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  • Review Article
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Single-molecule protein sequencing with nanopores

Nature Reviews Bioengineering volume 3, pages 303–316 (2025)Cite this article

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Abstract

Protein sequencing and the identification of post-translational modifications are key to understanding cellular signalling pathways and metabolic processes in health and disease. Nanopores, that is, nanometre-sized holes in a membrane, were previously put to use for DNA and RNA sequencing, offering single-molecule sensitivity and long read lengths. This prompted efforts to engineer nanopores for the high-throughput sequencing of peptides and proteins. In this Review, we discuss research towards single-molecule protein sequencing using biological nanopores, investigating how their sensitivity allows the discrimination of all 20 amino acids. We outline how fingerprinting of proteins is facilitated by using motor proteins and electro-osmotic flow to promote the slow translocation of proteins through nanopores. Moreover, we examine applications of nanopores to the identification of post-translational modifications, highlighting the potential of this technology for fundamental and clinical proteomic studies. Finally, we outline the advantages and limitations of nanopore systems for protein sequencing and the challenges that remain to be overcome for realizing de novo nanopore protein sequencing.

Key points

  • Twenty individual amino acids can be distinguished by biological nanopores.

  • Nanopores allow the discrimination of single-amino acid substitutions within proteins.

  • Electro-osmotic flow allows uncharged and heterogeneously charged proteins to translocate through nanopores.

  • Motor proteins reduce protein translocation speed, resulting in high-resolution signals.

  • Various post-translational modifications and their locations can be identified with nanopores.

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Fig. 1: Nanopore-based protein sequencing.
Fig. 2: Single amino acids can be discriminated using nanopores.
Fig. 3: Strategies to introduce electroosmotic flow in nanopores.
Fig. 4: Motor enzymes can control protein translocation speed.
Fig. 5: Detection of post-translational modifications with nanopores.

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Acknowledgements

We thank B. Albada, E. Bertosin, E. van der Sluis and L. Yu for a critical reading of the manuscript. This work was supported by funding from the Dutch Research Council (NWO) project NWO-I680 (SMPS), European Research Council Advanced Grant 883684 and US National Institutes of Health National Human Genome Research Institute project HG012544-01.

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  1. These authors contributed equally: Justas Ritmejeris, Xiuqi Chen.

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  1. Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands

    Justas Ritmejeris, Xiuqi Chen & Cees Dekker

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  1. Justas Ritmejeris
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  2. Xiuqi Chen
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Contributions

J.R. and X.C. surveyed the literature for this article. All authors contributed to writing the manuscript. C.D. supervised the work.

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Correspondence to Cees Dekker.

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C.D. is named inventor on a patent on protein sequencing with nanopores, which is licensed to Oxford Nanopore Technologies. Beyond that, the authors declare no competing interests.

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Ritmejeris, J., Chen, X. & Dekker, C. Single-molecule protein sequencing with nanopores. Nat Rev Bioeng 3, 303–316 (2025). https://doi.org/10.1038/s44222-024-00260-8

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