Summary
Two-dimensional electrophoresis (2DE) is an excellent technology for the analysis of complex protein mixtures, but it has drawbacks, such as hardly detecting very hydrophobic proteins. Shotgun protein analysis is one of the major technologies used to compensate for the weaknesses of 2DE. In this approach, total proteins are digested as a mixture and the digested peptides are separated by one-dimensional or multidimensional chromatography and introduced into a tandem mass spectrometer. Since the shotgun approach is the primary strategy in proteomics besides 2DE, a great number of related methodologies have been developed. In this chapter, we would like to introduce the simplest protocol, in which proteins are digested in solution and the digested peptides are analyzed by one-dimensional reversed-phase liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS), as a starting point for shotgun protein analysis.
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References
Rabilloud, T. (2002) Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains. Proteomics. 2, 3–10
Ong, S. E., and Pandey, A. (2001) An evaluation of the use of two-dimensional gel electrophoresis in proteomics. Biomol. Eng. 18, 195–205
Hunt, D. F., Henderson, R. A., Shabanowitz, J., Sakaguchi, K., Michel, H., Sevilir, N., et al (1992) Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science. 255, 1261–1263
McCormack, A. L., Schieltz, D. M., Goode, B., Yang, S., Barnes, G., Drubin, D., and Yates, J.R. 3rd (1997) Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level. Anal. Chem. 69, 767–776
Dongre, A. R., Eng, J. K., and Yates, J.R. 3rd (1997) Emerging tandem-mass-spectrometry techniques for the rapid identification of proteins. Trends. Biotechnol. 15, 418–425
Kubota, K., Kosaka, T., and Ichikawa, K. (2005) Combination of two-dimensional electrophoresis and shotgun peptide sequencing in comparative proteomics. J. Chromatogr. B. 815, 3–9
Link, A. J., Eng, J., Schieltz, D. M., Carmack, E., Mize, G. J., Morris, D. R., et al (1999) Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17, 676–682
Washburn, M. P., Wolters, D., and Yates, J.R. 3rd (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247
Liu, H., Sadygov, R. G., and Yates, J.R. 3rd (2004) A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal. Chem. 76, 4193–4201
Ishihama, Y., Oda, Y., Tabata, T., Sato, T., Nagasu, T., Rappsilber, J., and Mann, M. (2005) Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol. Cell. Proteomics. 4, 1265–1272
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
Aggarwal, K., Choe, L. H., and Lee, K. H. (2006) Shotgun proteomics using the iTRAQ isobaric tags. Brief. Funct. Genomic. Proteomic. 5, 112–120
Oda, Y., Huang, K., Cross, F. R., Cowburn, D., and Chait, B. T. (1999) Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 96, 6591–6596
Ong, S. E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A., and Mann, M. (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics. 1, 376–386
Wessel, D., and Flugge, U. I. (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143
Nesvizhskii, A. I. (2006) Protein identification by tandem mass spectrometry and sequence database searching. Methods Mol. Biol. 367, 87–120
Liska, A. J., and Shevchenko, A. (2003) Expanding the organismal scope of proteomics: cross-species protein identification by mass spectrometry and its implications. Proteomics. 3, 19–28
Kersey, P. J., Duarte, J., Williams, A., Karavidopoulou, Y., Birney, E., and Apweiler, R. (2004) The international protein index: an integrated database for proteomics experiments. Proteomics. 4, 1985–1988
Nesvizhskii, A. I., and Aebersold, R. (2005) Interpretation of shotgun proteomic data: the protein inference problem. Mol. Cell. Proteomics. 4, 1419–1440
MacCoss, M. J. (2005) Computational analysis of shotgun proteomics data. Curr. Opin. Chem. Biol. 9, 88–94
Acknowledgments
We would especially like to thank T. Yoneyama-Takazawa, R. Nakano, and Y. Fukushima for helping with the method optimization. We also acknowledge M. Kubota for her encouragement and helpful discussions.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Kubota, K., Kosaka, T., Ichikawa, K. (2009). Shotgun Protein Analysis by Liquid Chromatography-Tandem Mass Spectrometry. In: Tyther, R., Sheehan, D. (eds) Two-Dimensional Electrophoresis Protocols. Methods in Molecular Biology, vol 519. Humana Press. https://doi.org/10.1007/978-1-59745-281-6_32
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DOI: https://doi.org/10.1007/978-1-59745-281-6_32
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