Abstract
The 20S proteasome is a multicatalytic protein complex, present in all eukaryotic cells, that plays a major role in intracellular protein degradation. In mammalian cells, this symmetrical cylindrical complex is composed of two copies each of seven different α and β subunits arranged into four stacked rings (α7β7β7α7). Separation by two-dimensional (2D) gel electrophoresis of the human erythrocytes 20S proteasome subunits and mass spectrometry (MS) identification of all the observed spots reveal the presence of multiple isoforms for most of the subunits. These isoforms could correspond to protein variants and/or posttranslational modifications that may influence the 20S proteasome proteolytic activity. Their characterization is therefore important to establish the rules governing structure/activity relationships of the human 20S proteasome. This chapter describes the use of a combination of proteomic approaches to characterize the human 20S proteasome subunit isoforms separated by 2D gel electrophoresis. A “top-down” strategy was developed to determine by electrospray MS the molecular mass of the intact protein after its passive elution from the gel. Comparison of the experimental molecular mass to the theoretical one can reveal the presence of possible modifications. “Bottom-up” proteomic approaches are then performed and, after protein digestion, tandem MS analyses of the modified peptides allow the characterization and location of the modification. These methods are discussed for the study of the human erythrocytes 20S proteasome subunit isoforms.
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References
Unno, M., Mizushima, T., Morimoto, Y., Tomisugi, Y., Tanaka, K., Yasuoka, N., et al. (2002) The structure of the mammalian 20S proteasome at 2.75 Å resolution. Structure 10, 609–618.
Orlowski, M. and Wilk, S. (2000) Catalytic activities of the 20S proteasome, a multicatalytic proteinase complex. Arch. Biochem. Biophys. 383, 1–16.
Rock, K. L. and Goldberg, A. L. (1999) Degradation of cell proteins and the generation of MHC class I-presented peptides. Annu. Rev. Immunol. 17, 739–779.
Morel, S., Levy, F., Burlet-Schiltz, O., Brasseur, F., Probst-Kepper, M., Peitrequin, A. L., et al. (2000) Processing of some antigens by the standard proteasome but not by the immunoproteasome results in poor presentation by dendritic cells. Immunity 12, 107–117.
Van den Eynde, B. J. and Morel, S. (2001) Differential processing of class-I-restricted epitopes by the standard proteasome and the immunoproteasome. Curr. Opin. Immunol. 13, 147–153.
Burlet-Schiltz, O., Claverol, S., Gairin, J. E., and Monsarrat, B. (2005) The use of mass spectrometry to identify antigens from proteasome processing. Methods Enzymol. 405, 264–300.
Macagno, A., Gilliet, M., Sallusto, F., Lanzavecchia, A., Nestle, F. O., and Groettrup, M. (1999) Dendritic cells up-regulate immunoproteasomes and the proteasome regulator PA28 during maturation. Eur. J. Immunol. 29, 4037–4042.
Noda, C., Tanahashi, N., Shimbara, N., Hendil, K. B., and Tanaka, K. (2000) Tissue distribution of constitutive proteasomes, immunoproteasomes, and PA28 in rats. Biochem. Biophys. Res. Commun. 277, 348–354.
Husom, A. D., Peters, E. A., Kolling, E. A., Fugere, N. A., Thompson, L. V., and Ferrington, D. A. (2004) Altered proteasome function and subunit composition in aged muscle. Arch. Biochem. Biophys. 421, 67–76.
Drews, O., Zong, C., and Ping, P. (2007) Exploring proteasome complexes by proteomic approaches. Proteomics 7, 1047–1058.
Claverol, S., Burlet-Schiltz, O., Girbal-Neuhauser, E., Gairin, J. E., and Monsarrat, B. (2002) Mapping and structural dissection of human 20S proteasome using proteomic approaches. Mol. Cell. Proteomics 1, 567–578.
Iwafune, Y., Kawasaki, H., and Hirano, H. (2002) Electrophoretic analysis of phosphorylation of the yeast 20S proteasome. Electrophoresis 23, 329–338.
Kurucz, E., Ando, I., Sumegi, M., Holzl, H., Kapelari, B., Baumeister, W., et al. (2002) Assembly of the Drosophila 26S proteasome is accompanied by extensive subunit rearrangements. Biochem. J. 365, 527–536.
Yang, P., Fu, H., Walker, J., Papa, C. M., Smalle, J., Ju, Y. M., et al. (2004) Purification of the Arabidopsis 26S proteasome: biochemical and molecular analyses revealed the presence of multiple isoforms. J. Biol. Chem. 279, 6401–6413.
Huang, L. and Burlingame, A. L. (2005) Comprehensive mass spectrometric analysis of the 20S proteasome complex. Methods Enzymol. 405, 187–236.
Froment, C., Uttenweiler-Joseph, S., Bousquet-Dubouch, M. P., Matondo, M., Borges, J. P., Esmenjaud, C., et al. (2005) A quantitative proteomic approach using two-dimensional gel electrophoresis and isotope-coded affinity tag labeling for studying human 20S proteasome heterogeneity. Proteomics 5, 2351–2363.
Gomes, A. V., Zong, C., Edmondson, R. D., Li, X., Stefani, E., Zhang, J., et al. (2006) Mapping the murine cardiac 26S proteasome complexes. Circ. Res. 99, 362–371.
Schmidt, F., Dahlmann, B., Janek, K., Kloss, A., Wacker, M., Ackermann, R., et al. (2006) Comprehensive quantitative proteome analysis of 20S proteasome subtypes from rat liver by isotope coded affinity tag and 2-D gel-based approaches. Proteomics 6, 4622–4632.
Castano, J. G., Mahillo, E., Aritzi, P., and Arribas, J. (1996) Phosphorylation of C8 and C9 subunits of the multicatalytic proteinase by casein kinase II and identification of the C8 phosphorylation sites by direct mutagenesis. Biochemistry 35, 3782–3789.
Kimura, Y., Takaoka, M., Tanaka, S., Sassa, H., Tanaka, K., Polevoda, B., et al. (2000) N(alpha)-acetylation and proteolytic activity of the yeast 20S proteasome. J. Biol. Chem. 275, 4635–4639.
Wang, X., Chen, C. F., Baker, P. R., Chen, P. I., Kaiser, P., and Huang, L. (2007) Mass spectrometric characterization of the affinity-purified human 26S proteasome complex. Biochemistry 46, 3553–3565.
Bousquet-Dubouch, M. P., Uttenweiler-Joseph, S., Ducoux-Petit, M., Matondo, M., Monsarrat, M., and Burlet-Schiltz, O. (2008) Organelle proteomics. In: Pflieger, D., Rossier, J. (eds.). Methods Mol. Biol. 432, 301–320.
Claverol, S., Burlet-Schiltz, O., Gairin, J. E., and Monsarrat, B. (2003) Characterization of protein variants and post-translational modifications: ESI-MSn analyses of intact protein eluted from polyacrylamide gels. Mol. Cell. Proteomics 2, 483–493.
Lee, L. W., Moomaw, C. R., Orth, K., McGuire, M. J., DeMartino, G. N., and Slaughter, C. A. (1990) Relationship among the subunits of the high molecular weight proteinase, macropain (proteasome). Biochim. Biophys. Acta. 1037, 178–185.
Beausoleil, S. A., Jedrychowski, M., Schwartz, D., Elias, J. E., Villen, J., Li, J., et al. (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc. Natl. Acad. Sci. USA 101, 12130–12135.
Beausoleil, S. A., Villen, J., Gerber, S. A., Rush, J., and Gygi S. P. (2006) A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 24, 1285–1292.
Krokhin, O. V., Antonovici, M., Ens, W., Wilkins, J. A., and Standing, K. G. (2006) Deamidation of-Asn-Gly-sequences during sample preparation for proteomics: consequences for MALDI and HPLC-MALDI analysis. Anal. Chem. 78, 6645–6650.
Baumeister, W., Walz, J., Zühl, F., and Seemüller, E. (1998) The proteasome: paradigm of a self-compartmentalizing protease. Cell 92, 367–380.
Hamdan, M., Galvani, M., and Righetti, P. G. (2001) Monitoring 2-D gel-induced modifications of proteins by MALDI-TOF mass spectrometry. Mass Spectrom. Rev. 20, 121–141.
Shevchenko, A., Loboda, A., Ens, W., Schraven, B., and Standing, K. G. (2001) Archived polyacrylamide gels as a resource for proteome characterization by mass spectrometry. Electrophoresis 22, 1194–1203.
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Uttenweiler-Joseph, S., Claverol, S., Sylvius, L., Bousquet-Dubouch, MP., Burlet-Schiltz, O., Monsarrat, B. (2008). Toward a Full Characterization of the Human 20S Proteasome Subunits and Their Isoforms by a Combination of Proteomic Approaches. In: Thompson, J.D., Ueffing, M., Schaeffer-Reiss, C. (eds) Functional Proteomics. Methods in Molecular Biology, vol 484. Humana Press. https://doi.org/10.1007/978-1-59745-398-1_8
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DOI: https://doi.org/10.1007/978-1-59745-398-1_8
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