Summary
Two-dimensional (2-D) gel electrophoresis concerted with protein identification by mass spectrometry (MS) is an extremely powerful method for comparative expression profiling of complex protein samples such as cell lysates. The highly resolutive 2-D electrophoresis allows the separation of heterogeneous protein samples on the basis of isoelectric point (p I), molecular mass (Mr), solubility, and relative abundance ((1) J Biol Chem 250: 4007–4021, 1975; (2) Electrophoresis 14: 1067–1073, 1993). Consequently, it provides a comprehensive view of a proteome state ((3) Electrophoresis 21: 1037–1053, 2000), where variations in protein expression levels, isoforms, or post-translational modifications (e.g. phosphorylation) can be highlighted and investigated ((4) Electrophoresis 21: 2196–2208, 2000). Furthermore, this allows the identification of biological markers that characterize a specific physiological or pathological background of a cell or a tissue ((5) Proteomics 1: 397–408, 2001; (6) J Bacteriol 179: 7595–7599, 1997). In this way one can compare the effects of a stimulus or drug on cells or tissue, or more importantly, analyse the effects of disease on the expression level of proteins. Relatively recently, conventional 2-D gel electrophoresis has been combined with protein labelling strategies using up to three different fluorescent dyes to allow comparative analysis of different protein samples within a single 2-D gel platform. In this technique, termed differential in-gel electrophoresis (DIGE), samples are labelled separately then combined and run on the same 2D gel minimizing experimental variation and greatly facilitating spot matching. When three CyDyes (Cy2, Cy3, and Cy5) have been used, three images of the gel are captured then superposed to localize the differentially regulated spots on the 2-D gel using image analysis software. This is an extremely powerful tool in comparative proteomics as these dyes provide a linear response to protein concentration up to five orders of magnitude and great sensitivity with detection down to 125 pg of a single protein, which is less than needed for MS identification. In this chapter, we describe the basic methods for protein labelling, optimization of the isoelectrofocusing parameters for the first dimension (where proteins are separated according to their isoelectric point (p I)), sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) separation for the second dimension (based on molecular weight (MW)), and different post-staining protocols of the 2-D gel and protein preparation for mass spectrometry identification.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
O’Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250, 4007–4021.
Burstin, J., Zivy, M., de Vienne, D. & Damerval, C. (1993) Analysis of scaling methods to minimize experimental variations in two-dimensional electrophoresis quantitative data: application to the comparison of maize inbred lines. Electrophoresis 14, 1067–1073.
Görg, A., et al (2000) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 1037–1053.
Ducret, A., Desponts, C., Desmarais, S., Gresser, M.J. & Ramachandran, C. (2000) A general method for the rapid characterization of tyrosine-phosphorylated proteins by mini two-dimensional gel electrophoresis. Electrophoresis 21, 2196–2208.
Friso, G., Kaiser, L., Raud, J. & Wikstrom, L. (2001) Differential protein expression in rat trigeminal ganglia during inflammation. Proteomics 1, 397–408.
Lambert, L.A., Abshire, K., Blankenhorn, D. & Slonczewski, J.L. (1997) Proteins induced in Escherichia coli by benzoic acid. J Bacteriol 179, 7595–7599.
Blackstock, W.P. & Weir, M.P. (1999) Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotechnol 17, 121–127.
Aebersold, R. (2003) Constellations in a cellular universe. Nature 422, 115–116.
Aebersold, R. (2003) Quantitative proteome analysis: methods and applications. J Infect Dis 187 Suppl 2, S315–320.
Ducret, A., Van Oostveen, I., Eng, J.K., Yates, J.R., 3rd & Aebersold, R. (1998) High throughput protein characterization by automated reverse-phase chromatography/electrospray tandem mass spectrometry. Protein Sci 7, 706–719.
Figeys, D., Gygi, S.P., McKinnon, G. & Aebersold, R. (1998) An integrated microfluidics-tandem mass spectrometry system for automated protein analysis. Anal Chem 70, 3728–3734.
Gygi, S.P., Rist, B., Gerber, S.A., Turecek, F., Gelb, M.H., & Aebersold, R. (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 17, 994–999.
Haynes, P.A. & Yates, J.R., 3rd (2000) Proteome profiling-pitfalls and progress. Yeast 17, 81–87.
Link, A.J. Internal standards for 2-D. (1999) Methods Mol Biol 112, 281–284.
Link, A.J. Autoradiography of 2-D gels. (1999) Methods Mol Biol 112, 285–290.
Link, A.J. & Bizios, N. (1999) Measuring the radioactivity of 2-D protein extracts. Methods Mol Biol 112, 105–107.
Link, A.J., et al (1999) Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol 17, 676–682.
Washburn, M.P., Wolters, D. & Yates, J.R., 3rd (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19, 242–247.
Kalume, D.E., Molina, H. & Pandey, A. (2003) Tackling the phosphoproteome: tools and strategies. Curr Opin Chem Biol 7, 64–69.
Mann, M. & Jensen, O.N. (2003) Proteomic analysis of post-translational modifications. Nat Biotechnol 21, 255–261.
Packer, N.H., Ball, M.S. & Devine, P.L. (1999) Glycoprotein detection of 2-D separated proteins. Methods Mol Biol 112, 341–352.
Peters, E.C., Brock, A. & Ficarro, S.B. (2004) Exploring the phosphoproteome with mass spectrometry. Mini Rev Med Chem 4, 313–324.
Yan, J.X., Packer, N.H., Gooley, A.A. & Williams, K.L. (1998) Protein phosphorylation: technologies for the identification of phosphoamino acids. J Chromatogr A 808, 23–41.
Unlu, M., Morgan, M.E. & Minden, J.S. (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071–2077.
Chevallet, M., et al (1998) New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis. Electrophoresis 19, 1901–1909.
Gorg, A., Drews, O. & Weiss, W. (2004) Purifying Proteins for Proteomics, Simpson, R. J. (Ed.), Cold Spring Harbor Laboratory Press, New York, USA.
Rabilloud, T., Adessi, C., Giraudel, A. & Lunardi, J. (1997) Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18, 307–316.
Acknowledgements
We would like to thank the European Commission support for funding this work through the Marie Curie Actions Human Resources and Mobility Activity program.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science + Business Media, LLC
About this protocol
Cite this protocol
Larbi, N.B., Jefferies, C. (2009). 2D-DIGE: Comparative Proteomics of Cellular Signalling Pathways. In: McCoy, C.E., O’Neill, L.A.J. (eds) Toll-Like Receptors. Methods in Molecular Biology™, vol 517. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-541-1_8
Download citation
DOI: https://doi.org/10.1007/978-1-59745-541-1_8
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-72-5
Online ISBN: 978-1-59745-541-1
eBook Packages: Springer Protocols