Sample Preparation for 2D Electrophoresis and CE-Based Proteomics

  • Judit M. Nagy
  • Alexandria Lipka
  • Fiona Pereira
  • Nicky Marlin
  • Stuart HassardEmail author


This chapter describes sample preparation for gel-based proteomics, capillary electrophoresis and capillary electrophoresis coupled with mass spectrometry. Two dimensional gel electrophoresis (2D-PAGE) remains one of the most commonly used separation techniques for complex protein mixtures. Stringent and consistent sample preparation is essential for reproducible and high quality 2D-PAGE analysis of complex protein mixtures. The goal of this step is to produce homogeneous sample, free from contaminants and with consistent chemical parameters (pH, salt concentration etc) that will allow the user to generate reproducible results. Capillary electrophoresis (CE) separates analytes from a complex mixture with high resolution based on differential migration through a liquid filled capillary in a strong electric field. CE is easily automated and lends itself well to microfluidic device technology. The evolution of these microfluidic devices allows the potential for the hyphenation of CE to other separation and identification methods like gel chromatography, high performance liquid chromatography (HPLC) and mass spectrometry.


Capillary electrophoresis Consistent chemical parameters Proteomics 


  1. Ahmed, F.E. (2009). Sample preparation and fractionation for proteome analysis and cancer biomarker discovery by mass spectrometry. J Sep Sci 32, 771–798.CrossRefGoogle Scholar
  2. Antonioli, P., Bachi, A., Fasoli, E., and Righetti, P.G. (2009). Efficient removal of DNA from proteomic samples prior to two-dimensional map analysis. J Chromatogr A 1216, 3606–3612.CrossRefGoogle Scholar
  3. Beale, S.C. (1998). Capillary electrophoresis. Anal Chem 70, 279R–300R.CrossRefGoogle Scholar
  4. Bergkvist, J., Ekstrom, S., Wallman, L., Lofgren, M., Marko-Varga, G., Nilsson, J., and Laurell, T. (2002). Improved chip design for integrated solid-phase microextraction in on-line proteomic sample preparation. Proteomics 2, 422–429.CrossRefGoogle Scholar
  5. Bodzon-Kulakowska, A., Bierczynska-Krzysik, A., Dylag, T., Drabik, A., Suder, P., Noga, M., Jarzebinska, J., and Silberring, J. (2007). Methods for samples preparation in proteomic research. J Chromatogr B Anal Technol Biomed Life Sci 849, 1–31.CrossRefGoogle Scholar
  6. Camilleri, P. (1993). Capillary Electrophoresis: Theory and Practice (Boca Raton, FL, CRC Press).Google Scholar
  7. Canas, B., Pineiro, C., Calvo, E., Lopez-Ferrer, D., and Gallardo, J.M. (2007). Trends in sample preparation for classical and second generation proteomics. J Chromatogr A 1153, 235–258.CrossRefGoogle Scholar
  8. Cetin, B., and Li, D. (2008). Effect of Joule heating on electrokinetic transport. Electrophoresis 29, 994–1005.CrossRefGoogle Scholar
  9. Chambers, S.P. (2002). High-throughput protein expression for the post-genomic era. Drug Discov Today 7, 759–765.CrossRefGoogle Scholar
  10. Chevallet, M., Santoni, V., Poinas, A., Rouquie, D., Fuchs, A., Kieffer, S., Rossignol, M., Lunardi, J., Garin, J., and Rabilloud, T. (1998). New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis. Electrophoresis 19, 1901–1909.CrossRefGoogle Scholar
  11. Cilia, M., Fish, T., Yang, X., McLaughlin, M., Thannhauser, T.W., and Gray, S. (2009). A comparison of protein extraction methods suitable for gel-based proteomic studies of aphid proteins. J Biomol Tech 20, 201–215.Google Scholar
  12. Dong, M., Baggetto, L.G., Falson, P., Le Maire, M., and Penin, F. (1997). Complete removal and exchange of sodium dodecyl sulfate bound to soluble and membrane proteins and restoration of their activities, using ceramic hydroxyapatite chromatography. Anal Biochem 247, 333–341.CrossRefGoogle Scholar
  13. Dunn, M.J., and Bradd, S.J. (1993). Separation and analysis of membrane proteins by SDS-polyacrylamide gel electrophoresis. Methods Mol Biol 19, 203–210.Google Scholar
  14. Ekstrom, S., Malmstrom, J., Wallman, L., Lofgren, M., Nilsson, J., Laurell, T., and Marko-Varga, G. (2002). On-chip microextraction for proteomic sample preparation of in-gel digests. Proteomics 2, 413–421.CrossRefGoogle Scholar
  15. Engelhardt, H., Beck, W., Kohr, J., and Schmitt, T. (1993). Capillary electrophoresis – methods and scope. Angew Chem Int Ed 32, 629–649.CrossRefGoogle Scholar
  16. Englard, S., and Seifter, S. (1990). Precipitation techniques. Methods Enzymol 182, 285–300.CrossRefGoogle Scholar
  17. Fountoulakis, M., Juranville, J.F., Jiang, L., Avila, D., Roder, D., Jakob, P., Berndt, P., Evers, S., and Langen, H. (2004). Depletion of the high-abundance plasma proteins. Amino Acids 27, 249–259.CrossRefGoogle Scholar
  18. Freeman, W.M., and Hemby, S.E. (2004). Proteomics for protein expression profiling in neuroscience. Neurochem Res 29, 1065–1081.CrossRefGoogle Scholar
  19. Fu, Q., Bovenkamp, D.E., and Van Eyk, J.E. (2007). A rapid, economical, and reproducible method for human serum delipidation and albumin and IgG removal for proteomic analysis. Methods Mol Biol 357, 365–371.Google Scholar
  20. Gasper, A., Englmann, M., Fekete, A., Harir, M., and Schmitt-Kopplin, P. (2008). Trends in CE-MS 2005–2006. Electrophoresis 29, 66–79.CrossRefGoogle Scholar
  21. Ghosal, S. (2003). The effect of wall interactions in capillary-zone electrophoresis. J Fluid Mech 491: 285–300.CrossRefGoogle Scholar
  22. Gorg, A., Postel, W., Friedrich, C., Kuick, R., Strahler, J.R., and Hanash, S.M. (1991). Temperature-dependent spot positional variability in two-dimensional polypeptide patterns. Electrophoresis 12, 653–658.CrossRefGoogle Scholar
  23. Gorg, A., Weiss, W., and Dunn, M.J. (2004). Current two-dimensional electrophoresis technology for proteomics. Proteomics 4, 3665–3685.CrossRefGoogle Scholar
  24. Granier, F. (1988). Extraction of plant proteins for two-dimensional electrophoresis. Electrophoresis 9, 712–718.CrossRefGoogle Scholar
  25. Hannam, C., Lange, G.L., and Mellors, A. (1998). Synthesis of a radiolabeled zwitterionic detergent and its use in protein purification. Anal Biochem 258, 246–250.CrossRefGoogle Scholar
  26. Herbert, B.R., Molloy, M.P., Gooley, A.A., Walsh, B.J., Bryson, W.G., and Williams, K.L. (1998). Improved protein solubility in two-dimensional electrophoresis using tributyl phosphine as reducing agent. Electrophoresis 19, 845–851.CrossRefGoogle Scholar
  27. Hernández-Borges, J., Borges-Miquel, T.M., Rodríguez-Delgado, M.Á., and Cifuentes, A. (2007). Sample treatments prior to capillary electrophoresis-mass spectrometry. J Chromatogr A 1153, 214–226.CrossRefGoogle Scholar
  28. Hjerten, S. (1967). Free zone electrophoresis. Chromatogr Rev 9, 122 –219.CrossRefGoogle Scholar
  29. Jiang, L., He, L., and Fountoulakis, M. (2004). Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. J Chromatogr A 1023, 317–320.CrossRefGoogle Scholar
  30. Kang, Y., Techanukul, T., Mantalaris, A., and Nagy, J. (2009). Comparison of three commercially available DIGE analysis software packages using conditioned media samples. J Proteome Res 8, 1077–1084.CrossRefGoogle Scholar
  31. Karger, B.L., Cohen, A.S., and Guttman, A. (1989). High-performance capillary electrophoresis in the biological sciences. J Chromatogr 492, 585–614.CrossRefGoogle Scholar
  32. Klose, J., and Kobalz, U. (1995). Two-dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome. Electrophoresis 16, 1034–1059.CrossRefGoogle Scholar
  33. Knox, J. (1994). Terminology and nominclature in capillary electroseparation systems. J Chromatogr A 680, 3–13.CrossRefGoogle Scholar
  34. Lee, E.D., Muck, W., Henion, J.D., and Covey, T.R. (1988). On-line capillary zone electrophoresis-ion spray tandem mass spectrometry for the determination of dynorphins. J Chromatogr 458, 313–321.CrossRefGoogle Scholar
  35. Luche, S., Santoni, V., and Rabilloud, T. (2003). Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis. Proteomics 3, 249–253.CrossRefGoogle Scholar
  36. Mader, R.M., Rizovski, B., and Steger, G.G. (2002). On-line solid-phase extraction and determination of paclitaxel in human plasma. J Chromatogr B 769, 357–361.CrossRefGoogle Scholar
  37. Marina, M.L., and Torre, M. (1994). Capillary electrophoresis. Talanta 41, 1411–1433.CrossRefGoogle Scholar
  38. Mikus, P., and Marakova, K. (2009). Advanced CE for chiral analysis of drugs, metabolites, and biomarkers in biological samples. Electrophoresis 30, 2773–2802.CrossRefGoogle Scholar
  39. Minden, J.S., Dowd, S.R., Meyer, H.E., and Stuhler, K. 2009: Difference gel electrophoresis. Electrophoresis 30(Suppl 1): S156–161.Google Scholar
  40. Monnig, C.A., and Kennedy, R.T. (1994). Capillary electrophoresis. Anal Chem 66, R280–R314.CrossRefGoogle Scholar
  41. Monton, M.R.N., and Terabe, S. (2005). Recent developments in capillary electrophoresis-mass spectrometry of proteins and peptides. Anal Sci 21, 5–13.CrossRefGoogle Scholar
  42. Morita, I., and Sawada, J. (1993). Capillary electrophoresis with online sample pretreatment for the analysis of biological samples with direct-injection. J Chromatogr 641, 375–381.CrossRefGoogle Scholar
  43. Ohnesorge, J., Neususs, C., and Watzig, H. (2005). Quantitation in capillary electrophoresis-mass spectrometry. Electrophoresis 26, 3973–3987.CrossRefGoogle Scholar
  44. Paces, M., Kosek, J., Marek, M., Tallarek, U., and Seidel-Morgenstern, A. (2003). Mathematical modelling of adsorption and transport processes in capillary electrochromatography: Open-tubular geometry. Electrophoresis 24, 380–389.CrossRefGoogle Scholar
  45. Pelzing, M., and Neususs, C. (2005). Separation techniques hyphenated to electrospray-tandem mass spectrometry in proteomics: Capillary electrophoresis versus nanoliquid chromatography. Electrophoresis 26, 2717–2728.CrossRefGoogle Scholar
  46. Pieper, R., Su, Q., Gatlin, C.L., Huang, S.T., Anderson, N.L., and Steiner, S. (2003). Multi-component immunoaffinity subtraction chromatography: An innovative step towards a comprehensive survey of the human plasma proteome. Proteomics 3, 422–432.CrossRefGoogle Scholar
  47. Puig, P., Borrull, F., Calull, M., and Aguilar, C. (2008). Sorbent preconcentration procedures coupled to capillary electrophoresis for environmental and biological applications. Anal Chimica Acta 616, 1–18.CrossRefGoogle Scholar
  48. Rabilloud, T. (1996). Solubilization of proteins for electrophoretic analyses. Electrophoresis 17, 813–829.CrossRefGoogle Scholar
  49. Rabilloud, T., Adessi, C., Giraudel, A., and Lunardi, J. (1997). Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18, 307–316.CrossRefGoogle Scholar
  50. Rodrigues, S.P., Ventura, J.A., Zingali, R.B., and Fernandes, P.M. (2009). Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two-dimensional gel electrophoresis. Phytochem Anal 20, 456–464.CrossRefGoogle Scholar
  51. Schmitt-Kopplin, P., and Frommberger, M. (2003). Capillary electrophoresis – mass spectrometry: 15 years of developments and applications. Electrophoresis 24, 3837–3867.CrossRefGoogle Scholar
  52. Shaw, M.M., and Riederer, B.M. (2003). Sample preparation for two-dimensional gel electrophoresis. Proteomics 3, 1408–1417.CrossRefGoogle Scholar
  53. Shihabi, Z.K. (1999). Field amplified injection in the presence of salts for capillary electrophoresis. J Chromatogr A 853, 3–9.CrossRefGoogle Scholar
  54. Smith, R.D., and Udseth, H.R. (1988). Capillary zone electrophoresis-MS. Nature 331, 639–640.CrossRefGoogle Scholar
  55. Tamai, G., Edani, M., and Imai, H. (1991). Determination of ketoprofen enantiomers in plasma by soild-phase extraction and column switching high-perfomrnace liquid-chromatography. Anal Sci 7, 29–32.CrossRefGoogle Scholar
  56. 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.CrossRefGoogle Scholar
  57. Watzig, H., and Gunter, S. (2003). Capillary electrophoresis – A high performance analytical separation technique. Clin Chem Lab Med 41, 724–736.CrossRefGoogle Scholar
  58. Weiss, W., and Gorg, A. (2009). High-resolution two-dimensional electrophoresis. Methods Mol Biol 564, 13–32.CrossRefGoogle Scholar
  59. Zhang, X.M., Hu, H.L., Xu, S.Y., Yang, X.H., and Zhang, J. (2001). Comprehensive two-dimensional capillary LC and CE for resolution of neutral components in traditional Chinese medicines. J Sep Sci 24, 385–391.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Judit M. Nagy
    • 1
  • Alexandria Lipka
    • 2
  • Fiona Pereira
    • 3
  • Nicky Marlin
    • 4
  • Stuart Hassard
    • 5
    Email author
  1. 1.Department of ChemistryImperial College LondonLondonUK
  2. 2.deltaDOT QSTP-LLCQatar Science & Technology ParkDohaState of Qatar
  3. 3.Department of ChemistryImperial College LondonLondonUK
  4. 4.deltaDOT LtdLondonUK
  5. 5.London BioScience Innovation CentreLondonUK

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