Abstract
Wheat is a major crop in world agriculture and is consumed after processing into a range of food products. It is therefore of great importance to determine the consequences (intended and unintended) of transgenesis in wheat and whether genetically modified lines are substantially equivalent to those produced by conventional plant breeding. Proteomic analysis is one of several approaches which can be used to address these questions. Two-dimensional PAGE (2D PAGE) remains the most widely available method for proteomic analysis, but is notoriously difficult to reproduce between laboratories. We therefore describe methods which have been developed as standard operating procedures in our laboratory to ensure the reproducibility of proteomic analyses of wheat using 2D PAGE analysis of grain proteins.
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References
Safety Evaluation of Foods Derived Through Modern Biotechnology: Concepts and Principles (1993) OECD, Paris
Kuiper, H. A., Kleter, G. A., Noteborn, H. P. J. M. Kok, E. S. (2001) Assessment of the food safety issues related to genetically modified foods. Plant J. 27,503–528.
Shewry, P. R., Baudo, M., Lovegrove, A., Napier, J., Ward, J., Baker, J. Beale, M. (2007) Are GM and conventionally bred cereals really different? Trends Food Sci. Technol. 18,201–209.
Skylas, D. J., Mackintosh, J. A., Cordwell, S. J., Basseal, D. J., Walsh, B. J., Harry, J., Blumenthal, C., Copeland, L., Wrigley, C. W. Rathmell, W. (2000) Proteome approach to the characterisation of protein composition in the developing and mature wheat-grain endosperm. J. Cereal Sci. 32,169–188.
Amiour, N., Merlino, M., Leroy, P. Branlard, G. (2002) Proteomic analysis of amphiphilic proteins of hexaploid wheat. Proteomics 2,632–641.
Salt, L. J, Robertson, J. A., Jenkins, J. A., Mulholland, F. Mills, E. N. C. (2005) The identification of foam-forming soluble proteins from wheat (Triticum aestivum. ) dough Proteomics 5,1612–1623.
Vensel, W. H., Tanaka, C. K., Cari, N., Wong, J. H., Buchanan, B. B. Hurkman, W. J. (2005) Developmental changes in the metabolic protein profiles of wheat endosperm. Proteomics 5,1594–1611.
Ruebelt, M. C., Leimgruber, N. K., Lipp, M., Reynolds, T. L., Nemeth, M. A., Astwood, J. D., Engel, K.-H. Jany, K.-D. (2006) Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 1. Assessing analytical variation. J. Agr. Food Chem. 54,2154–2161.
Ruebelt, M. C., Lipp, M., Reynolds, T. L., Astwood, J. D., Engell, K.-H. Jany, K.-D. (2006) Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 1. Assessing natural variability. J. Agr. Food Chem. 54,2162–2168.
Ruebelt, M. C., Lipp, M., Reynolds, T. L., Schmuke, J. J., Astwood, J. D., DellaPenna, D., Engel, K.-H. Jany, K.-D. (2006) Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 1. Assessing unintended effects. J. Agr. Food Chem. 54,2169–2177.
Finnie, C., Mechior, S., Roepstorff, P. Svensson, B. (2002) Proteome analysis of grain filling and seed maturation in barley. Plant Physiol. 129,1308–1319.
Osborne, T. B. (1907) The Proteins of the Wheat Kernel. Publication No 84, Carnegie Institute.
Yan, J. X., Wait, R., Berkelman, T., Harry, R. A., Westbrook, J. A., Wheeler, C. H. Dunn, M. J. (2000) A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21,3666–3672.
Jensen, O. N., Wilm, M., Shevchenko, A. Mann, M. (1999) Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels, in. 2-D Proteome Analysis Protocols, (Link, A., ed.), Methods in Molecular Biology Series, Vol. 112,Humana Press, Totowa,513–530.
Kristensen, D. B., Imamura, K., Miyamoto, Y. Yoshizato, K. (2000) Mass spectrometric approaches for the characterisation of proteins on a hybrid quadrupole time-of-flight (Q-Tof) mass spectrometer. Electrophoresis 21,430–439.
Hall, N. P. Tolbert, N. E. (1978) A rapid procedure for the isolation of Rubisco from spinach leaves. FEBS Lett. 96167–169.
Acknowledgements
Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK. This work was supported by the Food Standards Agency under the GO2 programme.
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Lovegrove, A., Salt, L., Shewry, P.R. (2009). Establishing Substantial Equivalence: Proteomics. In: Jones, H., Shewry, P. (eds) Transgenic Wheat, Barley and Oats. Methods in Molecular Biology™, vol 478. Humana Press. https://doi.org/10.1007/978-1-59745-379-0_16
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DOI: https://doi.org/10.1007/978-1-59745-379-0_16
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