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.

Identification of cross-linked peptides from complex samples

Abstract

We have developed pLink, software for data analysis of cross-linked proteins coupled with mass-spectrometry analysis. pLink reliably estimates false discovery rate in cross-link identification and is compatible with multiple homo- or hetero-bifunctional cross-linkers. We validated the program with proteins of known structures, and we further tested it on protein complexes, crude immunoprecipitates and whole-cell lysates. We show that it is a robust tool for protein-structure and protein-protein–interaction studies.

Your institute does not have access to this article

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: The pLink program.
Figure 2: CXMS analysis of purified protein samples or crude immunoprecipitates.

Accession codes

Accessions

Protein Data Bank

References

  1. Singh, P., Panchaud, A. & Goodlett, D.R. Anal. Chem. 82, 2636–2642 (2010).

    CAS  Article  PubMed  Google Scholar 

  2. Zhang, H. et al. Mol. Cell. Proteomics 8, 409–420 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Petrotchenko, E.V., Serpa, J.J. & Borchers, C.H. Mol. Cell. Proteomics 10, M110.001420 (2011).

    Article  PubMed  Google Scholar 

  4. Kao, A. et al. Mol. Cell. Proteomics 10, M110.002212 (2011).

    Article  PubMed  Google Scholar 

  5. Hermanson, G.T. Bioconjugate Techniques 2nd edn Ch. 4, 241–243 (Academic, London, 2008).

  6. Li, S. et al. Genes Dev. 25, 2409–2421 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Dragon, F. et al. Nature 417, 967–970 (2002).

    CAS  Article  PubMed  Google Scholar 

  8. Grandi, P. et al. Mol. Cell 10, 105–115 (2002).

    CAS  Article  PubMed  Google Scholar 

  9. Krogan, N.J. et al. Mol. Cell 13, 225–239 (2004).

    CAS  Article  PubMed  Google Scholar 

  10. Champion, E.A., Lane, B.H., Jackrel, M.E., Regan, L. & Baserga, S.J. Mol. Cell. Biol. 28, 6547–6556 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Lee, L.W., Lo, H.W. & Lo, S.J. Gene 455, 16–21 (2010).

    CAS  Article  PubMed  Google Scholar 

  12. Aittaleb, M. et al. Nat. Struct. Biol. 10, 256–263 (2003).

    CAS  Article  PubMed  Google Scholar 

  13. Lin, J. et al. Nature 469, 559–563 (2011).

    CAS  Article  PubMed  Google Scholar 

  14. Rinner, O. et al. Nat. Methods 5, 315–318 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Xu, H., Hsu, P.H., Zhang, L., Tsai, M.D. & Freitas, M.A. J. Proteome Res. 9, 3384–3393 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Su, C. et al. Nucleic Acids Res. 36, D632–D636 (2008).

    CAS  Article  PubMed  Google Scholar 

  17. Kubala, M.H., Kovtun, O., Alexandrov, K. & Collins, B.M. Protein Sci. 19, 2389–2401 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Panchaud, A., Singh, P., Shaffer, S.A. & Goodlett, D.R. J. Proteome Res. 9, 2508–2515 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Brenner, S. Genetics 77, 71–94 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors wish to express gratitude to A.F. Hühmer and D. Horn (Thermo Fisher Scientific) for discussion; L.-L. Du (National Institute of Biological Sciences, Beijing (NIBS)) for critical suggestions; S.J. Lo (Chang Gung University) for the fib-1GFP strain; E.-Z. Shen, H.-Q. Wang, X.-D. He and G.-H. Cai (NIBS) for help with microscopy, GFP immunoprecipitation and peptide experiments; and R. Aebersold and T. Walzthöni (ETH Zurich) for help with xQuest search. We thank Z. Yuan, C. Liu, R.-X. Sun, Y. Fu and other members of the pFind team for discussion and support. This work was funded by the Ministry of Science and Technology of China (863 grant 2007AA02Z1A7 and 973 grant 2010CB835203 to M.-Q.D.; 863 grant 2008AA022310 and 973 grant 2010CB835402 to K.Y.; 973 grants 2012CB910602 and 2010CB912701 to S.-M.H.), the CAS Knowledge Innovation Program (KGCX1-YW-13) and an ICT basic research grant to S.-M.H. We thank the municipal government of Beijing for funds allocated to NIBS.

Author information

Authors and Affiliations

Authors

Contributions

B.Y. performed most wet-lab experiments and data analysis, and contributed to manuscript preparation; Y.-J.W. developed pLink; M.Z. performed peptide experiments (with B.Y.), non-BS3 cross-linking experiments and Y2H experiments; S.-B.F. fixed software bugs and maintained the system; J.L., S.L. and S.-K.L. purified the UTP-B, CNGP and F15E11.13-F15E11.14 protein complexes, respectively; K.Z. and L.-Y.X. developed pLabel for cross-links; H.C., H.-F.C. and L.-H.W. contributed to pLink development; Y.-X.L. contributed to data analysis; Y.-H.D. contributed to non-BS3 cross-linking experiments; Z.H. and S.C. contributed to MS analysis; K.Y. provided crucial samples and contributed to data interpretation and manuscript preparation; S.-M.H. directed software development and revised the manuscript; M.-Q.D. conceived the study, directed wet-lab experiments and wrote the manuscript.

Corresponding authors

Correspondence to Si-Min He or Meng-Qiu Dong.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–18, Supplementary Tables 1–20 and Supplementary Note (PDF 4178 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Yang, B., Wu, YJ., Zhu, M. et al. Identification of cross-linked peptides from complex samples. Nat Methods 9, 904–906 (2012). https://doi.org/10.1038/nmeth.2099

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.2099

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