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Affinity Chromatography pp 285–294Cite as

Affinity Chromatography of Phosphorylated Proteins

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 421))

Summary

This chapter covers the use of immobilized metal ion affinity chromatography (IMAC) for enrichment of phosphorylated proteins. Some requirements for successful enrichment of these types of proteins are discussed. An experimental protocol and a set of application data are included to enable the scientist to obtain high-yield results in a very short time with pre-packed phospho-specific metal ion affinity resin (PMAC).

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References

  1. Karr, D.B. and Emerich, D.W. (1989) Protein phosphorylation in Bradyrhizobium japonicum bacteroids and cultures. J. Bacteriol. 171(6), 3420–3426.

    CAS  PubMed  Google Scholar 

  2. Bourret, R.B., Hess J.F., Borkovich, K.A., Pakula, A.A., and Simon, M.I. (1989) Protein phosphorylation in chemotaxis and two-component regulatory systems of bacteria. J. Biol. Chem. 264(13), 7085–7088.

    CAS  PubMed  Google Scholar 

  3. Kennelly, P.J. and Potts, M. (1996) Fancy meeting you here! A fresh look at “prokaryotic” protein phosphorylation. J. Bacteriol. 178(16), 4759–4764.

    CAS  PubMed  Google Scholar 

  4. Klumpp, S. and Krieglstein, J. (2002) Phosphorylation and dephosphorylation of histidine residues in proteins. Eur. J. Biochem. 269(4), 1067–1071.

    Article  CAS  PubMed  Google Scholar 

  5. Eichler, J. and Adams, M.W.W. (2005) Posttranslational protein modification in archaea. Microbiol. Mol. Biol. Rev. 69(3), 393–425.

    Article  CAS  PubMed  Google Scholar 

  6. Ficarro, S.B., et al. (2003) Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation. J. Biol. Chem. 278(13), 11579–11589.

    Article  CAS  PubMed  Google Scholar 

  7. Ficarro, S.B., et al. (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20(3), 301–305.

    Article  CAS  PubMed  Google Scholar 

  8. Matthews, H.R. (1995) Protein kinases and phosphatases that act on histidine, lysine, or arginine residues in eukaryotic proteins: a possible regulator of the mitogen-activated protein kinase cascade. Pharmacol. Ther. 67(3), 323–350.

    Article  CAS  PubMed  Google Scholar 

  9. Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258, 598–599.

    Article  CAS  PubMed  Google Scholar 

  10. Pearson, R.G. (ed.) (1973) Hard and Soft Acids and Bases. Stroudsburg, PA: Hutchington 8 Ross; 53–85.

    Google Scholar 

  11. Andersson, L. and Porath, J. (1986) Isolation of phosphoproteins by Immobilized Metal (Fe3+) Affinity Chromatography. Anal. Biochem. 154, 250–254.

    Article  CAS  PubMed  Google Scholar 

  12. Muszynska, G., Andersson, L., and Porath, J. (1986) Selective adsorption of phosphoproteins on gel-immobilized ferric chelate. Biochemistry 25, 6850–6853.

    Article  CAS  PubMed  Google Scholar 

  13. Merryfield, M.L., Kramp, D.C., and Lardy, H.A. (1982) Purification and characterization of a rat liver ferroactivator with catalase activity. J. Biol. Chem. 257(8), 4646–4654.

    CAS  PubMed  Google Scholar 

  14. van Heusden, M.C., Fogarty, S., Porath, J., and Law, J.H. (1991) Purification of insect vitellogenin and vitellin by gel-immobilized ferric chelate. Protein Expr. Purif. 2, 24–28.

    Article  PubMed  Google Scholar 

  15. Kucerova, Z. (1989) Fractionation of human gastric proteinases by immobilized metal chelate (iron(3+)) affinity chromatography. J. Chromatogr. A 489(2), 390–393.

    CAS  Google Scholar 

  16. Vijayalakshmi, M.A. (1983) High performance liquid chromatography with immobilized metal adsorbents. In: Chaiken, I.M., Wilchek, M., and Parikh, I., eds. Affinity Chromatography and Biological Recognition. 1st ed. New York: Academic Press; 269–273.

    Google Scholar 

  17. Luong, C.B.H., Browner, M.F., Fletterick, R.J., and Haymore, B.L. (1992) Purification of glycogen phosphorylase isozymes by metal-affinity chromatography. J. Chromatogr. Biomed. Appl. 584(1), 77–84.

    Article  CAS  Google Scholar 

  18. Muszynska, G., Dobrowolska, G., Medin, A., Ekman, P., and Porath, J.O. (1992) Model studies on iron(III) ion affinity chromatography. II. Interaction of immobilized iron(III) ions with phosphorylated amino acids, peptides and proteins. J. Chromatogr. 604(1), 19–28.

    Article  CAS  PubMed  Google Scholar 

  19. Neville D.C., Rozanas C.R., Price E.M., Gruis D.B., Verkman A.S., and Townsend R.R. (1997) Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry. Protein Sci. 6(11), 2436–2445.

    Article  CAS  PubMed  Google Scholar 

  20. Zachariou M., Traverso I., and Hearn M.T. (1993) High-performance liquid chromatography of amino acids, peptides and proteins. CXXXI. O-phosphoserine as a new chelating ligand for use with hard Lewis metal ions in the immobilized-metal affinity chromatography of proteins. J. Chromatogr. A 646(1), 107–120.

    Article  CAS  Google Scholar 

  21. Smilenov L., Forsberg E., Zeligman I., Sparrman M., and Johansson S. (1992) Separation of fibronectin from a plasma gelatinase using immobilized metal affinity chromatography. FEBS Lett. 302(3), 227–230.

    Article  CAS  PubMed  Google Scholar 

  22. Bernos E., Girardet J.M., Humbert G., and Linden G. (1997) Role of the O-phosphoserine clusters in the interaction of the bovine milk alpha s1-, beta-, kappa-caseins and the PP3 component with immobilized iron (III) ions. Biochim. Biophys. Acta 1337(1), 149–159.

    Article  CAS  PubMed  Google Scholar 

  23. Anguenot R., Yelle S., and Nguyen-Quoc B. (1999) Purification of tomato sucrose synthase phosphorylated isoforms by Fe(III)-immobilized metal affinity chromatography. Arch. Biochem. Biophys. 365(1), 163–169.

    Article  CAS  PubMed  Google Scholar 

  24. Figeys D., Gygi S.P., Zhang Y., Watts J., Gu M., and Aebersold R. (1998) Electrophoresis combined with novel mass spectrometry techniques: powerful tools for the analysis of proteins and proteomes. Electrophoresis 19(10), 1811–1818.

    Article  CAS  PubMed  Google Scholar 

  25. Lin J.H. and Chiang B.H. (1996) A modified procedure for caseinophosphopeptide analysis. J. Chromatogr. Sci. 34(8), 358–361.

    CAS  PubMed  Google Scholar 

  26. Cao P. and Stults J.T. (1999) Phosphopeptide analysis by on-line immobilized metal-ion affinity chromatography-capillary electrophoresis-electrospray ionization mass spectrometry. J. Chromatogr. A 853(1), 225–235.

    Article  CAS  PubMed  Google Scholar 

  27. Posewitz M.C. and Tempst P. (1999) Immobilized gallium (III) affinity chromatography of phosphopeptides. Anal. Chem. 71(14), 2883–2892.

    Article  CAS  PubMed  Google Scholar 

  28. Barnouin K.N., Hart S.R., Thompson A.J., Okuyama M., Waterfield M., and Cramer R. (2005) Enhanced phosphopeptide isolation by Fe(III)-IMAC using 1,1,1,3,3,3-hexafluoroisopropanol. Proteomics 5(17), 4376–4388.

    Article  CAS  PubMed  Google Scholar 

  29. Wang J., Zhang Y., Jiang H., Cai Y., and Qian X. (2006) Phosphopeptide detection using automated online IMAC-capillary LC-ESI-MS/MS. Proteomics 6(2), 404–11.

    Article  CAS  PubMed  Google Scholar 

  30. Reinders J. and Sickmann A. (2005) State-of-the-art in phosphoproteomics. Proteomics 5(16), 4052–4061.

    Article  CAS  PubMed  Google Scholar 

  31. Shuai K., Horvath C.M., Huang L.H., Qureshi S.A., Cowburn D., and Darnell J.E. Jr. (1994) Interferon activation of the transcription factor Stat91 involves dimerization through SH2-phosphotyrosyl peptide interactions. Cell 76(5), 821–828.

    Article  CAS  PubMed  Google Scholar 

  32. Chen X., Vinkemeier U., Zhao Y., Jeruzalmi D., Darnell J.E. Jr., and Kuriyan J. (1998) Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA. Cell 93(5), 827–839.

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Clontech Laboratories, Inc., Mountain View, CA

    Grigoriy S. Tchaga

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  1. Grigoriy S. Tchaga
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Editors and Affiliations

  1. BioMarin Pharmaceutical Inc., CA

    Michael Zachariou (Director Project Management) (Director Project Management)

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

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Tchaga, G.S. (2008). Affinity Chromatography of Phosphorylated Proteins. In: Zachariou, M. (eds) Affinity Chromatography. Methods in Molecular Biology™, vol 421. Humana Press. https://doi.org/10.1007/978-1-59745-582-4_19

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  • DOI: https://doi.org/10.1007/978-1-59745-582-4_19

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-659-7

  • Online ISBN: 978-1-59745-582-4

  • eBook Packages: Springer Protocols

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