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Mapping native disulfide bonds at a proteome scale

Nature Methods volume 12, pages 329331 (2015) | Download Citation

Abstract

We developed a high-throughput mass spectrometry method, pLink-SS (http://pfind.ict.ac.cn/software/pLink/2014/pLink-SS.html), for precise identification of disulfide-linked peptides. Using pLink-SS, we mapped all native disulfide bonds of a monoclonal antibody and ten standard proteins. We performed disulfide proteome analyses and identified 199 disulfide bonds in Escherichia coli and 568 in proteins secreted by human endothelial cells. We discovered many regulatory disulfide bonds involving catalytic or metal-binding cysteine residues.

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Acknowledgements

We thank the National BioResource Project of Japan for providing bacterial strains; the antibody center of National Institute of Biological Sciences, Beijing, for the purified anti–DAF-16 IgG2 antibody; and X.-Z. Dong, A. Hühmer, D. Horn and Z. Hao for bringing the disulfide-bond problem to our attention and for encouragement. We also thank all members of the pFind group, X. Xiong, Y. Xia, S. Chen, K. Ye, Y. Zhou and members of the Dong lab for discussion and experimental support. This work was funded by the National Scientific Instrumentation Grant Program (2011YQ09000506 to M.-Q.D.), National Natural Science Foundation of China (grant no. 21475141 to S.-M.H.), Ministry of Science and Technology of China (973 grants 2013CB911203, 2012CB910602 to R.-X.S. and 2010CB912701 to S.-M.H.), CAS Knowledge Innovation Program (grant #KGCX1-YW-13 and ICT-20126033 to S.-M.H.), Strategic Priority Research Program of CAS (XDB13040600 to Y.F.), NCMIS CAS and municipal government of Beijing.

Author information

Author notes

    • Shan Lu
    • , Sheng-Bo Fan
    •  & Bing Yang

    These authors contributed equally to this work.

Affiliations

  1. College of Life Science, Beijing Normal University, Beijing, China.

    • Shan Lu
    •  & Meng-Qiu Dong
  2. National Institute of Biological Sciences, Beijing, China.

    • Shan Lu
    • , Bing Yang
    • , Yu-Xin Li
    • , Mei-Jun Zhang
    •  & Meng-Qiu Dong
  3. Key Lab of Intelligent Information Processing, Institute of Computing Technology, Chinese Academy of Sciences (CAS), Beijing, China.

    • Sheng-Bo Fan
    • , Jia-Ming Meng
    • , Long Wu
    • , Kun Zhang
    • , Rui-Xiang Sun
    •  & Si-Min He
  4. University of Chinese Academy of Sciences, Beijing, China.

    • Sheng-Bo Fan
    • , Jia-Ming Meng
    • , Long Wu
    •  & Kun Zhang
  5. Institute of Molecular Medicine, Peking University, Beijing, China.

    • Pin Li
    •  & Jincai Luo
  6. National Center for Mathematics and Interdisciplinary Sciences, Key Laboratory of Random Complex Structures and Data Science, Academy of Mathematics and Systems Science, CAS, Beijing, China.

    • Yan Fu

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Contributions

S.L. and B.Y. performed experiments, analyzed data and prepared the manuscript; S.-B.F. analyzed data and developed pLink-SS; J.-M.M., L.W., K.Z., Y.F. and R.-X.S. helped software development and data analysis; Y.-X.L. performed statistical analysis, pathway analysis and protein network analysis; M.-J.Z. helped with informatics analysis; P.L. and J.L. prepared the HUVEC samples; S.-M.H. directed software development and edited the manuscript; M.-Q.D. directed wet-lab experiments and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

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

Integrated supplementary information

Supplementary figures

  1. 1.

    HCD outperformed electron-transfer dissociation (ETD) in identification of disulfide-linked synthetic peptides.

  2. 2.

    Fragment-ion types that are specific for disulfide-linked peptides and their contribution to disulfide bond identification.

  3. 3.

    Nonspecific protease digestion allowed the identification of all native disulfide bonds in RNase A.

  4. 4.

    Workflows of protein disulfide bond identification for simple and complex samples.

  5. 5.

    Representative HCD spectra showing the identification of two disulfide bonds in the variable regions of a mouse monoclonal antibody (anti–DAF-16, IgG2).

  6. 6.

    Representative HCD spectra showing the identification of nine disulfide bonds in the constant regions of a mouse monoclonal antibody (anti-DAF-16, IgG2).

  7. 7.

    Mapping of disulfide bonds in a ten-protein mixture.

  8. 8.

    HCD spectra of disulfide-containing peptide(s) in a loop-linked form or a complex form.

  9. 9.

    Specific proteases outperformed nonspecific proteases in disulfide-bond analysis of complex protein samples.

  10. 10.

    Interprotein and intraprotein disulfide bonds identified from complex samples are filtered separately.

  11. 11.

    Known and novel protein disulfide bonds identified in the E. coli periplasmic fraction.

  12. 12.

    Eight purified E. coli proteins and the verification results of their disulfide bonds using undigested proteins.

  13. 13.

    The formation of the intersubunit disulfide bonds of LuxS is redox sensitive and may affect AI-2 production.

  14. 14.

    Changes in protein disulfide bonds and glutathionylation in human A549 cells upon induction of oxidative stress.

  15. 15.

    Enrichment of the CXXC-type disulfide bonds in E. coli.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15 and Supplementary Tables 1, 2 and 10

Excel files

  1. 1.

    Supplementary Table 3

    Disulfide bonds identified from the E. coli periplasmic fraction

  2. 2.

    Supplementary Table 4

    Potential substrates of DsbA

  3. 3.

    Supplementary Table 5

    Potential substrates of DsbC

  4. 4.

    Supplementary Table 6

    Disulfide bonds identified in human A549 cells

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    Supplementary Table 7

    Glutathionylation sites identified in human A549 cells

  6. 6.

    Supplementary Table 8

    Disulfide bonds identified in secreted HUVEC proteins

  7. 7.

    Supplementary Table 9

    Candidate allosteric disulfide bonds in HUVEC proteins

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DOI

https://doi.org/10.1038/nmeth.3283