Abstract
This chapter describes protocols for two-dimensional (2D) gel electrophoresis (isoelectric focusing [IEF] followed by sodium-dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis [PAGE]), staining of gels with the fluorescent dye Sypro Ruby, 2D gel image analysis, peptide mass fingerprint (PMF) analysis using matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS), liquid chromatography (LC)-tandem mass spectrometry (MS/MS), Western blot analysis of protein oxidations, and mass spectrometric mapping of sites of protein oxidations. Many of these methods were used to identify proteins affected in rat brain following ingestion of grape seed extract (GSE), a dietary supplement touted for anti-oxidant activity. Although beneficial actions in cell and animal models of chronic disease have been described for GSE, it has not been shown whether specific proteins were affected, or the nature of the effects. Applying 2D gel proteomics technology allowed discovery of proteins targeted by GSE without a priori knowledge of which one(s) might be affected. The newer 2D blue native (BN) electrophoresis methodology, which resolves protein complexes in a nondenaturing first dimension and then the components of these complexes in a denaturing second dimension, is discussed as a complementary approach. Analysis of protein oxidations and protein-protein interactions have special relevance to aging-related research, since oxidative stress and altered protein interactions may be at the heart of aging-related diseases. Finally, quality control issues related to implementation of high throughput technologies are addressed, to underscore the importance of minimizing bias and randomizing human and technical error in generating large datasets that are expensive and time-consuming to repeat.
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References
O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.
Bjellqvist, B., Ek, K., Righetti, P. G., et al. (1982) Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J. Biochem. Biophys. Methods 6, 317–339.
Gorg, A., Postel, W., and Gunther, S. (1988) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 9, 531–546.
Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, C. M. (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246, 64–71.
Tanaka, K., Waki, H., Ido, Y., Akita, S., Yoshida, Y., and Yoshida, T. (1988) Protein and polymer analyses up to m/z 100,000 by laser ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 2, 151–153.
Deshane, J., Chaves, L., Sarikonda, K. V., et al. (2004) Proteomics analysis of rat brain protein modulations by grape seed extract. J. Agric. Food Chem. 52, 7872–7883.
Schagger, H. and von Jagow, G. (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 199, 223–231.
Brookes, P. S., Pinner, A., Ramachandran, A., et al. (2002) High-throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of mitochondria and signalling complexes. Proteomics 2, 2969–2977.
Venkatraman, A., Landar, A., Davis, A. J., et al. (2004) Modification of the mitochondrial proteome in response to the stress of ethanol-dependent hepatoxicity. J. Biol. Chem. 279, 22,092–22,101.
Wilkins, M. R., Williams, K. L., Appel, R. D., and Hochstrasser, D. F. (eds) (2004) Proteome Research: New Frontiers in Functional Genomics. Springer, Berlin: p. 1997.
Bernard, K. R., Jonscher, K. R., Resing, K. A., and Ahn, N. G. (2004) Methods in functional proteomics: two-dimensional polyarylamide gel electrophoresis with immobilized pH gradients, in-gel digestion, and identification of proteins by mass spectrometry, in MAP Kinase Signaling Protocols (Seger, R., ed.). Humana, Totowa NJ: pp. 263–282.
Kim, H., Page, G., and Barnes, S. (2004) Proteomics and mass spectrometry in nutrition research. Nutrition 20, 155–165.
Washburn, M. P., Wolters, D., and Yates, J. R., III (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247.
Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R. (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999.
Yao, X., Freas, A., Ramirez, J., Demirev, P. A., and Fenselau, C. (2001) Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus. Anal. Chem. 73, 2836–2842.
Kelleher, N. L., Taylor, S. V., Grannis, D., et al. (1998) Efficient sequence analysis of the six gene products (7-74 kDa) from the Escherichia coli thiamin biosynthetic operon by tandem high-resolution mass spectrometry. Protein Sci. 7, 1796–1801.
Comisarow, M. B. and Marshall, A. G. (1996) The early development of Fourier transform ion cyclotron resonance (FT-ICR) spectroscopy. J. Mass Spectrom. 31, 581–585.
Senko, M. W., Hendrickson, C. L., Pasa-Tolic, L., et al. (1996) Electrospray ionization Fourier transform ion cyclotron resonance at 9.4 T. Rapid Commun. Mass Spectrom. 10, 1824–1828.
Carr, S., Aebersold, R., Baldwin, M., Burlingame, A., Clauser, K., and Nesvizhskii, A. (2004) The need for guidelines in publication of peptide and protein identification data. Mol. Cell. Proteomics 3, 531–533.
Meleth, S., Deshane, J., and Kim, H. (2005) The case for well-conducted experiments to validate statistical protocols for 2D gels: different pre-processing = different lists of significant proteins. BMC Biotechnology 5, 7.
Mardia, K. V., Kent, J. T., and Bibby, J. M. (1995) Chapter 8, in Multivariate Analysis. TJ Press (Padstow), Cornwall, UK.
Levine, R. L., Williams, J. A., and Stadtman, E. R. (1994) Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol. 233, 346–357.
Smith, C. D., Carney, J. M., Starke-Reed, P. E., et al. (1991) Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease. Proc. Natl. Acad. Sci. USA 88, 10,540–10,543.
Korolainen, M. A., Goldstein, G., Alafuzoff, I., Koistinaho, J. and Pirttila, T. (2002) Proteomic analysis of protein oxidation in Alzheimer’s disease brain. Electrophoresis 23, 3428–3433.
Abdul, H. M., Wenk, G. L., Gramling, M., Hauss-Wegrzyniak, B., and Butterfield, D. A. (2004) APP and PS-1 mutations induce brain oxidative stress independent of dietary cholesterol: implications for Alzheimer’s disease. J. Neurochem. 74, 2520–2527.
Kim, H., Eliuk, S., Deshane, J., Barnes, S., and Meleth, S. (2005) Nutriproteomics approach to understanding dementia-relevant brain protein changes in response to grape seed extract, a dietary anti-oxidant, in Oxidative Stress and Age-related Neurodegeneration (Luo, Y. and Packer, L., eds). CRC, Boca Raton, FL: Chapter 26.
Aksenov, M., Aksenova, M., Butterfield, D. A., and Markesbery, W. R. (2000) Oxidative modification of creatine kinase BB in Alzheimer’s disease brain. J. Neurochem. 74, 2520–2527.
Decker, L. A., ed. (1977) Worthington Enzyme Manual. Worthington Biochemical, Freehold, NJ.
Aslan, M., Ryan, T. M., Townes, T. M., et al. (2003) Nitric oxide dependent generation of reactive species in sickle cell disease. J. Biol. Chem. 278, 4194–4204.
Isom, A., Barnes, S., Wilson, L., et al. (2004) Modification of cytochrome c by 4-hydroxy-2-nonenal: evidence for histidine, lysine and arginine-aldehyde adducts. J. Am. Soc. Mass Spectrom. 15, 1136–1147.
Camacho-Carvajal, M. M., Wollscheid, B., Aebersold, R., Steimle, V., and Schamel, W. W. (2004) Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach. Mol. Cell. Proteomics 3, 176–182.
Culvenor, J. G., Ilaya, N. T., Ryan, M. T., et al. (2004) Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations. Eur. J. Biochem. 271, 375–385.
Galvani, M., Hamdan, M., Herbert, B., and Righetti, P. G. (2001) Alkylation kinetics of proteins in preparation for two-dimensional maps: a matrix-assisted laser desorption/ionization-mass spectrometry investigation. Electrophoresis 22, 2058–2065.
Bai, F., Liu, S., and Witzmann, F. A. (2005) A “destreaking” method for twodimensional electrophoresis using the reducing agent tris (2-carboxyethyl) phosphine hydrochloride and alkylating agent vinylpyridine. Proteomics 5, 2043–2047.
Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass Spectrometry sequencing of proteins from silver stained polyacrylamide gels. Anal. Chem. 68, 850–858.
Gharahdaghi, F., Weinberg, C. R., Meagher, D. A., Imai, B. S., and Mische, S. M. (1999) Mass Spectrometric identification of proteins from silver stained polyacrylamide gels: method for the removal of silver ions to enhance sensitivity. Electrophoresis 20, 601–605.
Lewis, T. S., Hunt, J. B., Aveline, L. D., et al. (2000) Identification of novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry. Mol. Cell. 6, 1343–1354.
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.
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.
Lilley, K. and Friedman, D. B. (2004) All about DIGE: quantification technology for differential-display 2D gel proteomics. Expert Rev. Proteomics 1, 1–9.
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Kim, H. et al. (2007). 2D Gel Proteomics. In: Tollefsbol, T.O. (eds) Biological Aging. Methods in Molecular Biology™, vol 371. Humana Press. https://doi.org/10.1007/978-1-59745-361-5_24
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DOI: https://doi.org/10.1007/978-1-59745-361-5_24
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