Cao, ChanMagalhaes, PedroKrapp, Lucien FabriceJuarez, Juan F. BadaMayer, Simon FinnRukes, VerenaChiki, AnassLashuel, Hilal ADal Peraro, Matteo2024-02-212024-02-212024-02-212023-12-1910.1021/acsnano.3c08623https://infoscience.epfl.ch/handle/20.500.14299/205089WOS:001144000200001Protein post-translational modifications (PTMs) play a crucial role in countless biological processes, profoundly modulating protein properties on both spatial and temporal scales. Protein PTMs have also emerged as reliable biomarkers for several diseases. However, only a handful of techniques are available to accurately measure their levels, capture their complexity at a single molecule level, and characterize their multifaceted roles in health and disease. Nanopore sensing provides high sensitivity for the detection of low-abundance proteins, holding the potential to impact single-molecule proteomics and PTM detection, in particular. Here, we demonstrate the ability of a biological nanopore, the pore-forming toxin aerolysin, to detect and distinguish alpha-synuclein-derived peptides bearing single or multiple PTMs, namely, phosphorylation, nitration, and oxidation occurring at different positions and in various combinations. The characteristic current signatures of the alpha-synuclein peptide and its PTM variants could be confidently identified by using a deep learning model for signal processing. We further demonstrate that this framework can quantify alpha-synuclein peptides at picomolar concentrations and detect the C-terminal peptides generated by digestion of full-length alpha-synuclein. Collectively, our work highlights the advantage of using nanopores as a tool for simultaneous detection of multiple PTMs and facilitates their use in biomarker discovery and diagnostics.Physical SciencesTechnologyBiological NanoporesProtein Post-Translational ModificationsDeep-LearningSingle-Molecule SensingAlpha-Synucleintext::journal::journal article::research article