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OFFGEL-Isoelectric Focusing Fractionation for the Analysis of Complex Proteome

  • Emilie Ernoult
  • Catherine GuetteEmail author
Protocol
Part of the Neuromethods book series (NM, volume 57)

Abstract

The proteomic analysis at the peptide level is increasingly becoming a method of choice for complex samples. The success, however, depends on the development of attractive peptide fractionation methodologies to decrease the sample complexity prior to mass analysis. Recently, the OFFGEL technology has emerged as being of great interest in shotgun proteomics. Using the Agilent 3100 OFFGEL fractionator, OFFGEL allows the in-solution separation of peptides from various biological sources by isoelectric focusing in up to highly resolved 24 fractions. In this chapter, we describe a detailed experimental protocol of a quantitative proteomic workflow including OFFGEL as a crucial step to improve the proteome ­coverage. We provide the technical details concerning sample preparation, trypsin digestion, iTRAQ labelling, OFFGEL-IEF, RP nano-LC separation, MALDI-TOF/TOF MS/MS, and pI analysis. We rely on our recently published data to illustrate the use of OFFGEL as a powerful method for peptide ­fractionation and as a filtering tool for pI-based validation of peptide identification.

Key words

OFFGEL Peptide fractionation Preparative IEF Shotgun proteomics Proteome coverage iTRAQ labelling Biomarker discovery Methodology 

References

  1. 1.
    Hannig, K. (1982) New Aspects in Preparative and Analytical Continuous Free-Flow Cell Electrophoresis Electrophoresis 3, 235–43.CrossRefGoogle Scholar
  2. 2.
    Bier, M. (1998) Recycling isoelectric focu-sing and isotachophoresis Electrophoresis 19, 1057–63.PubMedCrossRefGoogle Scholar
  3. 3.
    Righetti, P. G., Wenisch, E., Faupel, M. (1989) Preparative Protein-Purification in a Multi-Compartment Electrolyzer with Immobiline Membranes Journal of Chromato-graphy 475, 293–309.CrossRefGoogle Scholar
  4. 4.
    Righetti, P. G., Wenisch, E., Jungbauer, A., Katinger, H., Faupel, M. (1990) Preparative Purification of Human Monoclonal-Antibody Isoforms in a Multicompartment Electrolyzer with Immobiline Membranes Journal of Chromatography 500, 681–96.PubMedCrossRefGoogle Scholar
  5. 5.
    Ros, A., Faupel, M., Mees, H., van Oostrum, J., Ferrigno, R., Reymond, F., Michel, P., Rossier, J. S., Girault, H. H. (2002) Protein purification by Off-Gel electrophoresis Proteomics 2, 151–6.PubMedCrossRefGoogle Scholar
  6. 6.
    Michel, P. E., Reymond, F., Arnaud, I. L., Josserand, J., Girault, H. H., Rossier, J. S. (2003) Protein fractionation in a multicompartment device using Off-Gel (TM) isoelectric focusing Electrophoresis 24, 3–11.PubMedCrossRefGoogle Scholar
  7. 7.
    Hubner, N.C., Ren, S., Mann, M. (2008) Peptide separation with immobilized pI strips is an attractive alternative to in-gel protein digestion for proteome analysis Proteomics 8, 4862–72.PubMedCrossRefGoogle Scholar
  8. 8.
    Essader, A.S., Cargile, B.J., Bundy, J.L. Jr. (2005) A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics Proteomics 5, 24–34.PubMedCrossRefGoogle Scholar
  9. 9.
    Bjellgvist, B., Hughes, G.J., Pasquali, C., Paquet, N., Ravier, F., Frutiger, S., Hughes, G.J., Hochstrasser, D. (1993) The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences Electrophoresis 14, 1023–31.CrossRefGoogle Scholar
  10. 10.
    Krügsveld, J., Gauci, S., Dormever, W., Heck, A.J. (2006) In-gel isoelectric focusing of peptides as a tool for improved protein identification J. Proteome Res. 5, 1721–30.CrossRefGoogle Scholar
  11. 11.
    Cargile, B.J., Bundy, J.L., Freeman, T.W., Stephenson, J.L. Jr. (2004) Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of accuracy of pI predictability of peptides J. Proteome Res. 3, 112-9.PubMedCrossRefGoogle Scholar
  12. 12.
    Lengqvist, J., Uhlen, K., Lehtio, J. (2007) iTRAQ compatibility of peptide immobilized pH gradient isoelectric focusing Proteomics 7, 1746–52.PubMedCrossRefGoogle Scholar
  13. 13.
    Chenau, J., Michelland, S., Sidibe, J., Seve, M. (2008) Peptides OFFGEL electrophoresis: a suitable pre-analytical step for complex eukaryotic samples fractionation compatible with quantitative iTRAQ labeling Proteome Sci. 26, 6–9.Google Scholar
  14. 14.
    Ernoult, E., Gamelin, E., Guette, C. (2008) Improved proteome coverage by using iTRAQ labelling and peptide OFFGEL fractionation Proteome Sci. 6, 27–40.PubMedCrossRefGoogle Scholar
  15. 15.
    Ernoult, E., Bourreau, A., Gamelin, E., Guette, C. (2010) A proteomic approach for plasma biomarker discovery with iTRAQ labelling and OFFGEL fractionation J. Biomed. Biotechnol. 2010:927917. Google Scholar
  16. 16.
    Shilov, I.V., Seymour, S.L., Patel, A.A., Loboda, A. et al. (2007) The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra Mol. Cell. Proteomics 6, 1638–55.PubMedCrossRefGoogle Scholar
  17. 17.
    Cargile, B.J., Talley, D.L., Stephenson, J.L. (2004) Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pI predictability of peptides Electrophoresis 25, 936–45.Google Scholar
  18. 18.
    Arnaud, I.L., Josserand, J., Jensen, H., Lion, N., Roussel, C., Girault, H.H. (2005) Salt removal during Off-Gel electrophoresis of protein ­samples Electrophoresis 26, 1650–8.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Laboratory of Oncopharmacology-PharmacogeneticsCentre INSERM Régional de Recherche sur le Cancer U892, Centre Régional de Lutte Contre le Cancer Paul PapinAngersFrance

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