Nanopore characterisation of Post-Translational Modifications (PTMs) on protein concatemers

Abstract

Proteins exist as diverse proteoforms that carry distinct combinations of posttranslational modifications (PTMs). Reading these states at the single-molecule level would reveal biological phenomena inaccessible to genomics and transcriptomics. This thesis develops nanopore-based strategies that move towards full-length, residue-level proteoform analysis. First, we employ electro-osmotic flow (EOF) in an engineered charge-selective biological nanopore to achieve non-enzymatic capture, unfolding, and translocation of intact polypeptides exceeding ~1,200 residues. Underivatised thioredoxin polyproteins undergo directional, unit-by-unit cotranslocational unfolding from either terminus, and non-denaturing concentrations of chaotropes increase capture efficiency and reduce analysis times. Ionic current readout provides positional information on PTMs embedded deep within long polypeptide chains, establishing a framework for compiling inventories of cellular proteoforms. Second, we focus on phosphorylation, a central regulator of cellular signalling whose dysregulation underlies numerous diseases. We demonstrate binderassisted PTM detection within polypeptides (>700 residues): phosphate-recognising Phos-tag complexes are appended to target chains, EOF carries the tagged proteins through engineered nanopores, and distinct ionic current signatures localise phosphorylation sites within individual molecules. This approach establishes a foundation for single-molecule mapping of phosphorylation patterns and broader PTM profiling in intact proteins. Finally, we assess whether binder-assisted nanopore sensing can resolve distinct phosphorylation types (Ser-P, Thr-P, Tyr-P, and the noncanonical Cys-P). A methylated Phos-tag derivative complexed with Zn2+ (PMeZn2) distinguishes all four based on their current levels and kinetics, indicating that tailored binders can translate subtle chemical differences into distinctive electrical fingerprints. Together, electro-osmotically active nanopores, whether used alone or combined with tuneable, PTM-selective binders, enable single-molecule mapping of full-length proteoforms and their modification states. While further validation in complex samples is needed, these results advance nanopore proteomics towards more comprehensive, residue-level readouts with potential applications in mechanistic biology, biomarker discovery, and therapeutic target identification.