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Identification of SUMO-Conjugated Proteins and their SUMO Attachment Sites Using Proteomic Mass Spectrometry

  • James A. Wohlschlegel
Protocol
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 497)

Abstract

The covalent modification of cellular factors by the small ubiquitin-like modifier (SUMO) has emerged as a key regulatory pathway for many biological processes. One recent advance in the field of SUMO modification that has provided important insights into SUMO-mediated regulatory networks is the ability to use proteomic mass spectrometry to identify the substrates of SUMO modification as well as their sites of conjugation (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In this chapter, we describe a global strategy for affinity purifying and identifying a broad spectrum of SUMO-conjugated proteins and a focused approach for purifying a selected SUMO target and mapping its SUMO attachment site(s). Although both methods were initially developed for use in S. cerevisiae, they can be readily adapted to study the SUMO pathway in higher eukaryotes.

Key words

Mass spectrometry proteomics post-translational modifications Smt3∙SUMO. 

References

  1. 1.
    Denison, C., Kirkpatrick, D. S., and Gygi, S. P. (2005) Proteomic insights into ubiqui-tin and ubiquitin-like proteins. Curr. Opin. Chem. Biol. 9, 69–75.PubMedCrossRefGoogle Scholar
  2. 2.
    Hannich, J. T., Lewis, A., Kroetz, M. B., Li, S. J., Heide, H., Emili, A., and Hoch-strasser, M. (2005) Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae. J. Biol. Chem. 280, 4102–4110.PubMedCrossRefGoogle Scholar
  3. 3.
    Li, T., Evdokimov, E., Shen, R. F., Chao, C. C., Tekle, E., Wang, T., Stadtman, E. R., Yang, D. C., and Chock, P. B. (2004) Sumoylation of heterogeneous nuclear ribo-nucleoproteins, zinc finger proteins, and nuclear pore complex proteins: a proteomic analysis. Proc. Natl. Acad. Sci. USA 101, 8551–8556.PubMedCrossRefGoogle Scholar
  4. 4.
    Manza, L. L., Codreanu, S. G., Stamer, S. L., Smith, D. L., Wells, K. S., Roberts, R. L., and Liebler, D. C. (2004) Global shifts in protein sumoylation in response to elec-trophile and oxidative stress. Chem. Res. Toxicol. 17, 1706–1715.PubMedCrossRefGoogle Scholar
  5. 5.
    Panse, V. G., Hardeland, U., Werner, T., Kuster, B., and Hurt, E. (2004) A proteome-wide approach identifies sumoylated substrate proteins in yeast. J. Biol. Chem. 279, 41346–41351.PubMedCrossRefGoogle Scholar
  6. 6.
    Rosas-Acosta, G., Russell, W. K., Deyrieux, A., Russell, D. H., and Wilson, V. G. (2005) A universal strategy for proteomic studies of SUMO and other ubiquitin-like modifiers. Mol. Cell. Proteomics4, 56–72.Google Scholar
  7. 7.
    Vertegaal, A. C., Ogg, S. C., Jaffray, E., Rodriguez, M. S., Hay, R. T., Andersen, J. S., Mann, M., and Lamond, A. I. (2004) A proteomic study of SUMO-2 target proteins. J. Biol. Chem. 279, 33791–33798.PubMedCrossRefGoogle Scholar
  8. 8.
    Wohlschlegel, J. A., Johnson, E. S., Reed, S. I., and Yates, J. R., 3rd (2004) Global analysis of protein sumoylation in Saccharo-myces cerevisiae. J. Biol. Chem. 279, 45662–456628.PubMedCrossRefGoogle Scholar
  9. 9.
    Zhao, Y., Kwon, S. W., Anselmo, A., Kaur, K., and White, M. A. (2004) Broad spectrum identification of cellular small ubiq-uitin-related modifier (SUMO) substrate proteins. J. Biol. Chem. 279, 20999–21002.PubMedCrossRefGoogle Scholar
  10. 10.
    Zhou, W., Ryan, J. J., and Zhou, H. (2004) Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses. J. Biol. Chem. 279, 32262–32268.PubMedCrossRefGoogle Scholar
  11. 11.
    Florens, L., and Washburn, M. P. (2006) Proteomic analysis by multidimensional protein identification technology. Methods Mol. Biol. 328, 159–175.PubMedGoogle Scholar
  12. 12.
    Washburn, M. P., Wolters, D., and Yates, J. R., 3rd (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247.PubMedCrossRefGoogle Scholar
  13. 13.
    Denison, C., Rudner, A. D., Gerber, S. A., Bakalarski, C. E., Moazed, D., and Gygi, S. P. (2005) A proteomic strategy for gaining insights into protein sumoylation in yeast. Mol. Cell. Proteomics4, 246–254.PubMedCrossRefGoogle Scholar
  14. 14.
    Peng, J., Schwartz, D., Elias, J. E., Thoreen, C. C., Cheng, D., Marsischky, G., Roe-lofs, J., Finley, D., and Gygi, S. P. (2003) A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol. 21, 921–926.PubMedCrossRefGoogle Scholar
  15. 15.
    Wohlschlegel, J. A., Johnson, E. S., Reed, S. I., and Yates, J. R., 3rd (2006) Improved identification of SUMO attachment sites using C-terminal SUMO mutants and tailored protease digestion strategies. J. Pro-teome Res. 5, 761–770.CrossRefGoogle Scholar
  16. 16.
    Peng, J., Elias, J. E., Thoreen, C. C., Licklider, L. J., and Gygi, S. P. (2003) Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/ MS) for large-scale protein analysis: the yeast proteome. J. Proteome Res. 2, 43–50.PubMedCrossRefGoogle Scholar
  17. 17.
    Schirle, M., Heurtier, M. A., and Kuster, B. (2003) Profiling core proteomes of human cell lines by one-dimensional PAGE and liquid chromatography-tandem mass spectrometry. Mol. Cell. Proteomics2, 1297–1305.PubMedCrossRefGoogle Scholar
  18. 18.
    Tagwerker, C., Flick, K., Cui, M., Guerrero, C., Dou, Y., Auer, B., Baldi, P., Huang, L., and Kaiser, P. (2006) 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. Proteomics5, 737–748.PubMedGoogle Scholar
  19. 19.
    Tagwerker, C., Zhang, H., Wang, X., Larsen, L. S., Lathrop, R. H., Hatfield, G. W., Auer, B., Huang, L., and Kaiser, P. (2006) HB tag modules for PCR-based gene tagging and tandem affinity purification in Saccharomyces cerevisiae. Yeast23, 623–632.PubMedCrossRefGoogle Scholar
  20. 20.
    Kaiser, P., and Wohlschlegel, J. (2005) Identification of ubiquitination sites and determination of ubiquitin-chain architectures by mass spectrometry. Methods Enzymol. 399, 266–277.PubMedCrossRefGoogle Scholar
  21. 21.
    Florens, L., Carozza, M. J., Swanson, S. K., Fournier, M., Coleman, M. K., Workman, J. L., and Washburn, M. P. (2006) Analyzing chromatin remodeling complexes using shotgun proteomics and normalized spectral abundance factors. Methods40, 303–311.PubMedCrossRefGoogle Scholar
  22. 22.
    Li, S. J., and Hochstrasser, M. (1999) A new protease required for cell-cycle progression in yeast. Nature398, 246–251.PubMedCrossRefGoogle Scholar
  23. 23.
    Knuesel, M., Cheung, H. T., Hamady, M., Barthel, K. K., and Liu, X. (2005) A method of mapping protein sumoylation sites by mass spectrometry using a modified small ubiquitin-like modifier 1 (SUMO-1) and a computational program. Mol. Cell. P roteomics4, 1626–1636.CrossRefGoogle Scholar
  24. 24.
    Pedrioli, P. G., Raught, B., Zhang, X. D., Rogers, R., Aitchison, J., Matunis, M., and Aebersold, R. (2006) Automated identification of SUMOylation sites using mass spec-trometry and SUMmOn pattern recognition software. Nat. Methods3, 533–539.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • James A. Wohlschlegel
    • 1
  1. 1.Department of Biological ChemistryDavid Geffen School of Medicine, University of CaliforniaLos AngelesUSA

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