Isolation of Phosphoproteins

  • Lawrence G. Puente
  • Lynn A. Megeney
Part of the Methods in Molecular Biology™ book series (MIMB, volume 424)


Phosphorylation is one of the most abundant post-translational modifications on protein and one that frequently has functional biological consequences. For this reason, screening protein samples for phosphorylations has become an important tool in biochemical research. Affinity purification by immunological or chemical reagents can be used to isolate phosphoproteins from other cellular materials.

Key Words

Affinity purification immunoprecipitation protein phosphorylation 


  1. 1.
    Adamczyk, M., Gebler, J. C., and Wu, J. (2001) Selective analysis of phosphopeptides within a protein mixture by chemical modification, reversible biotinylation and mass spectrometry Rapid Commun Mass Spectrom 15, 1481–1488.CrossRefPubMedGoogle Scholar
  2. 2.
    Goodman, T., Schulenberg, B., Steinberg, T. H., and Patton, W. F. (2004) Detection of phosphoproteins on electroblot membranes using a small-molecule organic fluorophore Electrophoresis 25, 2533–2538.CrossRefPubMedGoogle Scholar
  3. 3.
    Schulenberg, B., Goodman, T. N., Aggeler, R., Capaldi, R. A., and Patton, W. F. (2004) Characterization of dynamic and steady-state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry Electrophoresis 25, 2526–2532.Google Scholar
  4. 4.
    Kocher, T., Allmaier, G., and Wilm, M. (2003) Nanoelectrospray-based detection and sequencing of substoichiometric amounts of phosphopeptides in complex mixtures J Mass Spectrom 38, 131–137.CrossRefPubMedGoogle Scholar
  5. 5.
    Bateman, R. H., Carruthers, R., Hoyes, J. B., Jones, C., Langridge, J. I., Millar, A., and Vissers, J. P. (2002) A novel precursor ion discovery method on a hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer for studying protein phosphorylation J Am Soc Mass Spectrom 13, 792–803.CrossRefPubMedGoogle Scholar
  6. 6.
    Andersson, L., and Porath, J. (1986) Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography Anal Biochem 154, 250–254.CrossRefPubMedGoogle Scholar
  7. 7.
    Muszynska, G., Andersson, L., and Porath, J. (1986) Selective adsorption of phosphoproteins on gel-immobilized ferric chelate Biochemistry 25, 6850–6853.CrossRefPubMedGoogle Scholar
  8. 8.
    Ficarro, S. B., McCleland, M. L., Stukenberg, P. T., Burke, D. J., Ross, M. M., Shabanowitz, J., Hunt, D. F., and White, F. M. (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae Nat Biotechnol 20, 301–305.CrossRefPubMedGoogle Scholar
  9. 9.
    Posewitz, M. C., and Tempst, P. (1999) Immobilized gallium(III) affinity chromatography of phosphopeptides Anal Chem 71, 2883–2892.CrossRefPubMedGoogle Scholar
  10. 10.
    Dubrovska, A., and Souchelnytskyi, S. (2005) Efficient enrichment of intact phosphorylated proteins by modified immobilized metal-affinity chromatography Proteomics 5, 4678–4683.CrossRefPubMedGoogle Scholar
  11. 11.
    Metodiev, M., Timanova, A., and Stone, D. E. (2004) Differential phosphoproteome profiling by affinity capture and tandem matrix-assisted laser desorption/ionization mass spectrometry Proteomics 4, 1433–1438.CrossRefPubMedGoogle Scholar
  12. 12.
    Puente, L. G., Borris, D. J., Carriere, J. F., Kelly, J. F., and Megeney, L. A. (2006) Identification of candidate regulators of embryonic stem cell differentiation by comparative phosphoprotein affinity profiling Mol Cell Proteomics 5, 57–67.PubMedGoogle Scholar
  13. 13.
    Puente, L. G., Carriere, J. F., Kelly, J. F., and Megeney, L. A. (2004) Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation FEBS Lett 574, 138–144.CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Lawrence G. Puente
    • 1
  • Lynn A. Megeney
    • 1
  1. 1.The Ottawa HospitalOttawaCanada

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