Enzyme-less nanopore detection of post-translational modifications within long polypeptides

作者：Pablo Martin-Baniandres, Wei-Hsuan Lan, Stephanie Board, Mercedes Romero‐Ruiz, Sergi Garcia-Manyes, Yujia Qing, Hagan Bayley · 发表于：Nature Nanotechnology · 年份：2023 · DOI：10.1038/s41565-023-01462-8 · 被引用次数：143 · 研究领域：Nanopore and Nanochannel Transport Studies、Ion-surface interactions and analysis、Microfluidic and Capillary Electrophoresis Applications

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.