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.

Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry

Nature volume 379, pages 466–469 (1996)Cite this article

Abstract

MOLECULAR analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry1 has been applied to the high-sensitivity sequencing of short peptides2, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electro-phoresis, using nano-electrospray3,4 tandem mass spectrometry5,6. As little as 5ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitroand thus may have potential antiangiogenic effects on solid tumours.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

Buy

All prices are NET prices.

References

  1. Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F. & Whitehouse, C. M. Science 246, 64–71 (1989).

    ADS  CAS  Article  Google Scholar 

  2. Hunt, D. F. et al. Science 255, 1261–1263 (1992).

    ADS  CAS  Article  Google Scholar 

  3. Wilm, M. S. & Mann, M. Int. J. Mass Spectrom. Ion Process. 136, 167–180 (1994).

    ADS  CAS  Article  Google Scholar 

  4. Wilm, M. & Mann, M. Analyt. Chem. 68, 1–8 (1996).

    CAS  Article  Google Scholar 

  5. Biemann, K. & Scoble, H. A. Science 237, 992–998 (1987).

    ADS  CAS  Article  Google Scholar 

  6. Hunt, D. F., Yates, J. R., Shabanowitz, J., Winston, S. & Hauer, C. R. Proc. natn. Acad. Sci. U.S.A. 83, 6233–6237 (1986).

    ADS  CAS  Article  Google Scholar 

  7. Mann, M. & Wilm, M. S. Analyt. Chem. 66, 4390–4399 (1994).

    CAS  Article  Google Scholar 

  8. Fotsis, T. et al. Proc. natn. Acad. Sci. U.S.A. 90, 2690–2694 (1993).

    ADS  CAS  Article  Google Scholar 

  9. Fotsis, T. et al. Nature 368, 237–239 (1994).

    ADS  CAS  Article  Google Scholar 

  10. Walker, J. E. et al. J. molec. Biol. 226, 1051–1072 (1992).

    CAS  Article  Google Scholar 

  11. Hunkapiller, M. et al. Nature 310, 105–111 (1984).

    ADS  CAS  Article  Google Scholar 

  12. Erdjument-Bromage, H., Lui, M., Sabatini, D. M., Snyder, S. H. & Tempst, P. Protein Sci. 3, 2435–2446 (1994).

    CAS  Article  Google Scholar 

  13. Matsudaira, P. (ed.) A Practical Guide to Protein and Peptide Purification for Microsequencing (Academic, San Diego, CA, 1993).

  14. Roepstorff, P. & Fohlman, J. Biomed. Mass Spectrom. 11, 601 (1984).

    CAS  Article  Google Scholar 

  15. Jenö, P., Mini, T., Hintermann, E. & Horst, M. Analyt. Biochem. 224, 451–455 (1995).

    Article  Google Scholar 

  16. Rosenfeld, J., Capdevielle, J., Guillemot, J. C. & Ferrara, P. Analyt. Biochem. 203, 173–179 (1992).

    CAS  Article  Google Scholar 

Download references

Author information

Author notes

  1. Lothar Schweigerer

    Present address: Children's Hospital, University of Marburg, Deutschhausstrasse 12, 35033, Marburg, Germany

  2. Theodore Fotsis

    Present address: Laboratory of Biological Chemistry, University of loannina Medical School, GR 45110, loannina, Greece

  3. Matthias Mann: To whom correspondence should be addressed.

Affiliations

  1. Protein & Peptide Group, EMBL, Meyerhofstrasse 1, 69117, Heidelberg, Germany

    Matthias Wilm, Andrej Shevchenko, Tony Houthaeve & Matthias Mann

  2. Division of Oncology and Haematology, Children's Hospital, University of Heidelberg, INF 150, 69120, Heidelberg, Germany

    Stephen Breit, Lothar Schweigerer & Theodore Fotsis

Authors
  1. Matthias Wilm
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrej Shevchenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tony Houthaeve
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephen Breit
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lothar Schweigerer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Theodore Fotsis
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Matthias Mann
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wilm, M., Shevchenko, A., Houthaeve, T. et al. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466–469 (1996). https://doi.org/10.1038/379466a0

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/379466a0

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced 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