Phosphoproteomics by Highly Selective IMAC Protocol

  • Chia-Feng Tsai
  • Yi-Ting Wang
  • Pei-Yi Lin
  • Yu-Ju ChenEmail author
Part of the Neuromethods book series (NM, volume 57)


Protein phosphorylation plays an important role in biological process such as cell differentiation, cell cycle control, metabolism, and apoptosis. Toward global analysis of the phosphoproteome, enrichment is an essential step to overcome analytical challenges associated with the nature of phosphoprotein, including their dynamic modification patterns, substoichiometric concentrations, heterogeneous forms of phosphoproteins, and low mass spectrometric response. Here, based on detailed evaluation of the capture and release mechanism in immobilized metal affinity chromatography (IMAC), we provide a pH/acid-controlled IMAC protocol for phosphopeptide purification with high specificity and lower sample loss. Based on a model study on non-small-cell lung cancer cell, better than 90% phosphopeptide enrichment specificity can be achieved without the use of commonly adapted methyl esterification procedure. In addition, the protocol is compatible to fractionation using SDS-PAGE. We have successfully employed the pH/acid-controlled IMAC enrichment strategy to characterize over 2,360 nondegenerate phosphopeptides and 2,747 phosphorylation sites in H1299 lung cancer cell line. We expect that the simple and reproducible IMAC protocol can be applied, fully automated or manual, for large-scale identification of the vastly under-explored phosphoproteome associated with neurodegenerative diseases.

Key words

IMAC SDS-PAGE Phosphoproteomics Mass spectrometry 



This work was supported by Academia Sinica and the National Science Council in Taiwan. We thank Dr. Jeou-Yuan Chen for providing human non-small cell lung carcinoma cell line (H1299).


  1. 1.
    Hunter, T. (1995) Protein kinases and ­phosphatases: The Yin and Yang of protein phosphorylation and signaling, Cell 80, 225–236.PubMedCrossRefGoogle Scholar
  2. 2.
    Mansuy, I. M., and Shenolikar, S. (2006) Protein serine/threonine phosphatases in neuronal plasticity and disorders of learning and memory, Trends in Neurosciences 29, 679–686.PubMedCrossRefGoogle Scholar
  3. 3.
    Ian, I. S., Ty, T., and Daniel, F. (2001) 18O Labeling: a tool for proteomics, Rapid Communications in Mass Spectrometry 15, 2456–2465.CrossRefGoogle Scholar
  4. 4.
    Mann, M., Ong, S.-E., Grønborg, M., Steen, H., Jensen, O. N., and Pandey, A. (2002) Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome, Trends in Biotechnology 20, 261–268.PubMedCrossRefGoogle Scholar
  5. 5.
    Andersson, L., and Porath, J. (1986) Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography, Analytical Biochemistry 154, 250–254.PubMedCrossRefGoogle Scholar
  6. 6.
    Posewitz, M. C., and Tempst, P. (1999) Immobilized Gallium(III) Affinity Chromatography of Phosphopeptides, Analytical Chemistry 71, 2883–2892.PubMedCrossRefGoogle Scholar
  7. 7.
    David, C. A. N., Townsend, R. R., Christine, R. R., Verkman, A. S., Elmer, M. P., and Darren, B. G. (1997) Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry, Protein Science 6, 2436–2445.Google Scholar
  8. 8.
    Gruhler, A., Olsen, J. V., Mohammed, S., Mortensen, P., Faergeman, N. J., Mann, M., and Jensen, O. N. (2005) Quantitative Phosphoproteomics Applied to the Yeast Pheromone Signaling Pathway, Mol Cell Proteomics 4, 310–327.PubMedCrossRefGoogle Scholar
  9. 9.
    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 Biotech 20, 301–305.CrossRefGoogle Scholar
  10. 10.
    Corthals, G. L., Aebersold, R., Goodlett, D. R., and Burlingame, A. L. (2005) Identification of Phosphorylation Sites Using Microimmobi­lized Metal Affinity Chromatography, in Abelson, J. N., Simon, M. I., Colowick, S. P., Kaplan, N. O. (eds.) Methods in Enzymology, pp 66–81, Academic Press.Google Scholar
  11. 11.
    Ndassa, Y. M., Orsi, C., Marto, J. A., Chen, S., and Ross, M. M. (2006) Improved Immobilized Metal Affinity Chromatography for Large-Scale Phosphoproteomics Applications, Journal of Proteome Research 5, 2789–2799.PubMedCrossRefGoogle Scholar
  12. 12.
    Kokubu, M., Ishihama, Y., Sato, T., Nagasu, T., and Oda, Y. (2005) Specificity of Immobilized Metal Affinity-Based IMAC/C18 Tip Enrichment of Phosphopeptides for Protein Phosphorylation Analysis, Analytical Chemistry 77, 5144–5154.PubMedCrossRefGoogle Scholar
  13. 13.
    Seeley, E. H., Riggs, L. D., and Regnier, F. E. (2005) Reduction of non-specific binding in Ga(III) immobilized metal affinity chromatography for phosphopeptides by using endoproteinase glu-C as the digestive enzyme, Journal of Chromatography B 817, 81–88.CrossRefGoogle Scholar
  14. 14.
    Salomon, A. R., Ficarro, S. B., Brill, L. M., Brinker, A., Phung, Q. T., Ericson, C., Sauer, K., Brock, A., Horn, D. M., Schultz, P. G., and Peters, E. C. (2003) Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry, Proceedings of the National Academy of Sciences of the United States of America 100, 443–448.PubMedCrossRefGoogle Scholar
  15. 15.
    Lee, J., Xu, Y., Chen, Y., Sprung, R., Kim, S. C., Xie, S., and Zhao, Y. (2007) Mitochondrial Phosphoproteome Revealed by an Improved IMAC Method and MS/MS/MS, Mol Cell Proteomics 6, 669–676.PubMedCrossRefGoogle Scholar
  16. 16.
    Kim, J.-E., Tannenbaum, S. R., and White, F. M. (2005) Global Phosphoproteome of HT-29 Human Colon Adenocarcinoma Cells, Journal of Proteome Research 4, 1339–1346.PubMedCrossRefGoogle Scholar
  17. 17.
    Speicher, K. D.; Kolbas, O.; Harper, S.; Speicher, D. W. (2000) Systematic analysis of peptide recoveries from in-gel digestions for protein identifications in proteome studies., J. Biomol. Tech. 11, 74–86.PubMedGoogle Scholar
  18. 18.
    Villén, J., Beausoleil, S. A., Gerber, S. A., and Gygi, S. P. (2007) Large-scale phosphorylation analysis of mouse liver, Proceedings of the National Academy of Sciences 104, 1488–1493.CrossRefGoogle Scholar
  19. 19.
    Nuhse, T. S., Stensballe, A., Jensen, O. N., and Peck, S. C. (2003) Large-scale Analysis of in Vivo Phosphorylated Membrane Proteins by Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry, Mol Cell Proteomics 2, 1234–1243.PubMedCrossRefGoogle Scholar
  20. 20.
    McNulty, D. E., and Annan, R. S. (2008) Hydrophilic Interaction Chromatography Reduces the Complexity of the Phosphoproteome and Improves Global Phosphopeptide Isolation and Detection, Mol Cell Proteomics 7, 971–980.PubMedCrossRefGoogle Scholar
  21. 21.
    Tsai, C.-F., Wang, Y.-T., Chen, Y.-R., Lai, C.-Y., Lin, P.-Y., Pan, K.-T., Chen, J.-Y., Khoo, K.-H., and Chen, Y.-J. (2008) Immobilized Metal Affinity Chromatography Revisited: pH/Acid Control toward High Selectivity in Phosphoproteomics, Journal of Proteome Research 7, 4058–4069.PubMedCrossRefGoogle Scholar
  22. 22.
    Han, C.-L., Chien, C.-W., Chen, W.-C., Chen, Y.-R., Wu, C.-P., Li, H., and Chen, Y.-J. (2008) A Multiplexed Quantitative Strategy for Membrane Proteomics: Opportunities for Mining Therapeutic Targets for Autosomal Dominant Polycystic Kidney Disease, Mol Cell Proteomics 7, 1983–1997.PubMedCrossRefGoogle Scholar
  23. 23.
    Elias, J. E., and Gygi, S. P. (2007) Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry, Nat Meth 4, 207–214.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Chia-Feng Tsai
  • Yi-Ting Wang
  • Pei-Yi Lin
  • Yu-Ju Chen
    • 1
    Email author
  1. 1.Graduate Institute of Medicine and Center for Research Resources and DevelopmentKaohsiung Medical UniversityKaohsiungTaiwan

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