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

Pablo Martin-Baniandres, Wei-Hsuan Lan, Stephanie Board, Mercedes Romero-Ruiz, Sergi Garcia-Manyes, Yujia Qing*, Hagan Bayley*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)

Abstract

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.
Original languageEnglish
Pages (from-to)1335-1340
JournalNature Nanotechnology
Volume18
Issue number11
Early online date27 Jul 2023
DOIs
Publication statusPublished - Nov 2023

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