from the computed current blockage profiles. Excitingly, with these recent breakthroughs, we have been able to demonstrate the theoretical feasibility of de novo nanopore protein sequencing. We have shown that 12 amino acid residues can be sequenced with >90% consensus accuracy, 2 residues with >85% accuracy, and the other 6 residue decoded as 3 pairs with >90% accuracy. In this project, we propose to implement these innovative approaches aiming to lay the foundation for the experimental realization of nanopore protein sequencing. The ability to sequence and enumerate proteins will enable routine proteome-scale identification and digital quantification of proteins with the ultimate single-molecule and single-cell sensitivity. The ability to sequence proteins at the single-molecule level will enable routine proteome-scale identification and digital quantification of proteins with the ultimate single- molecule sensitivity. If successful, such a disruptive technology will find broad general applications from basic research, drug target identification and precision clinical diagnosis of human diseases, will have potential to transform many aspects of biomedical research, personalized healthcare and medical practice.