Nanopore Direct Single-Molecule Protein Sequencing

项目总结/摘要： 蛋白质是细胞和有机体的主要组成部分。目前，没有技术可用于 常规蛋白质组规模测序和定量这类生理学上重要的分子。在 在这个项目中，我们建议开发一种纳米孔技术来直接从头合成单分子蛋白质 测序类似于纳米孔DNA测序，蛋白质分子的序列通过以下方式确定： 电子测量沿着线性多肽的残基对离子电流的改变 而完全变性的分子通过纳米孔一次一个地进行电泳移位。 为了实现该技术，我们需要克服三个主要挑战：（1）纳米孔的工程化， 尺寸（~1 nm直径和~0.5 nm厚度）需要区分20个氨基酸残基（~0.38 nm） 沿着线性多肽链间隔）;（2）用于控制未折叠多肽的单向易位的方法， （3）用于从当前阻断概况解码序列的算法。 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混合固态/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， 从头蛋白质测序。其次，我们还展示了一种赋予均匀电荷密度的策略 沿着多肽链以使蛋白质能够通过纳米孔单向移位。三是 开发了一种策略，使我们能够建模和计算所有207（=1.28x109）的当前阻塞 20个氨基酸的七聚体组合，以及因此任何蛋白质的电流阻断概况。我们有 还开发了从计算出的当前阻断概况中解码氨基酸序列的算法。 令人兴奋的是，随着这些最近的突破，我们已经能够证明de的理论可行性。 novo nanopore protein sequencing.我们已经表明，12个氨基酸残基可以测序与>90% 共有准确性，2个残基具有>85%的准确性，并且另外6个残基解码为3对，具有>90%的准确性。 精度在本项目中，我们建议实施这些创新方法，旨在为 用于纳米孔蛋白质测序的实验实现。排序和枚举的能力 蛋白质将使常规的蛋白质组规模的识别和蛋白质的数字定量， 单分子和单细胞灵敏度。在单分子水平上对蛋白质进行测序的能力 实现常规蛋白质组规模的鉴定和蛋白质的数字定量， 分子灵敏度如果成功，这种颠覆性的技术将从基本的 研究、药物靶点识别和人类疾病的精确临床诊断，将有潜力 改变了生物医学研究、个性化医疗保健和医疗实践的许多方面。

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.