Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hunter, T. (1995) Protein kinases and Âphosphatases: The Yin and Yang of protein phosphorylation and signaling, Cell 80, 225–236.
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.
Ian, I. S., Ty, T., and Daniel, F. (2001) 18O Labeling: a tool for proteomics, Rapid Communications in Mass Spectrometry 15, 2456–2465.
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.
Andersson, L., and Porath, J. (1986) Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography, Analytical Biochemistry 154, 250–254.
Posewitz, M. C., and Tempst, P. (1999) Immobilized Gallium(III) Affinity Chromatography of Phosphopeptides, Analytical Chemistry 71, 2883–2892.
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.
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.
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.
Corthals, G. L., Aebersold, R., Goodlett, D. R., and Burlingame, A. L. (2005) Identification of Phosphorylation Sites Using MicroimmobiÂlized 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Acknowledgments
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).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Tsai, CF., Wang, YT., Lin, PY., Chen, YJ. (2011). Phosphoproteomics by Highly Selective IMAC Protocol. In: Li, K. (eds) Neuroproteomics. Neuromethods, vol 57. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-111-6_14
Download citation
DOI: https://doi.org/10.1007/978-1-61779-111-6_14
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-110-9
Online ISBN: 978-1-61779-111-6
eBook Packages: Springer Protocols