Isolation of the Arabidopsis Phosphoproteome Using a Biotin-tagging Approach

  • Kwon, Sun Jae (School of Life Sciences and Biotechnology, Korea University) ;
  • Choi, Eun Young (School of Life Sciences and Biotechnology, Korea University) ;
  • Seo, Jong Bok (Seoul Branch, Korea Basic Science Institute) ;
  • Park, Ohkmae K. (School of Life Sciences and Biotechnology, Korea University)
  • Received : 2007.04.13
  • Accepted : 2007.06.20
  • Published : 2007.10.31


Protein phosphorylation plays a key role in signal transduction in cells. Since phosphoproteins are present in low abundance, enrichment methods are required for their purification and analysis. Chemical derivatization strategies have been devised for enriching phosphoproteins and phosphopeptides. In this report, we employed a strategy that replaces the phosphate moieties on serine and threonine residues with a biotin-containing tag via a series of chemical reactions. Ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO)-depleted protein extracts prepared from Arabidopsis seedlings were chemically modified for 'biotin-tagging'. The biotinylated (previously phosphorylated) proteins were then selectively isolated by avidin-biotin affinity chromatography, followed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). This led to the identification of 31 protein spots, representing 18 different proteins, which are implicated in a variety of cellular processes. Despite its current technical limitations, with further improvements in tools and techniques this strategy may be developed into a useful approach.


Supported by : Korea Science and Engineering Foundation, Korea Research Foundation


  1. Bykova, N. V., Egsgaard, H., and Moller, I. M. (2003) Identification of 14 new phosphoproteins involved in important plant mitochondrial processes. FEBS Lett. 540, 141−146
  2. Lad, L., Mewies, M., and Randaven, E. L. (2002) Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites. Biochemistry 41, 13774−13781
  3. Landry, F., Lombardo, C. R., and Smith, J. W. (2000) A method for application of samples to matrix-assisted laser desorption ionization time-of-flight targets that enhances peptide detection. Anal. Biochem. 279, 1−8
  4. Osanai, T., Magota, K., Tanaka, M., Shimada, M., Murakami, R., et al. (2005) Intracellular signaling for vasoconstrictor coupling factor 6: novel function of beta-subunit of ATP synthase as receptor. Hypertension 46, 1140−1146
  5. Pandey, A., Podtelejnikov, A. V., Blagoev, B., Bustelo, X. R., Mann, M., et al. (2000) Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proc. Natl. Acad. Sci. USA 97, 179−184
  6. Shimokawa, H., Fujii, Y., Furuichi, M., Sekiguchi, M., and Nakabeppu, Y. (2000) Functional significance of conserved residues in the phosphohydrolase module of Escherichia coli MutT protein. Nucleic Acids Res. 28, 3240−3249
  7. Sorrell, D. A., Marchbank, A. M., Chrimes, D. A., Dickinson, J. R., Rogers, H. J., et al. (2000) The Arabidopsis 14-3-3 protein, GF14omega, binds to the Schizosaccharomyces pombe Cdc25 phosphatase and rescues checkpoint defects in the rad24- mutant. Planta 218, 50−57
  8. You, M. K., Oh, S. I., Ok, S. H., Cho, S. K., Shin, H. Y., et al. (2007) Identification of putative MAPK kinases in Oryza minuta and O. sativa responsive to biotic stresses. Mol. Cells 23, 108−114
  9. Goshe, M. B., Conrads, T. P., Panisko, E. A., Angell, N. H., Veenstra, T. D., et al. (2001) Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses. Anal. Chem. 73, 2578−2586
  10. Lee, S., Lee, E. J., Yang, E. J., Lee, J. E., Park, A. R., et al. (2004) Proteomic identification of annexins, calcium-dependent membrane binding proteins that mediate osmotic stress and abscisic acid signal transduction in Arabidopsis. Plant Cell 16, 1378−1391
  11. Jensen, O. N., Wilm, M., Shevchenko, A., and Mann, M. (1999) Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels. Meth. Mol. Biol. 112, 513−530
  12. Nowitzki, U., Gelius-Dietrich, G., Schwieger, M., Henze, K., and Martin, W. (2004) Chloroplast phosphoglycerate kinase from Euglena gracilis: endosymbiotic gene replacement going against the tide. Eur. J. Biochem. 271, 4123−4131
  13. Baudry, A., Caboche, M., and Lepiniec, L. (2006) TT8 controls its own expression in a feedback regulation involving TTG1 and homologous MYB and bHLH factors, allowing a strong and cell-specific accumulation of flavonoids in Arabidopsis thaliana. Plant J. 46, 768−779
  14. Sugihara, K., Hanagata, N., Dubinsky, Z., Baba, S., and Karube, I. (2000) Molecular characterization of cDNA encoding oxygen evolving enhancer protein 1 increased by salt treatment in the mangrove Bruguiera gymnorrhiza. Plant Cell Physiol. 41, 1279−1285
  15. Gaberc-Porekar, V. and Menart, V. (2001) Perspectives of immobilized- metal affinity chromatography. J. Biochem. Biophys. Methods 49, 335−360
  16. Laugesen, S., Bergoin, A., and Rossignol, M. (2004) Deciphering the plant phosphoproteome: tools and strategies for a challenging task. Plant Physiol. Biochem. 42, 929−936
  17. Pawson, T. and Scott, J. D. (2005) Protein phosphorylation in signaling--50 years and counting. Trends Biochem. Sci. 30, 286−290
  18. Salomon, A. R., Ficarro, S. B., Brill, L. M., Brinker, A., Phung, Q. T., et al. (2003) Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry. Proc. Natl. Acad. Sci. USA 100, 443−448
  19. Herrmann, L., Bockau, U., Tiedtke, A., Hartmann, M. W., and Weide, T. (2006) The bifunctional dihydrofolate reductase thymidylate synthase of Tetrahymena thermophila provides a tool for molecular and biotechnology applications. BMC Biotechnol. 20, 6−21
  20. Gronborg, M., Kristiansen, T. Z., Stensballe, A., Andersen, J. S., Ohara, O., et al. (2002) A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, frigg, as a protein kinase a substrate. Mol. Cell. Proteomics 1, 517−527
  21. Fillingame, R. H. and Dmitriev, O. Y. (2000) The oligomeric subunit C rotor in the fo sector of ATP synthase: unresolved questions in our understanding of function. J. Bioenerg. Biomembr. 32, 433−439
  22. Leitner, A. and Lindner, W. (2004) Current chemical tagging strategies for proteome analysis by mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 813, 1−26
  23. Kalume, D. E., Molina, H., and Pandey, A. (2003) Tackling the phosphoproteome: tools and strategies. Curr. Opin. Chem. Biol. 7, 64−69
  24. Nuhse, T. S., Stensballe, A., Jensen, O. N., and Peck, S. C. (2004) Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database. Plant Cell 16, 2394−2405
  25. Zhou, H., Watts, J. D., and Aebersold, R. (2001) A systematic approach to the analysis of protein phosphorylation. Nat. Biotechnol. 19, 375−378
  26. Ficarro, S. B., McCleland, M. L., Stukenberg, P. T., Burke, D. J., Ross, M. M., et al. (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 30, 301−305
  27. Heese, A., Ludwig, A. A., and Jones, J. D. (2005) Rapid phosphorylation of a syntaxin during the Avr9/Cf-9-race-specific signaling pathway. Plant Physiol. 138, 2406−2416
  28. Tsuchisaka, A. and Theologis, A. (2004) Unique and overlapping expression patterns among the Arabidopsis 1-aminocyclopropane- 1-carboxylate synthase gene family members. Plant Physiol. 136, 2982−3000
  29. Igamberdiev, A. U., Bykova, N. V., and Hill, R. D. (2006) Nitric oxide scavenging by barley hemoglobin is facilitated by a monodehydroascorbate reductase-mediated ascorbate reduction of methemoglobin. Planta 223, 1033−1040
  30. Kim, S. T., Cho, K. S., Jang, Y. S., and Kang, K. Y. (2001) Twodimensional electrophoretic analysis of rice proteins by polyethylene glycol fractionation for protein arrays. Electrophoresis 22, 2103−2109
  31. Vener, A. V., Harms, A., Sussman, M. R., and Vierstra, R. D. (2001) Mass spectrometric resolution of reversible protein phosphorylation in photosynthetic membranes of Arabidopsis thaliana. J. Biol. Chem. 276, 6959−6966
  32. Oda, Y., Nagasu, T., and Chait, B. T. (2001) Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat. Biotechnol. 19, 379−382
  33. Sramko, M., Markus, J., Kabat, J., Wolff, L., and Bies, J. (2006) Stress-induced inactivation of the c-Myb transcription factor through conjugation of SUMO-2/3 proteins. J. Biol. Chem. 281, 40065−40075
  34. Zettl, R., Schell, J., and Palme, K. (1994) Photoaffinity labeling of Arabidopsis thaliana plasma membrane vesicles by 5- azido-[7-3H]indole-3-acetic acid: identification of a glutathione S-transferase. Proc. Natl. Acad. Sci. USA 91, 689− 693
  35. 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