Abstract
Because virtually all cellular processes are based on proteins, detailed knowledge of the mitochondrial proteome represents an integral part of understanding mitochondrial function. The analysis of very complex protein mixtures such as entire cell organelles makes high demands on analysis techniques in order to ensure integrity of the obtained data set. The use of Saccharomyces cerevisiae as a model system allows the isolation of mitochondria of utmost purity in large amounts. Various approaches can be accomplished in the subsequent analysis to achieve the most complete overview possible. Combinations of orthogonal proteomics techniques include two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-SDS-PAGE), and nano-LC-MS/MS (nano-liquid chromatography with tandem mass spectrometry) as well as multidimensional high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The inherent limitations of the individual methods can be countervailed by parallel application of these approaches.
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References
Sickmann, A., Reinders, J., Wagner, Y., et al. (2003) The proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl. Acad. Sci. U. S. A. 100, 13,207–13,212.
Stasyk, T. and Huber, L. A. (2004) Zooming in: fractionation strategies in proteomics. Proteomics 4, 3704–3716.
Meisinger, C., Sommer, T., and Pfanner, N. (2000) Purification of Saccharomcyes cerevisiae mitochondria devoid of microsomal and cytosolic contaminations. Anal. Biochem. 287, 339–342.
Klose, J. (1975) Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik 26, 231–243.
O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.
Simpson, R. J., Connolly, L. M., Eddes, J. S., Pereira, J. J., Moritz, R. L., and Reid, G. E. (2000) Proteomic analysis of the human colon carcinoma cell line (LIM 1215): development of a membrane protein database. Electrophoresis 21, 1707–1732.
Wagner, Y., Sickmann, A., Meyer, H. E., and Daum, G. (2003) Multidimensional nano-HPLC for analysis of protein complexes. J. Am. Soc. Mass. Spectrom. 14, 1003–1011.
Hartinger, J., Stenius, K., Hogemann, D., and Jahn, R. (1996) 16-BAC/SDS-PAGE: a two-dimensional gel electrophoresis system suitable for the separation of integral membrane proteins Anal. Biochem. 240, 126–133.
Heukeshoven, J. and Dernick, R. (1988) Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels Electrophoresis 9, 28–32.
Sickmann, A., Dormeyer, W., Wortelkamp, S., Woitalla, D., Kuhn, W., and Meyer, H. E. (2000) Identification of proteins from human cerebrospinal fluid, separated by two-dimensional polyacrylamide gel electrophoresis. Electrophoresis 21, 2721–2728.
Eng, J. K., McCormack, A. L., Yates, I., and John, R. (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass. Spectrom. 5, 976–989.
Olsson, I., Larsson, K., Palmgren, R., and Bjellqvist, B. (2002) Organic disulfides as a means to generate streak-free two-dimensional maps with narrow range basic immobilized pH gradient strips as first dimension. Proteomics 2, 1630–1632.
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© 2007 Humana Press Inc., Totowa, NJ
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Reinders, J., Sickmann, A. (2007). Proteomics of Yeast Mitochondria. In: Leister, D., Herrmann, J.M. (eds) Mitochondria. Methods in Molecular Biology™, vol 372. Humana Press. https://doi.org/10.1007/978-1-59745-365-3_37
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DOI: https://doi.org/10.1007/978-1-59745-365-3_37
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