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bioRxiv · 10.1101/2023.09.08.555904

Deep learning-assisted single-molecule detection of protein post-translational modifications with a biological nanopore

Cao, C.·Magalhaes, P.·Krapp, L.·Bada Juarez, J.·Mayer, S.·Rukes, V.·Chiki, A.·Lashuel, H.·Dal Peraro, M.
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Abstract

Protein post-translational modifications (PTMs) play a crucial role in countless biological processes, profoundly modulating protein properties on both the 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 -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 -synuclein peptide and its PTM variants could be confidently identified using a deep learning model for signal processing. We further demonstrate that this framework can quantify -synuclein peptides at picomolar concentration and detect the C-terminal peptides generated by digestion of full-length -synuclein. Collectively, our work highlights the unique advantage of using nanopore as a tool for simultaneous detection of multiple PTMs and paves the way for their use in biomarker discovery and diagnostics.

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Cao, C., Magalhaes, P., Krapp, L., Bada Juarez, J., Mayer, S., Rukes, V., Chiki, A., Lashuel, H., Dal Peraro, M.. 2023-09-08. Deep learning-assisted single-molecule detection of protein post-translational modifications with a biological nanopore. https://doi.org/10.1101/2023.09.08.555904

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