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High-stringency tandem affinity purification of proteins conjugated to ubiquitin-like moieties

Nature Protocols volume 5, pages 873–882 (2010)Cite this article

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Abstract

The post-translational modification of proteins with ubiquitin and ubiquitin-like proteins (Ubl) is vital to many cellular functions, and thus the identification of Ubl targets is key to understanding their function. In most cases, only a small proportion of the cellular pool of proteins is found conjugated to a particular Ubl, making identification of Ubl targets technically challenging. For the purposes of proteomic analyses, we have developed a protocol for the large-scale purification of Ubl-linked proteins that minimizes sample contamination with noncovalent interactors and prevents the cleavage of Ubl–substrate bonds catalyzed by Ubl-specific proteases. This is achieved by introducing a denaturing lysis step (in the presence of sodium dodecyl sulfate and alkylating agents that irreversibly inhibit Ubl proteases) before TAP (tandem affinity purification) that allows for efficient purification of putative Ubl-specific substrates in a form suitable for proteomic analysis. The timescale from cell lysis to purified protein sample is 5–6 d.

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Figure 1: The pEFIRES-P/TAP-SUMO-2 vector and its expression products.
Figure 2: Overview of the TAP procedure for SUMO-2.

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References

  1. Geiss-Friedlander, R. & Melchior, F. Concepts in sumoylation: a decade on. Nat. Rev. Mol. Cell Biol. 8, 947–956 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Girdwood, D.W., Tatham, M.H. & Hay, R.T. SUMO and transcriptional regulation. Semin. Cell Dev. Biol. 15, 201–210 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Hoege, C., Pfander, B., Moldovan, G.L., Pyrowolakis, G. & Jentsch, S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Muller, S., Ledl, A. & Schmidt, D. SUMO: a regulator of gene expression and genome integrity. Oncogene 23, 1998–2008 (2004).

    Article  PubMed  Google Scholar 

  5. Pichler, A. & Melchior, F. Ubiquitin-related modifier SUMO1 and nucleocytoplasmic transport. Traffic 3, 381–387 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Stelter, P. & Ulrich, H.D. Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 425, 188–191 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Hay, R.T. Role of ubiquitin-like proteins in transcriptional regulation. Ernst Schering Res. Found. Workshop 173–192 (2006).

  8. Herrmann, J., Lerman, L.O. & Lerman, A. Ubiquitin and ubiquitin-like proteins in protein regulation. Circ. Res. 100, 1276–1291 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Jentsch, S. & Pyrowolakis, G. Ubiquitin and its kin: how close are the family ties? Trends Cell Biol. 10, 335–342 (2000).

    Article  CAS  PubMed  Google Scholar 

  10. Bergink, S. & Jentsch, S. Principles of ubiquitin and SUMO modifications in DNA repair. Nature 458, 461–467 (2009).

    Article  CAS  PubMed  Google Scholar 

  11. Tagwerker, C. et al. A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking. Mol. Cell Proteomics 5, 737–748 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Saitoh, H., Pu, R.T. & Dasso, M. SUMO-1: wrestling with a new ubiquitin-related modifier. Trends Biochem. Sci. 22, 374–376 (1997).

    Article  CAS  PubMed  Google Scholar 

  13. Hay, R.T. SUMO: a history of modification. Mol. Cell 18, 1–12 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Saitoh, H. & Hinchey, J. Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3. J. Biol. Chem. 275, 6252–6258 (2000).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  16. Andersen, J.S., Matic, I. & Vertegaal, A.C. Identification of SUMO target proteins by quantitative proteomics. Methods Mol. Biol. 497, 19–31 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. Rosas-Acosta, G., Russell, W.K., Deyrieux, A., Russell, D.H. & Wilson, V.G. A universal strategy for proteomic studies of SUMO and other ubiquitin-like modifiers. Mol. Cell Proteomics 4, 56–72 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. 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  PubMed  Google Scholar 

  19. Chung, T.L. et al. In vitro modification of human centromere protein CENP-C fragments by small ubiquitin-like modifier (SUMO) protein: definitive identification of the modification sites by tandem mass spectrometry analysis of the isopeptides. J. Biol. Chem. 279, 39653–39662 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Cooper, H.J. et al. Fourier transform ion cyclotron resonance mass spectrometry for the analysis of small ubiquitin-like modifier (SUMO) modification: identification of lysines in RanBP2 and SUMO targeted for modification during the E3 autoSUMOylation reaction. Anal. Chem. 77, 6310–6319 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Pedrioli, P.G. et al. Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software. Nat. Methods 3, 533–539 (2006).

    Article  CAS  PubMed  Google Scholar 

  22. Zhao, Y., Kwon, S.W., Anselmo, A., Kaur, K. & White, M.A. Broad spectrum identification of cellular small ubiquitin-related modifier (SUMO) substrate proteins. J. Biol. Chem. 279, 20999–21002 (2004).

    Article  CAS  PubMed  Google Scholar 

  23. Li, T. et al. Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: a proteomic analysis. Proc. Natl. Acad. Sci. USA 101, 8551–8556 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Vertegaal, A.C. et al. A proteomic study of SUMO-2 target proteins. J. Biol. Chem. (2004).

  25. Gregan, J. et al. Tandem affinity purification of functional TAP-tagged proteins from human cells. Nat. Protoc. 2, 1145–1151 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Puig, O. et al. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229 (2001).

    Article  CAS  PubMed  Google Scholar 

  27. Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032 (1999).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  29. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. 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  PubMed  Google Scholar 

  31. Mann, M. Functional and quantitative proteomics using SILAC. Nat. Rev. Mol. Cell Biol. 7, 952–958 (2006).

    Article  CAS  PubMed  Google Scholar 

  32. Ong, S.E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell Proteomics 1, 376–386 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Schimmel, J. et al. The ubiquitin-proteasome system is a key component of the SUMO-2/3 cycle. Mol. Cell Proteomics 7, 2107–2122 (2008).

    Article  CAS  PubMed  Google Scholar 

  34. Xirodimas, D.P. Novel substrates and functions for the ubiquitin-like molecule NEDD8. Biochem. Soc. Trans. 36, 802–806 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Wisniewski, J.R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009).

    Article  CAS  PubMed  Google Scholar 

  36. Ros, A. et al. Protein purification by off-gel electrophoresis. Proteomics 2, 151–156 (2002).

    Article  CAS  PubMed  Google Scholar 

  37. Nielsen, M.L. et al. Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nat. Methods 5, 459–460 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Hobbs, S., Jitrapakdee, S. & Wallace, J.C. Development of a bicistronic vector driven by the human polypeptide chain elongation factor 1alpha promoter for creation of stable mammalian cell lines that express very high levels of recombinant proteins. Biochem. Biophys. Res. Commun. 252, 368–372 (1998).

    Article  CAS  PubMed  Google Scholar 

  39. Wang, M., You, J., Bemis, K.G., Tegeler, T.J. & Brown, D.P. Label-free mass spectrometry-based protein quantification technologies in proteomic analysis. Brief Funct. Genomic Proteomic 7, 329–339 (2008).

    Article  CAS  PubMed  Google Scholar 

  40. Ong, S.E., Foster, L.J. & Mann, M. Mass spectrometric-based approaches in quantitative proteomics. Methods 29, 124–130 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Ong, S.E. & Mann, M. Mass spectrometry-based proteomics turns quantitative. Nat. Chem. Biol. 1, 252–262 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Gygi, S.P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 (1999).

    Article  CAS  PubMed  Google Scholar 

  43. Ishihama, Y., Rappsilber, J. & Mann, M. Modular stop and go extraction tips with stacked disks for parallel and multidimensional peptide fractionation in proteomics. J. Proteome Res. 5, 988–994 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Cox, J. et al. A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat. Protoc. 4, 698–705 (2009).

    Article  CAS  PubMed  Google Scholar 

  45. Pan, C., Kumar, C., Bohl, S., Klingmueller, U. & Mann, M. Comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions. Mol. Cell Proteomics 8, 443–450 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Park, S.K., Venable, J.D., Xu, T. & Yates, J.R. III A quantitative analysis software tool for mass spectrometry-based proteomics. Nat. Methods 5, 319–322 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

F.G. is supported by the Marie Curie Fellowship and Biotechnology and Biological Sciences Research Council. M.H.T. is supported by a Cancer Research UK program grant.

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Authors and Affiliations

  1. Welcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, Scotland, UK

    Filip Golebiowski, Michael H Tatham & Ronald T Hay

  2. Department of Molecular and Cellular Pharmacology, Faculty of Medicine, Graduate School of Medicine, Gunma University, Gunma, Japan

    Akihiro Nakamura

Authors
  1. Filip Golebiowski
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  2. Michael H Tatham
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  3. Akihiro Nakamura
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  4. Ronald T Hay
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Contributions

F.G. and M.H.T. were responsible for optimization of the protocol's efficiency and specificity, as well as for its integration with quantitative proteomic applications. A.N. originally applied and modified the standard TAP procedure to facilitate purification of Ubl covalent conjugates. R.T.H. provided valuable advice and discussion.

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Correspondence to Filip Golebiowski.

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The authors declare no competing financial interests.

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Golebiowski, F., Tatham, M., Nakamura, A. et al. High-stringency tandem affinity purification of proteins conjugated to ubiquitin-like moieties. Nat Protoc 5, 873–882 (2010). https://doi.org/10.1038/nprot.2010.40

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