Advertisement

Fractionation of Complex Protein Mixtures by Liquid-Phase Isoelectric Focusing

  • Julie Hey
  • Anton Posch
  • Andrew Cohen
  • Ning Liu
  • Adrianna Harbers
Part of the Methods in Molecular Biology™ book series (MIMB, volume 424)

Summary

Protein fractionation is essential to uncovering low-abundance proteins in complex protein mixtures. Many common methods and techniques are used to fractionate proteins, including chromatography (size exclusion, affinity, ion exchange, etc.), electrophoresis, and solution chemistry. Regardless of the method employed, the ultimate goal of protein fractionation is to enable more protein analysis by today’s current proteomics technologies, such as one- (1-DGE) or two-dimensional gel electrophoresis (2-DGE) and liquid-chromatography and tandem mass spectrometry (LC-MS/MS).

The MicroRotofor\(^{\rm TM}\) isoelectric focusing (IEF) cell fractionates proteins in free solution according to their isoelectric point (pI). We demonstrate the ability of the MicroRotofor to enrich low-abundance proteins in mouse brain tissue, thus enabling further identification of potential biomarker candidates.

Key Words

Carrier ampholytes 1-D electrophoresis 2-D electrophoresis fractionation liquid phase isoelectric focusing low-abundance proteins mass spectrometry protein purification proteomics Rotofor 

References

  1. Bier, F. F., Egen, N. B., Allgyer, T. T., Twitty, G. E., and Mosher, R. A. (1979) New developments in isoelectric focusing, inStructure and biological function (Gross, E., and Meierhofer, J., Eds.), pp. 79–89, Pierce Chemical Company.Google Scholar
  2. Bier, F. F., and Egen, N. B. (1979) Large-scale recycling electrofocusing inElectrofocus/78 (Haglund, H., and Westerfield, J. G., Eds.), Elsevier, New York.Google Scholar
  3. 3.
    Righetti, P. G., Castagna, A., Herbert, B., and Candiano, G. (2005) How to bring the “unseen” proteome to the limelight via electrophoretic pre-fractionation techniques.Biosci Rep 25, 3–17.CrossRefPubMedGoogle Scholar
  4. 4.
    Davidsson, P., Folkesson, S., Christiansson, M., Lindbjer, M., Dellheden, B., Blennow, K., and Westman-Brinkmalm, A. (2002) Identification of proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing as a prefractionation step followed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation mass spectrometry.Rapid Commun Mass Spectrom 16, 2083–8.CrossRefPubMedGoogle Scholar
  5. 5.
    Davidsson, P., Paulson, L., Hesse, C., Blennow, K., and Nilsson, C. L. (2001) Proteome studies of human cerebrospinal fluid and brain tissue using a preparative two-dimensional electrophoresis approach prior to mass spectrometry.Proteomics 1, 444–52.CrossRefPubMedGoogle Scholar
  6. 6.
    Davidsson, P., and Nilsson, C. L. (1999) Peptide mapping of proteins in cerebrospinal fluid utilizing a rapid preparative two-dimensional electrophoretic procedure and matrix-assisted laser desorption/ionization mass spectrometry.Biochim Biophys Acta 1473, 391–9.PubMedGoogle Scholar
  7. 7.
    Davidsson, P., Puchades, M., and Blennow, K. (1999) Identification of synaptic vesicle, pre- and postsynaptic proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing.Electrophoresis 20, 431–7.CrossRefPubMedGoogle Scholar
  8. 8.
    Peirce M.J., Wait R., Begum S., Saklatvala J. and Cope A.P. (2004) Expression profiling of lymphocyte plasma membrane proteins.Mol.Cell. Proteomics 3: 56–65.PubMedGoogle Scholar
  9. 9.
    Hamler R.L., Zhu K., Buchanan N.S., Kreunin P., Kachman M.T., Miller F.R. and Lubman D.M. (2004) A two-dimensional liquid-phase separation method coupled with mass spectrometry for proteomic studies of breast cancer and biomarker identification. Proteomics 4: 562–577.CrossRefPubMedGoogle Scholar
  10. 10.
    Wang M.Z., Howard B., Campa M.J., Patz, E.F. and Fitzgerald M.C. (2003) Analysis of human serum proteins by liquid-phase isoelectric focusing and matrix-assisted laser desorption/ionization-mass spectrometry.Proteomics 3: 1661–1666.CrossRefPubMedGoogle Scholar
  11. 11.
    Harper R.G., Workman S.R., Schuetzner S., Timperman A.T. and Sutton J.N. (2004) Low-molecular-weight human serum proteome using ultrafiltration, isoelectric focusing, and mass spectrometry.Electrophoresis 25: 128–133.CrossRefGoogle Scholar
  12. 12.
    Xiao Z., Conrads T.P., Lucas D.A., Janini G.M., Schaefer C.F., Buetow K.H., Issaq H.J. and Veenstra T.D. (2004) Direct ampholyte-free liquid-phase isoelectric peptide focusing: application to the human serum proteome.Electrophoresis 25: 128–133.CrossRefPubMedGoogle Scholar
  13. 13.
    Janini G.M., Conrads T.P., Veenstra T.D. and Issaq H.J. (2003) Development of a two-dimensional protein-peptide separation protocol for comprehensive proteome measurements.J Chromatography B 787:43–51.Google Scholar
  14. Bindschedler L.V., Palmblad M. and Cramer R. (2006) First-dimension separation with the MicroRotofor cell prior to SDS-PAGE and LC-MS/MS analysis. Bulletin 5451, Bio-Rad Laboratories, Inc.Google Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Julie Hey
    • 1
  • Anton Posch
    • 2
  • Andrew Cohen
    • 1
  • Ning Liu
    • 1
  • Adrianna Harbers
    • 1
  1. 1.Bio-Rad Laboratories, Inc.Hercules
  2. 2.Bio-Rad Laboratories GmbHMunichGermany

Personalised recommendations