Enzymeless nanopore proteoform identification

快速而廉价地对DNA和RNA进行测序的方法已经彻底改变了生物学和医学。分析细胞蛋白质及其数百万种变体（蛋白质型）的能力将是一个具有可比影响的进步。最先进的蛋白质组学技术主要集中在短肽片段上，它们无法再现蛋白质组群体的动态和复杂的图像。纳米孔传感技术已经实现了超长读段的DNA和RNA测序，是一种具有潜在能力的全长多肽链测序的解决方案。该项目旨在建立无酶方法来捕获、解折叠和驱动单个全长多肽通过蛋白质纳米孔的移位，以定位变异位点（例如翻译后修饰，PTM）。已经收集了两种平行但互补的策略的大量初步数据：电泳驱动的易位和化学控制的多肽通过蛋白质纳米孔的步进。在这项初步工作的基础上，我的团队将优化无酶系统，用于在全长多肽中快速检测PTM，并建立一种通用的方法来识别和计数单个蛋白质。我们建议设计新的电泳活性纳米孔，并筛选它们容纳长分子轨道的能力，以进行化学步进。通过这些方法，我们将检测在多肽易位过程中的PTM，无论是直接还是在它们与特定配体结合之后。各种偏向蛋白质展开的方法将被内置到系统中，包括电压和温度的调节，以及一系列变性剂的使用。最后，无酶系统将被整合到商业纳米孔阵列中，以有意义的通量测定具有生物学意义的蛋白质。我们的研究结果将为单细胞中蛋白质组的分类以及最终的细胞或组织的蛋白质组比较奠定基础

Means to sequence DNA and RNA quickly and cheaply have revolutionized biology and medicine. The ability to analyse cellularproteins and their millions of variants, "proteoforms", would be an advance of comparable impact. The state-of-the-art proteomictechnologies largely focus on short peptide fragments; they are unable to recapitulate the dynamic and complex picture ofproteoform populations. Nanopore sensing, which has enabled ultra-long-read DNA and RNA sequencing, is emerging as a promisingsolution with the potential ability to characterise full-length polypeptide chains.This project aims to establish enzymeless means to capture, unfold, and drive the translocation of individual full-length polypeptidesthrough protein nanopores to map sites of variation (e.g. post-translational modifications, PTMs). A strong body of preliminary datahas been collected for two parallel but complementary strategies: electroosmosis-driven translocation and chemically-controlledstepping of polypeptides through protein nanopores. Building on this initial work, my group will optimise the enzymeless systems forrapid PTM detection within full-length polypeptides and establish a general approach to identify and count individual proteoforms.We propose to engineer new electroosmotically active nanopores and screen their ability to accommodate long molecular tracksforchemical stepping. By these means, we will detect PTMs during polypeptide translocation, either directly or after they have boundto specific ligands. Various means to bias protein unfolding will be built into the systems, including the modulation of voltage andtemperature, and the use of a range of denaturants. Finally, the enzymeless systems will be integrated into commercial nanoporearrays to assay proteoforms of biological significance with a meaningful throughput. Our results will lay the groundwork forcataloguing proteoforms in single cells and ultimately, the proteome-wide comparison of cells or tissues