Summary
Mitochondria are complex organelles, and their proteomics analysis requires a combination of techniques. The emphasis in this chapter is made first on mitochondria preparation from cultured mammalian cells, then on the separation of the mitochondrial proteins with two-dimensional electrophoresis (2DE), showing some adjustment over the classical techniques to improve resolution of the mitochondrial proteins. This covers both the protein solubilization, the electrophoretic part per se, and the protein detection on the gels, which makes the interface with the protein identification part relying on mass spectrometry.
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References
Mokranjac, D., Neupert, W. (2005) Protein import into mitochondria. Biochem. Soc. Trans. 33, 1019–1023.
Iwata, S., Lee, J. W., Okada, K., Lee, J. K., Iwata, M., Rasmussen, B., et al. (1998) Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex. Science 281, 64–71.
Tsukihara, T., Aoyama, h., Yamashita, E., Tomizaki, T., Yamaguchi, h., Shinzawa-Itoh, K., et al. (1996) The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A. Science 272, 1136–1144.
Santoni, V., Molloy, M. P., Rabilloud, T. (2000) Membrane proteins and proteomics: un amour impossible? Electrophoresis 21, 1054–1070.
Eschenbruch, M., Bürk, R. R. (1982) Experimentally improved reliability of ultrasensitive silver staining of protein in polyacrylamide gels. Anal. Biochem. 125, 96–99.
Righetti, P. G. (1990) Immobilized pH gradients. Theory and methodology. Elsevier, Amsterdam.
Sanchez, J. C., Rouge, V., Pisteur, M., Ravier, F., Tonella, L., Moosmayer, M., et al. (1997) Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients. Electrophoresis 18, 324–327.
Gorg, A., Postel, W., Friedrich, C., Kuick, R., Strahler, J. R., Hanash, S. M. (1991) Temperature-dependent spot positional variability in two-dimensional polypeptide patterns. Electrophoresis 12, 653–658.
Tastet, C., Lescuyer, P., Diemer, h., Luche, S., van Dorsselaer, A., Rabilloud, T. (2003) A versatile electrophoresis system for the analysis of high- and low-molecular-weight proteins. Electrophoresis 24, 1787–1794.
Richert, S., Luche, S., Chevallet, M., Van Dorsselaer, A., Leize-Wagner, E., Rabilloud, T. (2004) About the mechanism of interference of silver staining with peptide mass spectrometry. Proteomics 4, 909–916.
Gharahdaghi, F., Weinberg, C. R., Meagher, D. A., Imai, B. S., Mische, S. M. (1999) Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 20, 601–605.
Rabilloud, T., Kieffer, S., Procaccio, V., Louwagie, M., Courchesne, P. L., Patterson, S. D., et al. (1998) Two-dimensional electrophoresis of human placental mitochondria and protein identification by mass spectrometry: toward a human mitochondrial proteome. Electrophoresis 19, 1006–1014.
Lescuyer, P., Strub, J. M., Luche, S., Diemer, h., Martinez, P., Van Dorsselaer, A., et al. (2003) Progress in the definition of a reference human mitochondrial proteome. Proteomics 3, 157–167.
Chevallet, M., Lescuyer, P., Diemer, h., van Dorsselaer, A., Leize-Wagner, E., Rabilloud T. (2006) Alterations of the mitochondrial proteome caused by the absence of mitochondrial DNA: a proteomic view. Electrophoresis 27, 1574–1583.
Zischka, h., Weber, G., Weber, P. J., Posch, A., Braun, R. J., Buhringer, D., et al. (2003) Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis. Proteomics 3, 906–916.
Luche, S., Santoni, V., Rabilloud, T. (2003) Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis. Proteomics 3, 249–253.
Luche, S., Diemer, h., Tastet, C., Chevallet, M., Van Dorsselaer, A., Leize-Wagner, E., et al. (2004) About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis. Proteomics 4, 551–561.
Granier, F., De Vienne, D. (1986) Silver staining of proteins: standardized procedure for two-dimensional gels bound to polyester sheets. Anal. Biochem. 155, 45–50.
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Rabilloud, T. (2008). Mitochondrial Proteomics. In: Pflieger, D., Rossier, J. (eds) Organelle Proteomics. Methods in Molecular Biology™, vol 432. Humana Press. https://doi.org/10.1007/978-1-59745-028-7_6
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DOI: https://doi.org/10.1007/978-1-59745-028-7_6
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