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:

A protease for 'middle-down' proteomics

Abstract

We developed a method for restricted enzymatic proteolysis using the outer membrane protease T (OmpT) to produce large peptides (>6.3 kDa on average) for mass spectrometry–based proteomics. Using this approach to analyze prefractionated high-mass HeLa proteins, we identified 3,697 unique peptides from 1,038 proteins. We demonstrated the ability of large OmpT peptides to differentiate closely related protein isoforms and to enable the detection of many post-translational modifications.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: OmpT-based platform for middle-down proteomics and characterization of OmpT peptides from digestion of a standard protein.
Figure 2: Proteotypic OmpT peptides, peptide size distribution and iceLogo of OmpT recognition site.

Similar content being viewed by others

References

  1. de Godoy, L.M. et al. Nature 455, 1251–1254 (2008).

    Article  CAS  Google Scholar 

  2. Tran, J.C. et al. Nature 480, 254–258 (2011).

    Article  CAS  Google Scholar 

  3. Chait, B.T. Science 314, 65–66 (2006).

    Article  CAS  Google Scholar 

  4. Nesvizhskii, A.I. & Aebersold, R. Mol. Cell. Proteomics 4, 1419–1440 (2005).

    Article  CAS  Google Scholar 

  5. Forbes, A.J., Mazur, M.T., Patel, H.M., Walsh, C.T. & Kelleher, N.L. Proteomics 1, 927–933 (2001).

    Article  CAS  Google Scholar 

  6. Tran, J.C. & Doucette, A.A. Anal. Chem. 80, 1568–1573 (2008).

    Article  CAS  Google Scholar 

  7. Wu, S.L., Kim, J., Hancock, W.S. & Karger, B. J. Proteome Res. 4, 1155–1170 (2005).

    Article  CAS  Google Scholar 

  8. Taouatas, N., Drugan, M.M., Heck, A.J. & Mohammed, S. Nat. Methods 5, 405–407 (2008).

    Article  CAS  Google Scholar 

  9. Cannon, J. et al. J. Proteome Res. 9, 3886–3890 (2010).

    Article  CAS  Google Scholar 

  10. Kramer, R.A., Zandwijken, D., Egmond, M.R. & Dekker, N. Eur. J. Biochem. 267, 885–893 (2000).

    Article  CAS  Google Scholar 

  11. White, C.B., Chen, Q., Kenyon, G.L. & Babbitt, P.C. J. Biol. Chem. 270, 12990–12994 (1995).

    Article  CAS  Google Scholar 

  12. Dekker, N., Cox, R.C., Kramer, R.A. & Egmond, M.R. Biochemistry 40, 1694–1701 (2001).

    Article  CAS  Google Scholar 

  13. Okuno, K. et al. Biosci. Biotechnol. Biochem. 66, 127–134 (2002).

    Article  CAS  Google Scholar 

  14. Vandeputte-Rutten, L. et al. EMBO J. 20, 5033–5039 (2001).

    Article  CAS  Google Scholar 

  15. McCarter, J.D. et al. J. Bacteriol. 186, 5919–5925 (2004).

    Article  CAS  Google Scholar 

  16. Olsen, J.V. et al. Mol. Cell. Proteomics 8, 2759–2769 (2009).

    Article  CAS  Google Scholar 

  17. Dekker, N., Merck, K., Tommassen, J. & Verheij, H.M. Eur. J. Biochem. 232, 214–219 (1995).

    Article  CAS  Google Scholar 

  18. Lee, J.E. et al. J. Am. Soc. Mass Spectrom. 20, 2183–2191 (2009).

    Article  CAS  Google Scholar 

  19. Tran, J.C. & Doucette, A.A. Anal. Chem. 81, 6201–6209 (2009).

    Article  CAS  Google Scholar 

  20. Kramer, R.A. et al. Eur. J. Biochem. 269, 1746–1752 (2002).

    Article  CAS  Google Scholar 

  21. Wessel, D. & Flugge, U.I. Anal. Biochem. 138, 141–143 (1984).

    Article  CAS  Google Scholar 

  22. Elias, J.E. & Gygi, S.P. Nat. Methods 4, 207–214 (2007).

    Article  CAS  Google Scholar 

  23. Meng, F. et al. Nat. Biotechnol. 19, 952–957 (2001).

    Article  CAS  Google Scholar 

  24. Benjamini, Y. & Hochberg, Y. J. R. Stat. Soc. Series B Stat. Methodol. 57, 289–300 (1995).

    Google Scholar 

  25. Storey, J.D. & Tibshirani, R. Proc. Natl. Acad. Sci. USA 100, 9440–9445 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We sincerely thank current and former members of the Kelleher and Sweedler research groups, especially B. Evans, P. Chu, P. Compton, A. Catherman, S. Sweet, I. Ntai, J. Lee, A. Vellaichamy and K. Catherman, for technical support and insightful suggestions, P. Yau and B. Imai from the Protein Sciences Facility at the University of Illinois for the synthesis of the fluorogenic substrate and M. Burke for the access to a fluorimeter. The project was supported by the US National Institutes of Health through awards R01 GM067193, P30 DA018310 and F30 DA026672, by the National Science Foundation through award DMS 0800631 and by the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust.

Author information

Authors and Affiliations

Authors

Contributions

C.W. designed research, conducted experiments, analyzed data and wrote the paper; J.C.T., L.Z., K.R.D. and B.P.E. analyzed data; M.L., K.R.D. and D.R.A. conducted cell culture; P.M.T. analyzed data and wrote the paper; J.V.S. interpreted data and wrote the paper; N.L.K. designed research, interpreted data and wrote the paper.

Corresponding author

Correspondence to Neil L Kelleher.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Note (PDF 2307 kb)

Supplementary Table 1

Identified unique OmpT peptide lists and unique protein counts from Protein Center report. Unique OmpT peptides identified from absolute mass and biomarker searches are listed separately as well as in a combined list after removing the redundant overlapped hits from both search modes. (XLS 9183 kb)

Supplementary Table 2

Pooled unique OmpT peptide identifications from the nanoLC-MS/MS injections for the CID and ETD comparisons. (XLS 2739 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wu, C., Tran, J., Zamdborg, L. et al. A protease for 'middle-down' proteomics. Nat Methods 9, 822–824 (2012). https://doi.org/10.1038/nmeth.2074

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.2074

This article is cited by

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