Enzymeless nanopore proteoform identification

快速、廉价地对DNA和RNA进行测序的方法已经给生物学和医学带来了革命性的变化。分析细胞蛋白质及其数百万种变种--“蛋白质形式”--的能力，将是一项具有同等影响的进步。最先进的蛋白质组学技术主要集中在短肽片段上；它们无法概括蛋白质形式种群的动态和复杂图景。纳米孔传感使超长DNA和RNA测序成为可能，成为一种有希望的解决方案，具有表征全长多肽链的潜在能力。该项目旨在建立无酶手段，通过蛋白质纳米孔捕获、展开和驱动单个全长多肽的移位，以绘制变异位点(例如，翻译后修饰，PTM)。已经为两种平行但互补的策略收集了大量的初步数据：电渗驱动的移位和通过蛋白质纳米孔的化学控制多肽的步进。在这项初步工作的基础上，我的团队将优化用于全长多肽中快速PTM检测的无酶系统，并建立一种识别和计算单个蛋白质形式的通用方法。我们建议设计新的电渗透活性纳米孔，并筛选它们适应化学步进的长分子轨迹的能力。通过这些手段，我们将在多肽转位过程中直接或在它们与特定配体结合后检测到PTMS。系统中将内置各种偏向蛋白质展开的方法，包括电压和温度的调节，以及一系列变性剂的使用。最后，无酶系统将被集成到商业纳米阵列中，以有意义的吞吐量检测具有生物学意义的蛋白质形式。我们的结果将为在单细胞中进行蛋白质组分析奠定基础，并最终在细胞或组织的蛋白质组学范围内进行比较

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