Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

Detection of phosphorylation post-translational modifications along single peptides with nanopores

Nature Biotechnology (2023)Cite this article

Subjects

Abstract

Current methods to detect post-translational modifications of proteins, such as phosphate groups, cannot measure single molecules or differentiate between closely spaced phosphorylation sites. We detect post-translational modifications at the single-molecule level on immunopeptide sequences with cancer-associated phosphate variants by controllably drawing the peptide through the sensing region of a nanopore. We discriminate peptide sequences with one or two closely spaced phosphates with 95% accuracy for individual reads of single molecules.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Nanopore PTM detection experimental schematic and data workflow.
Fig. 2: MCMC calculations of phosphate-containing peptides.

Similar content being viewed by others

Data availability

The raw nanopore data for all of the reads used in this study are publicly available at https://doi.org/10.57760/sciencedb.0833831.

References

  1. Kim, M. S., Zhong, J. & Pandey, A. Common errors in mass spectrometry-based analysis of post-translational modifications. Proteomics 16, 700–714 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Khoury, G. A., Baliban, R. C. & Floudas, C. A. Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database. Sci. Rep. 1, 1–5 (2011).

    Article  Google Scholar 

  3. Xu, H. et al. ‘PTMD: a database of human disease-associated post-translational modifications’. Genomics Proteomics Bioinformatics 16, 244–251 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Engelhard, V. H. et al. MHC-restricted phosphopeptide antigens: preclinical validation and first-in-humans clinical trial in participants with high-risk melanoma. J. Immunother. Cancer 8, e000262 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Rosen, C. B. et al. Single-molecule site-specific detection of protein phosphorylation with a nanopore. Nat. Biotechnol. 32, 179–181 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Restrepo-Pérez, L., Wong, C. H., Maglia, G., Dekker, C. & Joo, C. Label-free detection of post-translational modifications with a nanopore. Nano Lett. 19, 7957–7964 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Huo, M. Z., Hu, Z. L., Ying, Y. L. & Long, Y. T. Enhanced identification of tau acetylation and phosphorylation with an engineered aerolysin nanopore. Proteomics 22, 2100041 (2022).

    Article  CAS  Google Scholar 

  8. Li, S. et al. T232K/K238Q aerolysin nanopore for mapping adjacent phosphorylation sites of a single tau peptide. Small Methods 4, 2000014 (2020).

    Article  CAS  Google Scholar 

  9. Wloka, C. et al. Label-free and real-time detection of protein ubiquitination with a biological nanopore. ACS Nano 11, 4387–4394 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Shorkey, S. A., Du, J., Pham, R., Strieter, E. R. & Chen, M. Real-time and label-free measurement of deubiquitinase activity with a MspA nanopore. ChemBioChem 22, 2688–2692 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Nir, I., Huttner, D. & Meller, A. Direct sensing and discrimination among ubiquitin and ubiquitin chains using solid-state nanopores. Biophys. J. 108, 2340–2349 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Fahie, M. A. & Chen, M. Electrostatic interactions between OmpG nanopore and analyte protein surface can distinguish between glycosylated isoforms. J. Phys. Chem. B 119, 10198–10206 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Versloot, R. C. A. et al. Quantification of protein glycosylation using nanopores. Nano Lett. 22, 5357–5364 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Brinkerhoff, H., Kang, A. S., Liu, J., Aksimentiev, A. & Dekker, C. Multiple rereads of single proteins at single–amino acid resolution using nanopores. Science 374, 1509–1513 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Yan, S. et al. Single molecule ratcheting motion of peptides in a Mycobacterium smegmatis porin A (MspA) nanopore. Nano Lett. 21, 6703–6710 (2021).

    Article  CAS  PubMed  Google Scholar 

  16. Chen, Z. et al. Controlled movement of ssDNA conjugated peptide through Mycobacterium smegmatis porin A (MspA) nanopore by a helicase motor for peptide sequencing application. Chem. Sci. 12, 15750–15756 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Butler, T. Z., Pavlenok, M., Derrington, I. M., Niederweis, M. & Gundlach, J. H. Single-molecule DNA detection with an engineered MspA protein nanopore. Proc. Natl Acad. Sci. USA 105, 20647–20652 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Derrington, I. M. et al. Subangstrom single-molecule measurements of motor proteins using a nanopore. Nat. Biotechnol. 33, 1073–1075 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Manrao, E. A. et al. Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase. Nat. Biotechnol. 30, 349–353 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cherf, G. M. et al. Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision. Nat. Biotechnol. 30, 344–348 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Laszlo, A. H. et al. Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA. Proc. Natl Acad. Sci. USA 110, 18904–18909 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Laszlo, A. H. et al. Decoding long nanopore sequencing reads of natural DNA. Nat. Biotechnol. 32, 829–833 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Swaminathan, J. et al. Highly parallel single-molecule identification of proteins in zeptomole-scale mixtures. Nat. Biotechnol. 36, 1076–1082 (2018).

    Article  CAS  Google Scholar 

  24. Requião, R. D. et al. Protein charge distribution in proteomes and its impact on translation. PLoS Comput. Biol. 13, e1005549 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Huang, G. et al. Electro-osmotic capture and ionic discrimination of peptide and protein biomarkers with FraC nanopores. Nat. Commun. 8, 935 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Metropolis, N., Rosenbluth, A. W., Rosenbluth, M. N., Teller, A. H. & Teller, E. Equation of state calculations by fast computing machines. J. Chem. Phys. 21, 1087–1092 (1953).

    Article  CAS  Google Scholar 

  27. Wiggins, P. A. An information-based approach to change-point analysis with applications to biophysics and cell biology. Biophys. J. 109, 346–354 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Craig, J. M. et al. Determining the effects of DNA sequence on Hel308 helicase translocation along single-stranded DNA using nanopore tweezers. Nucleic Acids Res. 47, 2506–2513 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Noakes, M. T. et al. Increasing the accuracy of nanopore DNA sequencing using a time-varying cross membrane voltage. Nat. Biotechnol. 37, 651–656 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Bhattacharya, S., Yoo, J. & Aksimentiev, A. Water mediates recognition of DNA sequence via ionic current blockade in a biological nanopore. ACS Nano 10, 4644–4651 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Nova, I. C. Nanopore data traces for PTM detection on peptides. Science Data Bank https://doi.org/10.57760/sciencedb.08338 (2023).

Download references

Acknowledgements

We thank A. Laszlo for discussions on the MCMC calculations, J. van der Torre for help in troubleshooting POC construction, E. van der Sluis and A. Goutou for Hel308 purification, and A. Aksimentiev for discussions. The work was supported by funding from the Dutch Research Council (NWO) project NWO-I680 (SMPS) (C.D.); European Research Council Advanced Grant 883684 (C.D.); European Commission Marie Skłodowska-Curie Fellowship 897672 (H.B.); and NIH NHGRI project HG012544-01 (J.G. and C.D.).

Author information

Author notes

  1. These authors contributed equally: Ian C. Nova, Justas Ritmejeris, Henry Brinkerhoff.

Authors and Affiliations

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

    Ian C. Nova, Justas Ritmejeris, Henry Brinkerhoff, Theo J. R. Koenig & Cees Dekker

  2. Department of Physics, University of Washington, Seattle, WA, USA

    Henry Brinkerhoff & Jens H. Gundlach

Authors
  1. Ian C. Nova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Justas Ritmejeris
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Henry Brinkerhoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Theo J. R. Koenig
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jens H. Gundlach
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Cees Dekker
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.B. and C.D. conceived of and designed the study. I.C.N., J.R. and T.J.R.K. performed nanopore experiments. J.R. established and troubleshooted the method for POC construction. I.C.N. performed computational analyses of the experimental data. H.B. performed the simulations. C.D. and J.H.G. supervised the work. I.C.N. wrote the initial manuscript draft, and all authors contributed to the writing of the final manuscript.

Corresponding author

Correspondence to Cees Dekker.

Ethics declarations

Competing interests

H.B. and C.D. have filed a provisional patent for the nanopore peptide measurement method (NL patent N2024579 P1600131NL00). The remaining authors declare no competing interests.

Peer review

Peer review information

Nature Biotechnology thanks Meni Wanunu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Figs. 1–16 and Text 1 and 2.

Reporting Summary

Supplementary Video 1

Video of mean phosphate position within pore for betacatenin variants across many hel308 steps. Derived from MCMC simulation and described in Supplementary Text.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nova, I.C., Ritmejeris, J., Brinkerhoff, H. et al. Detection of phosphorylation post-translational modifications along single peptides with nanopores. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-023-01839-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41587-023-01839-z

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research