Abstract
The lack of an efficient technique to enrich for phosphorylated proteins is limiting of phosphoproteomes studies. Enrichment with antiphospho serine, antiphospho threonine, or anti-phospho tyrosine antibodies depends on the affinity and specificity of antibodies that limit comprehensiveness of the analysis (1–3). Metabolic labeling of cells with inorganic (32P) phosphate, followed by an analysis of the whole proteome, requires efficient separation to exclude comigration with nonphosphorylated proteins (4,5). Chemical modifications of phosphorylated residues in the peptides, followed by MS, allow identification of phosphorylation sites (1,5–8). However, these techniques do not provide information about full length proteins, e.g., molecular mass of intact proteins. Immobilized metal-affinity chromatography (IMAC) has been successfully used for enrichment of phosphopeptides (1,5,9,10), but the efficiency of purification of phosphoproteins has been low, indicating significant losses of phosphoproteins (5,9,10). Another important limitation of available IMAC methods is that bound proteins are eluted into a solution which is incompatible with further separation by 2-DE. A modified IMAC technique developed in our laboratory makes possible efficient and comprehensive analysis of phosphoproteins, and is compatible with 2-DE (11).
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References
Figeys, D. (2002) Proteomics approaches in drug discovery. Anal. Chem. 74, 412–419.
Stancato, L. F. and Petricoin, E. F. III (2001) Fingerprinting of signal transduc-tion pathways using a combination of anti-phosphotyrosine immunoprecipitations and two-dimensional polyacrylamide gel electrophoresis. Electrophoresis 22, 2120–2124.
Imam-Sghiouar, N., Laude-Lemaire, I., Labas, V., and Pflieger, D. et al. (2002) Subproteomics analysis of phosphorylated proteins: application to the study of B-lymphoblasts from a patient with Scott syndrome. Proteomics 2, 828–838.
Stasyk, T., Dubrovska, A., Lomnytska, M., Yakymovych, I. et al. (2005) Phosphopro-teome profiling of transforming growth factor (TGF)-beta signaling: abrogation of TGFbeta1-dependent phosphorylation of transcription factor-II-I (TFII-I) enhances cooperation of TFII-I and Smad3 in transcription. Mol. Biol. Cell. 16, 4765–4780.
Adam, G. C., Sorensen, E. J., and Cravatt, B. F. (2002) Mapping enzyme active sites in complex proteomes. Mol. Cell. Proteomics, 1, 781–790.
Zhou, H., Watts, J. D., and Aebersold, R. (2001) Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry. Nature Biotech. 19, 375–378.
Oda, Y., Nagasu, T., and Chait, B. T. (2001) Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nature Biotech. 19, 379–382.
Knight, Z. A., Schilling, B., Row, R. H., and Kenski, D. M. et al. (2003) Phosphos-pecific proteolysis for mapping sites of protein phosphorylation. Nature Biotech. 21, 1047–1054.
Ficarro, S. B., McCleland, M. L., Stukenberg, P. T., and Burke, D. J. et al. (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharo-myces cerevisiae. Nature Biotech. 20, 301–305.
Phosphoprotein Purification Handbook, Vol. 12, Qiagen, Crawley 2002, pp. 7–19.
Dubrovska, A. and Souchelnytskyi, S. (2005) Efficient enrichment of intact phos-phorylated proteins by modified immobilized metal-affinity chromatography. Proteomics 5, 4678–4683.
Souchelnytskyi, S., Moustakas, A., and Heldin, C.-H. (2002) TGF-beta signaling from a three-dimensional perspective: insight into selection of partners. Trends Cell Biol. 12, 304–307.
Kanamoto, T., Hellman, U., Heldin, C.-H., Souchelnytskyi, S. (2002) Functional proteomics of transforming growth factor-beta1-stimulated Mv1Lu epithelial cells: Rad51 as a target of TGFbeta1-dependent regulation of DNA repair. EMBO J. 21, 1219–1230.
Yan, X., Li, F., Liang, Y., and Shen, Y. et al. (2003) Human Nudel and NudE as regulators of cytoplasmic dynein in poleward protein transport along the mitotic spindle. Mol. Cell. Biol. 23, 1239–1250.
Vodermaier, H. C. (2004) APC/C and SCF: controlling each other and the cell cycle. Curr. Biol. 14, R787–R796.
Matousek, P., Durchankova, D., Svandova, I., Novotny, J., and Svoboda, P. (2005) Agonist-induced tyrosine phosphorylation of Gq/G11 alpha requires the intact structure of membrane domains. Biochem. Biophys. Res. Comm. 328, 526–532.
Mizukoshi, E., Suzuki, M., Misono, T., and Loupatov, A. et al. (2001) Fibroblast growth factor-1 interacts with the glucose-regulated protein GRP75/mortalin. Biochem. Biophys. Res. Commun. 280, 1203–1209.
Dierks, H., Kolanus, J., and Kolanus, W. (2001) Actin cytoskeletal association of cytohesin-1 is regulated by specific phosphorylation of its carboxyl-terminal polybasic domain. J. Biol. Chem. 276, 37472–37481.
Shen, T. L. and Guan, J. L. (2004) Grb7 in intracellular signaling and its role in cell regulation. Front. Biosci. 9, 192–200.
Acknowledgments
The author would like to thank Serhiy Souchelnytskyi for his advices and encouragement and J. Ericsson, V. Lukiyanchuk, C.-H. Heldin, N. Bhaskaran for comments, U. Hellman for discussions and access to a mass spectrometer. This work was supported in part by grants from the Swedish Cancer Society, the Swedish Research Council, the Hiroshima University, and Merck KGaA to Serhiy Souchelnytskyi.
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Dubrovska, A. (2009). Efficient Enrichment of Intact Phosphorylated Proteins by Modified Immobilized Metal-Affinity Chromatography. In: Walker, J.M. (eds) The Protein Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-198-7_159
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DOI: https://doi.org/10.1007/978-1-59745-198-7_159
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