Abstract
The identification of target proteins for small ubiquitin-like modifiers (SUMOs) is a critical step towards a detailed understanding of the cellular functions of SUMOs. Substrate protein identification for SUMOs is hampered by the low abundance of SUMO targets, the finding that only a small fraction of these target proteins is sumoylated, and the high activity of deconjugating enzymes. Quantitative proteomics is a powerful tool to overcome these challenges, because it allows discrimination between contaminating proteins in SUMO-enriched preparations and true target proteins. In this chapter, the methodological details of the application of stable isotope labeling of amino acids in cell culture (SILAC) for the identification of target proteins for SUMOs are described. In addition to steady state sumoylation, the sumoylated proteome undergoes dynamic rearrangements in response to a diverse array of stimuli. SILAC also allows the study of sumoylation dynamics in response to these stimuli.
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References
Hay, R. T. (2005) SUMO: a history of modification. Mol. Cell 18, 1–12.
Xu, P., and Peng, J. (2006) Dissecting the ubiquitin pathway by mass spectrometry. Biochim. Biophys. Acta 1764, 1940–1947.
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.
Vertegaal, A. C., Andersen, J. S., Ogg, S. C., Hay, R. T., Mann, M., and Lamond, A. I. (2006) Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics. Mol. Cell Proteomics 5, 2298–2310.
Ong, S. E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A., and Mann, M. (2002) 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.
Ong, S. E., Kratchmarova, I., and Mann, M. (2003) Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC). J. Proteome Res. 2, 173–181.
Ong, S. E., and Mann, M. (2006) A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat. Protoc. 1, 2650–2660.
Andersen, J. S., Lam, Y. W., Leung, A. K., Ong, S. E., Lyon, C. E., Lamond, A. I., and Mann, M. (2005) Nucleolar proteome dynamics. Nature 433, 77–83.
Kratchmarova, I., Blagoev, B., Haack-Sorensen, M., Kassem, M., and Mann, M. (2005) Mechanism of divergent growth fac- tor effects in mesenchymal stem cell differentiation. Science 308, 1472–1477.
Trinkle-Mulcahy, L., Andersen, J., Lam, Y. W., Moorhead, G., Mann, M., and Lamond, A. I. (2006) Repo-Man recruits PP1 gamma to chromatin and is essential for cell viability. J. Cell Biol. 172, 679–692.
Saitoh, H., and Hinchey, J. (2000) Functional heterogeneity of small ubiqui-tin-related protein modifiers SUMO-1 versus SUMO-2/3. J. Biol. Chem. 275, 6252–6258.
Rappsilber, J., Ishihama, Y., and Mann, M. (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pre-treatment in proteomics. Anal. Chem. 75, 663–670.
Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R. (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999.
Mann, M. (2006) Functional and quantitative proteomics using SILAC. Nat. Rev. Mol. Cell Biol. 7, 952–958.
Matic, I., van Hagen, M., Schimmel, J., Macek, B., Ogg, S. C., Tatham, M. H., Hay, R. T., Lamond, A. I., Mann, M., and Verte-gaal, A. C. (2008) In vivo identification of human SUMO polymerization sites by high accuracy mass spectrometry and an in-vitro to in vivo strategy. Mol. Cell. Proteomics 7, 132–144.
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.
Acknowledgments
The authors would like to thank Professor Matthias Mann for comments on the manuscript. This work was supported by the Netherlands Organisation for Scientific Research (NWO) to ACOV as part of the Innovational Research Incentives Scheme and by a generous grant from the Danish National Research Foundation to Matthias Mann and JSA.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Andersen, J.S., Matic, I., Vertegaal, A.C.O. (2009). Identification of SUMO Target Proteins by Quantitative Proteomics. In: Ulrich, H.D. (eds) SUMO Protocols. METHODS IN MOLECULAR BIOLOGY™, vol 497. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-566-4_2
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DOI: https://doi.org/10.1007/978-1-59745-566-4_2
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