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.

Rapid empirical discovery of optimal peptides for targeted proteomics

Abstract

We report a method for high-throughput, cost-efficient empirical discovery of optimal proteotypic peptides and fragment ions for targeted proteomics applications using in vitro–synthesized proteins. We demonstrate the approach using human transcription factors, which are typically difficult, low-abundance targets and empirically derived proteotypic peptides for 98% of the target proteins. We show that targeted proteomic assays developed using our approach facilitate robust in vivo quantification of human transcription factors.

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: Development of targeted proteomics assays using enriched in vitro–synthesized full-length proteins.
Figure 2: Targeted assays can be efficiently developed using in vitro–synthesized proteins and applied to measure proteins in vivo.

References

  1. Lange, V., Picotti, P., Domon, B. & Aebersold, R. Mol. Syst. Biol. 4, 222 (2008).

    Article  Google Scholar 

  2. Carr, S.A. & Anderson, L. Clin. Chem. 54, 1749–1752 (2008).

    CAS  Article  Google Scholar 

  3. Picotti, P. et al. Nat. Methods 5, 913–914 (2008).

    CAS  Article  Google Scholar 

  4. Prakash, A. et al. J. Proteome Res. 8, 2733–2739 (2009).

    CAS  Article  Google Scholar 

  5. Mallick, P. et al. Nat. Biotechnol. 25, 125–131 (2007).

    CAS  Article  Google Scholar 

  6. Fusaro, V.A., Mani, D.R., Mesirov, J.P. & Carr, S.A. Nat. Biotechnol. 27, 190–198 (2009).

    CAS  Article  Google Scholar 

  7. Picotti, P. et al. Nat. Methods 7, 43–46 (2010).

    CAS  Article  Google Scholar 

  8. Keshishian, H., Addona, T., Burgess, M., Kuhn, E. & Carr, S.A. Mol. Cell. Proteomics 6, 2212–2229 (2007).

    CAS  Article  Google Scholar 

  9. Goshima, N. et al. Nat. Methods 5, 1011–1017 (2008).

    CAS  Article  Google Scholar 

  10. Ramachandran, N. et al. Nat. Methods 5, 535–538 (2008).

    CAS  Article  Google Scholar 

  11. Vaquerizas, J.M., Kummerfeld, S.K., Teichmann, S.A. & Luscombe, N.M. Nat. Rev. Genet. 10, 252–263 (2009).

    CAS  Article  Google Scholar 

  12. Rolfs, A. et al. Proc. Natl. Acad. Sci. USA 105, 4364–4369 (2008).

    CAS  Article  Google Scholar 

  13. MacLean, B. et al. Bioinformatics 26, 966–968 (2010).

    CAS  Article  Google Scholar 

  14. Lee, M.E., Temizer, D.H., Clifford, J.A. & Quertermous, T. J. Biol. Chem. 266, 16188–16192 (1991).

    CAS  PubMed  Google Scholar 

  15. Klenova, E.M. et al. Mol. Cell. Biol. 13, 7612–7624 (1993).

    CAS  Article  Google Scholar 

  16. Dorschner, M.O. et al. Nat. Methods 1, 219–225 (2004).

    CAS  Article  Google Scholar 

  17. Maclean, B. et al. Anal. Chem. 82, 10116–10124 (2010).

    CAS  Article  Google Scholar 

  18. Eng, J.K., McCormack, A.L. & Yates, J.R. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994).

    CAS  Article  Google Scholar 

  19. Kall, L., Canterbury, J.D., Weston, J., Noble, W.S. & MacCoss, M.J. Nat. Methods 4, 923–925 (2007).

    Article  Google Scholar 

  20. Frewen, B.E., Merrihew, G.E., Wu, C.C., Noble, W.S. & MacCoss, M.J. Anal. Chem. 78, 5678–5684 (2006).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank P. von Haller, M. Bereman, E. Hommema and J. Rogers for their discussions and technical assistance. This work was supported in part by the University of Washington's Proteomics Resource (UWPR95794), the Thermo Scientific Pierce Human In vitro Translation Research Grant, and US National Institutes of Health grants P41RR011823 (M.J.M.) and U54HG004592 (J.A.S.).

Author information

Authors and Affiliations

Authors

Contributions

A.B.S., J.A.S. and M.J.M. conceived and designed the experiments, and wrote the paper. A.B.S. and K.L. performed the wet laboratory experiments. A.B.S. and B.M. analyzed the data.

Corresponding authors

Correspondence to John A Stamatoyannopoulos or Michael J MacCoss.

Ethics declarations

Competing interests

The authors received financial support from ThermoFisher Scientific.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Data 1–2, Supplementary Note 1 (PDF 4072 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Stergachis, A., MacLean, B., Lee, K. et al. Rapid empirical discovery of optimal peptides for targeted proteomics. Nat Methods 8, 1041–1043 (2011). https://doi.org/10.1038/nmeth.1770

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1770

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