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.

Single-molecule site-specific detection of protein phosphorylation with a nanopore

Abstract

We demonstrate single-molecule, site-specific detection of protein phosphorylation with protein nanopore technology. A model protein, thioredoxin, was phosphorylated at two adjacent sites. Analysis of the ionic current amplitude and noise, as the protein unfolds and moves through an α-hemolysin pore, enables the distinction between unphosphorylated, monophosphorylated and diphosphorylated variants. Our results provide a step toward nanopore proteomics.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Single-molecule nanopore detection of phosphorylation of a model substrate.
Figure 2: Single-molecule nanopore detection of four phosphorylation states.

References

  1. Prabakaran, S., Lippens, G., Steen, H. & Gunawardena, J. Wiley Interdiscip. Rev. Syst. Biol. Med 4, 565–583 (2012).

    CAS  Article  Google Scholar 

  2. Khoury, G.A., Baliban, R.C. & Floudas, C.A. Sci. Rep. 1, 90 (2011).

    CAS  Article  Google Scholar 

  3. Choudhary, C. & Mann, M. Nat. Rev. Mol. Cell Biol. 11, 427–439 (2010).

    CAS  Article  Google Scholar 

  4. Howorka, S. & Siwy, Z. Chem. Soc. Rev. 38, 2360–2384 (2009).

    CAS  Article  Google Scholar 

  5. Pennisi, E. Science 336, 534–537 (2012).

    CAS  Article  Google Scholar 

  6. Cherf, G.M. et al. Nat. Biotechnol. 30, 344–348 (2012).

    CAS  Article  Google Scholar 

  7. Manrao, E.A. et al. Nat. Biotechnol. 30, 349–353 (2012).

    CAS  Article  Google Scholar 

  8. Rodriguez-Larrea, D. & Bayley, H. Nat. Nanotechnol. 8, 288–295 (2013).

    CAS  Article  Google Scholar 

  9. Nivala, J., Marks, D.B. & Akeson, M. Nat. Biotechnol. 31, 247–250 (2013).

    CAS  Article  Google Scholar 

  10. Wallace, E.V. et al. Chem. Commun. (Camb.) 46, 8195–8197 (2010).

    CAS  Article  Google Scholar 

  11. Tam, J.P., Yu, Q. & Miao, Z. Biopolymers 51, 311–332 (1999).

    CAS  Article  Google Scholar 

  12. Hinner, M.J. & Johnsson, K. Curr. Opin. Biotechnol. 21, 766–776 (2010).

    CAS  Article  Google Scholar 

  13. Howorka, S., Cheley, S. & Bayley, H. Nat. Biotechnol. 19, 636–639 (2001).

    CAS  Article  Google Scholar 

  14. Witus, L.S. et al. J. Am. Chem. Soc. 132, 16812–16817 (2010).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank E. Mikhailova for the preparation of α-hemolysin pores. We thank L. Harrington for the PKA-CAT plasmid and the purification protocol for the catalytic subunit of PKA. This work has been supported by the National Institutes of Health, Oxford Nanopore Technologies and an European Research Council Advanced Grant. C.B.R. is funded by the Danish National Research Foundation (grant number DNRF78) and Aarhus University, Faculty of Science and Technology.

Author information

Authors and Affiliations

Authors

Contributions

C.B.R. performed experiments, analyzed data and wrote the paper. D.R.-L. planned the research, performed experiments, analyzed data and wrote the paper. H.B. planned the research and wrote the paper.

Corresponding authors

Correspondence to David Rodriguez-Larrea or Hagan Bayley.

Ethics declarations

Competing interests

H.B. is the founder, and a director and shareholder of Oxford Nanopore Technologies, a company engaged in the development of nanopore sequencing technology. Work in the Bayley laboratory at the University of Oxford is supported in part by Oxford Nanopore Technologies.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Table 1 (PDF 1704 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Rosen, C., Rodriguez-Larrea, D. & Bayley, H. Single-molecule site-specific detection of protein phosphorylation with a nanopore. Nat Biotechnol 32, 179–181 (2014). https://doi.org/10.1038/nbt.2799

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2799

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing