Abstract
The covalent attachment of the small protein ubiquitin to other proteins is known to control a host of biological pathways and is emerging as an important regulatory factor in various processes specific to the nervous system. Ubiquitination is also tightly linked to most neurodegenerative disorders. A quantitative, proteome-wide view of the dynamic changes in ubiquitin modification associated with neuronal activity states and various stages of neurodegenerative disorders is therefore desired. Advances in quantitative mass spectrometry and the development of new biological tools make these approaches feasible for many laboratories. We describe here a combination of SILAC-based (stable isotope labeling by amino acids in cell culture) quantitative mass spectrometry and tandem-affinity purification to detect system-wide changes in ubiquitination and ubiquitin chain topologies that will be useful to probe the role of ubiquitin in the nervous system.
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References
Patrick, G. N. (2006) Synapse formation and plasticity: recent insights from the perspective of the ubiquitin proteasome system. Curr Opin Neurobiol 16, 90–4.
Paul, S. (2008) Dysfunction of the ubiquitin-proteasome system in multiple disease conditions: therapeutic approaches. Bioessays 30, 1172–84.
Segref, A., and Hoppe, T. (2009) Think locally: control of ubiquitin-dependent protein degradation in neurons. EMBO Rep 10, 44–50.
Hershko, A., and Ciechanover, A. (1998) The ubiquitin system. Annu Rev Biochem 67, 425–79.
Pickart, C. M. (2004) Back to the future with ubiquitin. Cell 116, 181–90.
Kim, I., and Rao, H. (2006) What’s Ub chain linkage got to do with it? Sci STKE 330, 18.
Kaiser, P., and Wohlschlegel, J. (2005) Identification of Ubiquitination Sites and Determination of Ubiquitin-Chain Architectures by Mass Spectrometry. Methods Enzymol 399C, 266–77.
Peng, J., Schwartz, D., Elias, J. E., Thoreen, C. C., Cheng, D., Marsischky, G., Roelofs, J., Finley, D., and Gygi, S. P. (2003) A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 21, 921–6.
Nijman, S. M., Luna-Vargas, M. P., Velds, A., Brummelkamp, T. R., Dirac, A. M., Sixma, T. K., and Bernards, R. (2005) A genomic and functional inventory of deubiquitinating enzymes. Cell 123, 773–86.
Xu, P., Duong, D. M., Seyfried, N. T., Cheng, D., Xie, Y., Robert, J., Rush, J., Hochstrasser, M., Finley, D., and Peng, J. (2009) Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137, 133–45.
Meierhofer, D., Wang, X., Huang, L., and Kaiser, P. (2008) Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry. J Proteome Res 7, 4566–76.
Mayor, T., Graumann, J., Bryan, J., MacCoss, M. J., and Deshaies, R. J. (2007) Quantitative profiling of ubiquitylated proteins reveals proteasome substrates and the substrate repertoire influenced by the Rpn10 receptor pathway. Mol Cell Proteomics 6, 1885–95.
Bennett, E. J., Shaler, T. A., Woodman, B., Ryu, K. Y., Zaitseva, T. S., Becker, C. H., Bates, G. P., Schulman, H., and Kopito, R. R. (2007) Global changes to the ubiquitin system in Huntington’s disease. Nature 448, 704–8.
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 vivocross-linking. Mol Cell Proteomics 5, 737–48.
Kaiser, P., and Huang, L. (2005) Global approaches to understanding ubiquitination. Genome Biol 6, 233.
Guerrero, C., Tagwerker, C., Kaiser, P., and Huang, L. (2006) An integrated mass spectrometry-based proteomic approach: quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network. Mol Cell Proteomics 5, 366–78.
Cronan, J. E., Jr. (1990) Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins. J Biol Chem 265, 10327–33.
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. Yeast 23, 623–32.
Savage, D., Mattson, G., Desai, S., Niedlander, G., Morgensen, S., and Conklin, E. (1994) Avidin-Biotin Chemistry: A Handbook, 2nd ed., Pierce Chemical, Rockford.
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, 173–81.
Ong, S. E., Foster, L. J., and Mann, M. (2003) Mass spectrometric-based approaches in quantitative proteomics. Methods 29, 124–30.
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, 376–86.
Mann, M. (2006) Functional and quantitative proteomics using SILAC. Nat Rev Mol Cell Biol 7, 952–8.
Blagoev, B., Ong, S. E., Kratchmarova, I., and Mann, M. (2004) Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat Biotechnol 22, 1139–45.
Acknowledgments
This work was supported by NIH (GM66164 and CA113823). David Meierhofer was an Erwin Schrödinger fellow supported by the FWF Austria.
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Meierhofer, D., Kaiser, P. (2011). Global Analysis of Ubiquitination. In: Li, K. (eds) Neuroproteomics. Neuromethods, vol 57. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-111-6_15
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DOI: https://doi.org/10.1007/978-1-61779-111-6_15
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