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

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posted on 2023-11-23, 11:17 authored by Pablo Martin-Baniandres, Wei-Hsuan Lan, Stephanie Board, Mercedes Romero-Ruiz, Sergi Garcia-Manyes, Yujia Qing, Hagan Bayley
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.

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