Abstract
In this report we present a detailed protocol for the analysis of differential protein expression between two plant tissue samples. The protocol involves harvesting of leaves and roots from mature tomato plants, preparing protein extracts from the harvested tissues, fluorescent labeling of each sample prior to differential in-gel electrophoresis (DIGE), first- and second-dimension electrophoretic separations, and image analysis to visualize and quantify differential protein expression. This protocol is adaptable for use with a wide variety of plant materials and can be used to measure protein expression changes occurring in response to abiotic stress, biotic stress, genetic manipulation, selective breeding, and many other conditions. In addition to the detailed protocol, we also present the results of a representative experiment analyzing subtle changes in protein expression in the roots of tomato plants grown under control and salt-stress conditions.
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References
O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.
Gorg, A., Obermaier, C., Boguth, G., et al. (2000) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 1037–1053.
Haynes, P. A., Fripp, N., and Aebersold, R. (1998) Identification of gel separated proteins by liquid chromatography-electrospray tandem mass spectrometry: comparison of methods and their limitations. Electrophoresis 19, 939–945.
Haynes, P., Miller, I., Aebersold, R., et al. (1998) Proteins of rat serum I: establishing a reference 2-DE map by immunodetection and microbore high performance liquid chromatography-electrospray mass spectrometry. Electrophoresis 19, 1484–1492.
Cooper, B., Eckert, D., Andon, N. L., Yates, J. R., and Haynes, P. A. (2003) Investigative proteomics: identification of an unknown plant virus from infected plants using mass spectrometry. J. Am. Soc. Mass Spectrom. 14, 736–741.
Andon, N. L., Hollingworth, S., Koller, A., Greenland, A. J., Yates, J. R. III, and Haynes, P. A. (2002) Proteomic characterization of wheat amyloplasts using identification of proteins by tandem mass spectrometry. Proteomics 2, 1156–1168.
Unlu, M., Morgan, M. E., and Minden, J. S. (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071–2077.
Tonge, R., Shaw, J., Middleton, B., et al. (2001) Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics 1, 377–396.
Von Eggeling, F., Gawriljuk, A., Fiedler, W., et al. (2001) Fluorescent dual colour 2D-protein gel electrophoresis for rapid detection of differences in protein pattern with standard image analysis software. Int. J. Mol. Med. 8, 373–377.
Zhou, G., Li, H., DeCamp, D., et al. (2002) 2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers. Mol. Cell. Proteomics 1, 117–124.
Shaw, J., Rowlinson, R., Nickson, J., et al. (2003) Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes. Proteomics 3, 1181–1195.
Hu, Y., Wang, G., Chen, G. Y., Fu, X., and Yao, S. Q. (2003) Proteome analysis of Saccharomyces cerevisiae under metal stress by two-dimensional differential gel electrophoresis. Electrophoresis 24, 1458–1470.
Kreil, D. P., Karp, N. A., and Lilley, K. S. (2004) DNA microarray normalization methods can remove bias from differential protein expression analysis of 2D difference gel electrophoresis results. Bioinformatics 20, 2026–2034.
Karp, N. A., Kreil, D. P., and Lilley, K. S. (2004) Determining a significant change in protein expression with DeCyder during a pair-wise comparison using two-dimensional difference gel electrophoresis. Proteomics 4, 1421–1432.
Alban, A., David, S. O., Bjorkesten, L., et al. (2003) A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3, 36–44.
Lee, J. R., Baxter, T. M., Yamaguchi, H., Wang, T. C., Goldenring, J. R., and Anderson, M. G. (2003) Differential protein analysis of spasomolytic polypeptide expressing metaplasia using laser capture microdissection and two-dimensional difference gel electrophoresis. Appl. Immunohistochem. Mol. Morphol. 11, 188–193.
Friedman, D. B., Hill, S., Keller, J. W., et al. (2004) Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics 4, 793–811.
Somiari, R. I., Sullivan, A., Russell, S., et al. (2003) High-throughput proteomic analysis of human infiltrating ductal carcinoma of the breast. Proteomics 3, 1863–1873.
Wang, D., Jensen, R., Gendeh, G., Williams, K., and Pallavicini, M. G. (2004) Proteome and transcriptome analysis of retinoic acid-induced differentiation of human acute promyelocytic leukemia cells, NB4. J. Proteome Res. 3, 627–635.
Ruepp, S. U., Tonge, R. P., Shaw, J., Wallis, N., and Pognan, F. (2002) Genomics and proteomics analysis of acetaminophen toxicity in mouse liver. Toxicol. Sci. 65, 135–150.
Kleno, T. G., Leonardsen, L. R., Kjeldal, H. O., Laursen, S. M., Jensen, O. N., and Baunsgaard, D. (2004) Mechanisms of hydrazine toxicity in rat liver investigated by proteomics and multivariate data analysis. Proteomics 4, 868–880.
Swatton, J. E., Prabakaran, S., Karp, N. A., Lilley, K. S., and Bahn, S. (2004) Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE). Mol. Psychiatry 9, 128–143.
Van den Bergh, G., Clerens, S., Vandesande, F., and Arckens, L. (2003) Reversed-phase high-performance liquid chromatography prefractionation prior to two-dimensional difference gel electrophoresis and mass spectrometry identifies new differentially expressed proteins between striate cortex of kitten and adult cat. Electrophoresis 24, 1471–1481.
Yan, J. X., Devenish, A. T., Wait, R., Stone, T., Lewis, S., and Fowler, S. (2002) Fluorescence two-dimensional difference gel electrophoresis and mass spectrometry based proteomic analysis of Escherichia coli. Proteomics 2, 1682–1698.
Damerval, C., de Vienne, 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.
Koller, A., Washburn, M. P., Lange, B. M., et al. (2002) Proteomic survey of metabolic pathways in rice. Proc. Natl. Acad. Sci. U. S. A. 99, 11969–11974.
Thiellement, H., Zivy, M., Colas des Francs, C., Bahrman, N., and Granier, F. (1987) Two-dimensional gel electrophoresis of proteins as a tool in wheat genetics. Biochimie 69, 781–787.
Tsugita, A., Kawakami, T., Uchiyama, Y., Kamo, M., Miyatake, N., and Nozu, Y. (1994) Separation and characterization of rice proteins. Electrophoresis 15, 708–720.
Wilm, M., Shevchenko, A., Houthaeve, T., et al. (1996) Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466–469.
Andon, N. L., Eckert, D., Yates, J. R. III, and Haynes, P. A. (2003) High-throughput functional affinity purification of mannose binding proteins from Oryza sativa. Proteomics 3, 1270–1278.
Berggren, K., Chernokalskaya, E., Steinberg, T. H., et al. (2000) Background-free, high sensitivity staining of proteins in one-and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex. Electrophoresis 21, 2509–2521.
Mackintosh, J. A., Choi, H. Y., Bae, S. H., et al. (2003) A fluorescent natural product for ultra sensitive detection of proteins in one-dimensional and two-dimensional gel electrophoresis. Proteomics 3, 2273–2288.
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© 2007 Humana Press Inc., Totowa, NJ
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Keeler, M., Letarte, J., Hattrup, E., Hickman, F., Haynes, P.A. (2007). Two-Dimensional Differential In-Gel Electrophoresis (DIGE) of Leaf and Roots of Lycopersicon esculentum . In: Thiellement, H., Zivy, M., Damerval, C., Méchin, V. (eds) Plant Proteomics. Methods in Molecular Biology, vol 355. Humana Press. https://doi.org/10.1385/1-59745-227-0:157
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DOI: https://doi.org/10.1385/1-59745-227-0:157
Publisher Name: Humana Press
Print ISBN: 978-1-58829-635-1
Online ISBN: 978-1-59745-227-4
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