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.

  • Protocol
  • Published:

Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal antibodies

Nature Protocols volume 9, pages 896–909 (2014)Cite this article

Subjects

Abstract

SUMOylation is a protein modification that regulates the function of hundreds of proteins. Detecting endogenous SUMOylation is challenging: most small ubiquitin-related modifier (SUMO) targets are low in abundance, and only a fraction of a protein's cellular pool is typically SUMOylated. Here we present a step-by-step protocol for the enrichment of endogenous SUMO targets from mammalian cells and tissues (specifically, mouse liver), based on the use of monoclonal antibodies that are available to the scientific community. The protocol comprises (i) production of antibodies and affinity matrix, (ii) denaturing cell lysis, and (iii) SUMO immunoprecipitation followed by peptide elution. Production of affinity matrix and cell lysis requires ∼1 d. The immunoprecipitation with peptide elution can be performed in 2 d. As SUMO proteins are conserved, this protocol should also be applicable to other organisms, including many vertebrates and Drosophila melanogaster.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Experimental design of the endogenous SUMO immunoprecipitation.
Figure 2: Monitoring the production of affinity matrix (Steps 9–23).
Figure 3: Monitoring SUMO immunoprecipitation (Steps 36–47).
Figure 4: Analysis of the SUMO-targets RanGAP1 and TFII-I.

Similar content being viewed by others

References

  1. Flotho, A. & Melchior, F. SUMOylation: a regulatory protein modification in health and disease. Annu. Rev. Biochem. 82, 357–385 (2013).

    Article  CAS  Google Scholar 

  2. Jentsch, S. & Psakhye, I. Control of nuclear activities by substrate-selective and protein-group SUMOylation. Annu. Rev. Genet. 47, 167–186 (2013).

    Article  CAS  Google Scholar 

  3. Gareau, J.R. & Lima, C.D. The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. Nat. Rev. Mol. Cell Biol. 11, 861–871 (2010).

    Article  CAS  Google Scholar 

  4. Cubenas-Potts, C. & Matunis, M.J. SUMO: a multifaceted modifier of chromatin structure and function. Dev. Cell 24, 1–12 (2013).

    Article  CAS  Google Scholar 

  5. Hickey, C.M., Wilson, N.R. & Hochstrasser, M. Function and regulation of SUMO proteases. Nat. Rev. Mol. Cell Biol. 13, 755–766 (2012).

    Article  CAS  Google Scholar 

  6. Hecker, C.M., Rabiller, M., Haglund, K., Bayer, P. & Dikic, I. Specification of SUMO1- and SUMO2-interacting motifs. J. Biol. Chem. 281, 16117–16127 (2006).

    Article  CAS  Google Scholar 

  7. Song, J., Durrin, L.K., Wilkinson, T.A., Krontiris, T.G. & Chen, Y. Identification of a SUMO-binding motif that recognizes SUMO-modified proteins. Proc. Natl. Acad. Sci. USA 101, 14373–14378 (2004).

    Article  CAS  Google Scholar 

  8. Song, J., Zhang, Z., Hu, W. & Chen, Y. Small ubiquitin-like modifier (SUMO) recognition of a SUMO binding motif: a reversal of the bound orientation. J. Biol. Chem. 280, 40122–40129 (2005).

    Article  CAS  Google Scholar 

  9. Watts, F.Z. Starting and stopping SUMOylation: What regulates the regulator? Chromosoma 122, 451–463 (2013).

    Article  CAS  Google Scholar 

  10. Da Silva-Ferrada, E., Lopitz-Otsoa, F., Lang, V., Rodriguez, M.S. & Matthiesen, R. Strategies to identify recognition signals and targets of SUMOylation. Biochem. Res. Int. 2012, 875148 (2012).

    Article  Google Scholar 

  11. Filosa, G., Barabino, S.M. & Bachi, A. Proteomics strategies to identify SUMO targets and acceptor sites: a survey of RNA-binding proteins SUMOylation. Neuromolecular. Med. 15, 661–676 (2013).

    Article  CAS  Google Scholar 

  12. Desterro, J.M., Rodriguez, M.S. & Hay, R.T. SUMO-1 modification of IκBα inhibits NF-κB activation. Mol. Cell 2, 233–239 (1998).

    Article  CAS  Google Scholar 

  13. Mahajan, R., Gerace, L. & Melchior, F. Molecular characterization of the SUMO-1 modification of RanGAP1 and its role in nuclear envelope association. J. Cell Biol. 140, 259–270 (1998).

    Article  CAS  Google Scholar 

  14. Rodriguez, M.S. et al. SUMO-1 modification activates the transcriptional response of p53. EMBO J. 18, 6455–6461 (1999).

    Article  CAS  Google Scholar 

  15. Sternsdorf, T., Jensen, K., Reich, B. & Will, H. The nuclear dot protein sp100, characterization of domains necessary for dimerization, subcellular localization, and modification by small ubiquitin-like modifiers. J. Biol. Chem. 274, 12555–12566 (1999).

    Article  CAS  Google Scholar 

  16. Pichler, A. et al. SUMO modification of the ubiquitin-conjugating enzyme E2-25K. Nat. Struct. Mol. Biol. 12, 264–269 (2005).

    Article  CAS  Google Scholar 

  17. Rodriguez, M.S., Dargemont, C. & Hay, R.T. SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J. Biol. Chem. 276, 12654–12659 (2001).

    Article  CAS  Google Scholar 

  18. Denison, C. et al. A proteomic strategy for gaining insights into protein SUMOylation in yeast. Mol. Cell Proteomics 4, 246–254 (2005).

    Article  CAS  Google Scholar 

  19. Panse, V.G., Hardeland, U., Werner, T., Kuster, B. & Hurt, E. A proteome-wide approach identifies SUMOylated substrate proteins in yeast. J. Biol. Chem. 279, 41346–41351 (2004).

    Article  CAS  Google Scholar 

  20. Zhou, W., Ryan, J.J. & Zhou, H. Global analyses of SUMOylated proteins in Saccharomyces cerevisiae. Induction of protein SUMOylation by cellular stresses. J. Biol. Chem. 279, 32262–32268 (2004).

    Article  CAS  Google Scholar 

  21. Nie, M., Xie, Y., Loo, J.A. & Courey, A.J. Genetic and proteomic evidence for roles of Drosophila SUMO in cell cycle control, Ras signaling, and early pattern formation. PLoS ONE 4, e5905 (2009).

    Article  Google Scholar 

  22. Kaminsky, R. et al. SUMO regulates the assembly and function of a cytoplasmic intermediate filament protein in C. elegans. Dev. Cell 17, 724–735 (2009).

    Article  CAS  Google Scholar 

  23. Miller, M.J., Barrett-Wilt, G.A., Hua, Z. & Vierstra, R.D. Proteomic analyses identify a diverse array of nuclear processes affected by small ubiquitin-like modifier conjugation in Arabidopsis. Proc. Natl. Acad. Sci. USA 107, 16512–16517 (2010).

    Article  CAS  Google Scholar 

  24. Tirard, M. et al. In vivo localization and identification of SUMOylated proteins in the brain of His6-HA-SUMO1 knock-in mice. Proc. Natl. Acad. Sci. USA 109, 21122–21127 (2012).

    Article  CAS  Google Scholar 

  25. Vertegaal, A.C. et al. Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics. Mol. Cell Proteomics 5, 2298–2310 (2006).

    Article  CAS  Google Scholar 

  26. Vertegaal, A.C. et al. A proteomic study of SUMO-2 target proteins. J. Biol. Chem. 279, 33791–33798 (2004).

    Article  CAS  Google Scholar 

  27. Blomster, H.A. et al. Novel proteomics strategy brings insight into the prevalence of SUMO-2 target sites. Mol. Cell Proteomics 8, 1382–1390 (2009).

    Article  CAS  Google Scholar 

  28. Galisson, F. et al. A novel proteomics approach to identify SUMOylated proteins and their modification sites in human cells. Mol. Cell Proteomics 10, M110 004796 (2011).

    Article  Google Scholar 

  29. Matic, I. et al. In vivo identification of human small ubiquitin-like modifier polymerization sites by high-accuracy mass spectrometry and an in vitro–to–in vivo strategy. Mol. Cell Proteomics 7, 132–144 (2008).

    Article  CAS  Google Scholar 

  30. Golebiowski, F. et al. System-wide changes to SUMO modifications in response to heat shock. Sci. Signal 2, ra24 (2009).

    Article  Google Scholar 

  31. Golebiowski, F., Tatham, M.H., Nakamura, A. & Hay, R.T. High-stringency tandem affinity purification of proteins conjugated to ubiquitin-like moieties. Nat. Protoc. 5, 873–882 (2010).

    Article  CAS  Google Scholar 

  32. Bruderer, R. et al. Purification and identification of endogenous polySUMO conjugates. EMBO Rep. 12, 142–148 (2011).

    Article  CAS  Google Scholar 

  33. Matafora, V., D'Amato, A., Mori, S., Blasi, F. & Bachi, A. Proteomics analysis of nucleolar SUMO-1 target proteins upon proteasome inhibition. Mol. Cell Proteomics 8, 2243–2255 (2009).

    Article  CAS  Google Scholar 

  34. Sarge, K.D. & Park-Sarge, O.K. Detection of proteins SUMOylated in vivo and in vitro. Methods Mol. Biol. 590, 265–277 (2009).

    Article  CAS  Google Scholar 

  35. Becker, J. et al. Detecting endogenous SUMO targets in mammalian cells and tissues. Nat. Struct. Mol. Biol. 20, 525–531 (2013).

    Article  CAS  Google Scholar 

  36. Matunis, M.J., Coutavas, E. & Blobel, G. A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J. Cell Biol. 135, 1457–1470 (1996).

    Article  CAS  Google Scholar 

  37. Zhang, X.D. et al. SUMO-2/3 modification and binding regulate the association of CENP-E with kinetochores and progression through mitosis. Mol. Cell 29, 729–741 (2008).

    Article  CAS  Google Scholar 

  38. Ong, S.E. & Mann, M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat. Protoc. 1, 2650–2660 (2006).

    Article  CAS  Google Scholar 

  39. Davis, J.M. Basic Cell Culture (Practical Approach Series) 2nd edn. (Oxford University Press, 2002).

  40. Vlasak, J. & Ionescu, R. Fragmentation of monoclonal antibodies. MAbs 3, 253–263 (2011).

    Article  Google Scholar 

Download references

Acknowledgements

We are grateful to M. Matunis (Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University) for making the hybridomas 21C7 and 8A2 available to the community. We thank all Melchior and Herzig laboratory members for sharing reagents and advice. This work was supported by the Deutsche Forschungsgemeinschaft (SPP1365, ME 2279/3 to F.M.), by the EU Network of Excellence Rubicon (to F.M.) and by a fellowship of the Excellence Cluster CellNetworks (to S.V.B.).

Author information

Author notes

  1. Sina V Barysch and Claudia Dittner: These authors contributed equally to this work.

Authors and Affiliations

  1. Zentrum für Molekulare Biologie Heidelberg (ZMBH), Deutsches Krebsforschungszentrum (DKFZ)-ZMBH Alliance, Heidelberg, Germany

    Sina V Barysch, Claudia Dittner, Annette Flotho, Janina Becker & Frauke Melchior

  2. Joint Division Molecular Metabolic Control, Zentrum für Molekulare Biologie Heidelberg, Deutsches Krebsforschungszentrum (DKFZ) and University Hospital Heidelberg, DKFZ-ZMBH Alliance, Heidelberg, Germany

    Claudia Dittner

Authors
  1. Sina V Barysch
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Claudia Dittner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Annette Flotho
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Janina Becker
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Frauke Melchior
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.B. and S.V.B. established the initial protocol; C.D. and A.F. contributed to its final version. C.D. carried out the experiments shown here. S.V.B., C.D., A.F. and F.M. wrote the manuscript. F.M. guided the development of this method.

Corresponding author

Correspondence to Frauke Melchior.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Barysch, S., Dittner, C., Flotho, A. et al. Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal antibodies. Nat Protoc 9, 896–909 (2014). https://doi.org/10.1038/nprot.2014.053

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2014.053

This article is cited by

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