Summary
Because the dynamic range of most cell or tissue proteomes is enormous, separation of such complex protein samples by two-dimensional electrophoresis (2-DE) on broad pH gradients often results in the visualization of only the most abundantly expressed proteins. It is, therefore, often beneficial to first subdivide the proteome in smaller, less complex fractions before 2-DE. This enables the analysis of a larger number of proteins. One approach to prefractionate protein samples is by reversed-phase high-performance liquid chromatography (RP-HPLC), separating proteins according to their hydrophobicity. This effectively introduces a third separation dimension, increasing the spatial resolution of the experiment. Here, we will describe a procedure for separating whole protein lysates by RP-HPLC, before their analysis by 2-DE or 2-D difference gel electrophoresis
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 subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
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.
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.
Ünlü, 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., Rowlinson, R., Rayner, S., Young, J., Pognan, F., Hawkins, E., Currie, I., and Davison, M. (2001) Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics. 1, 377–396.
Alban, A., David, S. O., Bjorkesten, L., Andersson, C., Sloge, E., Lewis, S., and Currie, I. (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.
Knowles, M. R., Cervino, S., Skynner, H. A., Hunt, S. P., de Felipe, C., Salim, K., Meneses-Lorente, G., McAllister, G., and Guest, P. C. (2003) Multiplex proteomic analysis by two-dimensional differential in-gel electrophoresis. Proteomics 3, 1162–1171.
Van den Bergh, G. and Arckens, L. (2004) Fluorescent two-dimensional difference gel electrophoresis unveils the potential of gel-based proteomics. Curr. Opin. Biotechnol. 15, 38–43.
Rabilloud, T., Kieffer, S., Procaccio, V., Louwagie, M., Courchesne, P. L., Patterson, S. D., Martinez, P., Garin, J., and Lunardi, J. (1998) Two-dimensional electrophoresis of human placental mitochondria and protein identification by mass spectrometry: towards a human mitochondrial proteome. Electrophoresis 19, 1006–1014.
Murayama, K., Fujimura, T., Morita, M., and Shindo, N. (2001) One-step subcellular fractionation of rat liver tissue using a Nycodenz density gradient prepared by freezing-thawing and two-dimensional sodium dodecyl sulfate electrophoresis profiles of the main fraction of organelles. Electrophoresis 22, 2872–2880.
Gronborg, M., Kristiansen, T. Z., Stensballe, A., Andersen, J. S., Ohara, O., Mann, M., Jensen, O. N., and Pandey, A. (2002) A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho- specific antibodies: identification of a novel protein, Frigg, as a protein kinase A substrate. Mol. Cell Proteomics. 1, 517–527.
Molloy, M. P., Herbert, B. R., Walsh, B. J., Tyler, M. I., Traini, M., Sanchez, J. C., Hochstrasser, D. F., Williams, K. L., and Gooley, A. A. (1998) Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 19, 837–844.
Badock, V., Steinhusen, U., Bommert, K., and Otto, A. (2001) Prefractionation of protein samples for proteome analysis using reversed-phase high-performance liquid chromatography. Electrophoresis 22, 2856–2864.
Van den Bergh, G., Clerens, S., Vandesande, F., and Arckens, L. (2003) Reversed-phase high performance liquid chromatography pre-fractionation prior to 2D difference gel electrophoresis and mass spectrometry identifies new differentially expressed proteins between striate cortex of kitten and adult cat. Electrophoresis 24, 1471–1481.
Van den Bergh, G., Clerens, S., Cnops, L., Vandesande, F., and Arckens, L. (2003) Fluorescent two-dimensional difference gel electrophoresis and mass spectrometry identify age-related protein expression differences for the primary visual cortex of kitten and adult cat. J. Neurochem. 85, 193–205.
Qu, Y., Moons, L., and Vandesande, F. (1997) Determination of serotonin, catecholamines and their metabolites by direct injection of supernatants from chicken brain tissue homogenate using liquid chromatography with electrochemical detection. J. Chromatogr. B Biomed. Sci. Appl. 704, 351–358.
Klose, J. (1999) Large-gel 2-D electrophoresis. Methods Mol. Biol. 112, 147–172.
Görg, 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.
Görg, A., Weiss, W., and Dunn, M. J. (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4, 3665–3685.
Stasyk, T. and Huber, L. A. (2004) Zooming in: fractionation strategies in proteomics. Proteomics 4, 3704–3716.
Acknowledgments
Gert Van den Bergh is a postdoctoral fellow of the Fund for Scientific Research Flanders (FWO-Vlaanderen), Belgium. We thank Lieve Geenen for critically reading the manuscript.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Van den Bergh, G., Arckens, L. (2008). Reducing Sample Complexity by RP-HPLC: Beyond the Tip of the Protein Expression Iceberg. In: Posch, A. (eds) 2D PAGE: Sample Preparation and Fractionation. Methods in Molecular Biology™, vol 424. Humana Press. https://doi.org/10.1007/978-1-60327-064-9_13
Download citation
DOI: https://doi.org/10.1007/978-1-60327-064-9_13
Publisher Name: Humana Press
Print ISBN: 978-1-58829-722-8
Online ISBN: 978-1-60327-064-9
eBook Packages: Springer Protocols