Abstract
An understanding of gene function requires a complementation of gene and gene expression analysis by the systematic analysis of proteins. Progress in plant proteomics has been lagging behind animal and microbial proteomics due to the lack of plant genome data and the problems involved in successful protein extraction from plant material. With the sequencing of more and more plant genomes, this slow progress will soon be overcome. The moss Physcomitrella patens is a model organism in the field of plant functional genomics. P. patens is the first seedless plant for which the complete genome was sequenced. Genome annotation is currently in progress. While identification of proteins requires knowledge of all coding genes of the organism under study, gene annotation and functional characterization benefit greatly from the findings of proteome analysis. The proteome of P. patens is accessible and approaches are under way to increase the spectrum of proteomic methods applied to this plant. Here we provide a protocol for the extraction of proteins from P. patens and describe the basic and still most important method of proteome analysis, two-dimensional polyacrylamide electrophoresis of proteins. As this technique (not entirely unjustifiably) has the reputation of being unpredictably complicated, we provide a detailed protocol intended to reduce the reluctance that many scientists may have in using this technique.
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References
Rossignol, M., Peltier, J. B., Mock, H. P., Matros, A., Maldonado, A. M., and Jorrin, J. V. (2006) Plant proteome analysis: A 2004–2006 update. Proteomics 6, 5529–5548.
Pasquali, C., Frutiger, S., Wilkins, M. R., Hughes, G. J., Appel, R. D., Bairoch, A., Schaller, D., Sanchez, J. C., and Hochstrasser, D. F. (1996) Two-dimensional gel electrophoresis of Escherichia coli homogenates: the Escherichia coli SWISS-2DPAGE database. Electrophoresis 17, 547–555.
Gorg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R., and Weiss, W. (2000) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 1037–1053.
Cho, K., Torres, N. L., Subramanyam, S., Deepak, S. A., Sardesai, N., Han, O., Williams, C. E., Ishii, H., Iwahashi, H., and Rakwal, R. (2006) Protein extraction/solubilization protocol for monocot and dicot plant gel-based proteomics. J. Plant Biol. 49, 413–420.
Rose, J. K. C., Bashir, S., Giovannoni, J. J., Jahn, M. M., and Saravanan, R. S. (2004) Tackling the plant proteome: practical approaches, hurdles and experimental tools. Plant J. 39, 715–733.
Damerval, C., Devienne, D., Zivy, M., and Thiellement, H. (1986) Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat seedling proteins. Electrophoresis 7, 52–54.
Hurkman, W. J., and Tanaka, C. K. (1986) Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 81, 802–806.
Sarhan, F. and Perras, M. (1987) Accumulation of a high molecular weight protein during cold hardening of wheat (Triticum aestivum L). Plant Cell Physiol. 28, 1173–1179.
Granier, F. (1988) Extraction of plant proteins for two-dimensional electrophoresis. Electrophoresis 9, 712–718.
Saravanan, R. S. and Rose, J. K. C. (2004) A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics 4, 2522–2532.
Schaefer, D. G. and Zryd, J. P. (1997) Efficient gene targeting in the moss Physcomitrella patens. Plant J. 11, 1195–1206.
Frank, W., Holtorf, H., and Reski, R. (2005) Functional genomics in Physcomitrella. In Plant Functional Genomics (Leister, D., ed.). The Harworth Press, Binghamton, NY, pp. 203–234.
Reski, R. and Cove, D. J. (2004) Quick guide: Physcomitrella patens. Curr. Biol. 14, R261–R262.
Chen, S. X. and Harmon, A. C. (2006) Advances in plant proteomics. Proteomics 6, 5504–5516.
Bjellqvist, B. Ek, K. Righetti, P. G., Gianazza, E., Gorg, A., Westermeier, R., and Postel, W. (1982) Isoelectric focusing in immobilized pH gradients—principle, methodology and some applications. J. Biochem. Biophys. Methods 6, 317–339.
Ramagli, L. S. and Rodriguez, L. V. (1985) Quantitation of microgram amounts of protein in two-dimensional polyacrylamide gel electrophoresis sample buffer. Electrophoresis 6, 559–563.
Sarnighausen, E., Wurtz, V., Heintz, D., Van Dorsselaer, A., and Reski, R. (2004) Mapping of the Physcomitrella patens proteome. Phytochemistry 65, 1589–1607.
O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.
Hochstrasser, D. F., Patchornik, A., and Merril, C. R. (1988) Development of polyacrylamide gels that improve the separation of proteins and their detection by silver staining. Anal. Biochem. 173, 412–423.
Herbert, B., Galvani, M., Hamdan, M., Olivieri, E., MacCarthy, J., Pedersen, S., and Righetti, P. G. (2001) Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when and how? Electrophoresis 22, 2046–2057.
Herbert, B. R., Molloy, M. P., Gooley, A. A., Walsh, B. J., Bryson, W. G., and Williams, K. L. (1998) Improved protein solubility in two-dimensional electrophoresis using tributyl phosphine as reducing agent. Electrophoresis 19, 845–851.
Gallagher, S. R. (1995) One-dimensional SDS gel electrophoresis of proteins. In Current Protocols in Protein Science (Coligan, J. E. et al., eds.), John Wiley & Sons, Inc., New York, pp. 10.1.1–10.1.34.
Jacobs, D. I., van Rijssen, M. S., van der Heijden, R., and Verpoorte, R. (2001) Sequential solubilization of proteins precipitated with trichloroacetic acid in acetone from cultured Catharanthus roseus cells yields 52% more spots after two-dimensional electrophoresis. Proteomics 1, 1345–1350.
Jin, L. T., Hwang, S. Y., Yoo, G. S., and Choi, J. K. (2006) A mass spectrometry compatible silver staining method for protein incorporating a new silver sensitizer in sodium dodecyl sulfate-polyacrylamide electrophoresis gels. Proteomics 6, 2334–2337.
Chevallet, M., Diemer, H., Luche, S., Van Dorsselaer, A., Rabilloud, T., and Leize-Wagner, E. (2006) Improved mass spectrometry compatibility is afforded by ammoniacal silver staining. Proteomics 6, 2350–2354.
Cho, S. H., Hoang, Q. T., Kim, Y. T., Shin, H. Y., Ok, S. H., Bae, J. M., and Shin, J. S. (2006) Proteome analysis of gametophores identified a metallothionein involved in various abiotic stress responses in Physcomitrella patens. Plant Cell Rep. 25, 475–488.
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Sarnighausen, E., Reski, R. (2008). Plant Proteomics. 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_3
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DOI: https://doi.org/10.1007/978-1-59745-398-1_3
Publisher Name: Humana Press
Print ISBN: 978-1-58829-971-0
Online ISBN: 978-1-59745-398-1
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