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bioRxiv · 10.64898/2026.10.21.760613

Sample preparation for direct nanopore based protein identification via tryptic peptide recognition

Hensel, C.·Kleber, C.·Suppanz, I.·Akbari, A.·Gutsch, S.·Hutzenlaub, T.·Ensslen, T.·Behrends, J. C.·von Stetten, F.·Mittler, G.·Klatt, N.·Schreiber, A.
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Abstract

Rapid, cost-efficient and miniaturized protein analysis is essential for point-of-care clinical diagnostics. Nanopores offer a promising alternative to mass spectrometry, offering high-resolution, single-molecule sensing with a minimal footprint. However, peptide-level protein identification remains challenging due to difficulties in assigning nanopore signals to peptide sequences and the incompatibility of conventional proteolysis workflows with nanopore sensing. Here we integrate a single-pot, solid-phase-enhanced sample preparation (SP3) workflow with nanopore sensing. The SP3 protocol decouples proteolysis from interfering components via magnetic bead cleanup, yielding a digest compatible with nanopore sensing. As a proof of concept, tryptic digests of myelin basic protein measured with an engineered aerolysin variant (AeL-R220S) generated a reproducible five-peak peptide fingerprint. Cross-validation against a synthetic peptide library and LC-MS/MS enabled reference-based assignment of blockade signatures to four MBP-derived peptides. This approach establishes SP3 as a sample-preparation workflow for nanopore sensing, paving the way for label-free identification and characterization of protein targets.

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Hensel, C., Kleber, C., Suppanz, I., Akbari, A., Gutsch, S., Hutzenlaub, T., Ensslen, T., Behrends, J. C., von Stetten, F., Mittler, G., Klatt, N., Schreiber, A.. 2026-10-23. Sample preparation for direct nanopore based protein identification via tryptic peptide recognition. https://doi.org/10.64898/2026.10.21.760613

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