Department of Chemistry, University of Oxford, Oxford, UK.
Department of Physics, Randall Centre for Cell and Molecular Biophysics and London Centre for Nanotechnology, King's College London, London, UK.
Nat Nanotechnol. 2023 Nov;18(11):1335-1340. doi: 10.1038/s41565-023-01462-8. Epub 2023 Jul 27.
Means to analyse cellular proteins and their millions of variants at the single-molecule level would uncover substantial information previously unknown to biology. Nanopore technology, which underpins long-read DNA and RNA sequencing, holds potential for full-length proteoform identification. We use electro-osmosis in an engineered charge-selective nanopore for the non-enzymatic capture, unfolding and translocation of individual polypeptides of more than 1,200 residues. Unlabelled thioredoxin polyproteins undergo transport through the nanopore, with directional co-translocational unfolding occurring unit by unit from either the C or N terminus. Chaotropic reagents at non-denaturing concentrations accelerate the analysis. By monitoring the ionic current flowing through the nanopore, we locate post-translational modifications deep within the polypeptide chains, laying the groundwork for compiling inventories of the proteoforms in cells and tissues.
在单分子水平上分析细胞蛋白及其数百万种变体的方法将揭示生物学以前未知的大量信息。纳米孔技术是长读长 DNA 和 RNA 测序的基础，具有鉴定全长蛋白异构体的潜力。我们在工程化的电荷选择性纳米孔中利用电渗流，实现了单个超过 1200 个残基的多肽的非酶捕获、展开和易位。未标记的硫氧还蛋白多聚体通过纳米孔进行转运，从 C 端或 N 端逐个单元发生定向共转运展开。非变性浓度的变构试剂加速了分析。通过监测流过纳米孔的离子电流，我们可以在多肽链的深处定位翻译后修饰，为在细胞和组织中编制蛋白异构体目录奠定基础。
