Quantitative Analysis of Protein Phosphorylation Using Two-Dimensional Difference Gel Electrophoresis

  • Zhiping Deng
  • Shuolei Bu
  • Zhi-Yong Wang
Part of the Methods in Molecular Biology book series (MIMB, volume 876)


Posttranslational modifications of proteins, especially phosphorylation and dephosphorylation, play an important role in signal transduction and cellular regulation in plants. Both 2-DE gel-based and non-gel-based proteomic technologies can monitor the changes in phosphorylation state of proteins. In this chapter, we describe two protocols for discovery and validation of differential protein phosphorylation using affinity enrichment of phosphoproteins by immobilized metal affinity chromatography (IMAC) or protein immunoprecipitation (IP) followed by two-dimensional difference gel electrophoresis (2-D DIGE). We name these methods IMAC-DIGE and IP-DIGE. For IMAC-DIGE, phosphoproteins are enriched from tissue extract using GaCl3-based IMAC and then analyzed by 2-D DIGE, which reveals changes of protein phosphorylation as protein spot shifts. IMAC enrichment improves detection of low-abundance regulatory phosphoproteins. For IP-DIGE, proteins of interest can be immunopurified and then analyzed by 2-D DIGE to confirm changes of posttranslational modifications that alter the charge or size of the proteins.

Key words

Phosphoprotein Immobilized metal affinity chromatography Proteomics 2-D DIGE Phosphorylation Posttranslational modification Brassinosteroid BZR1 



This work was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy through Grant DE-FG02-08ER15973 (to Z.-Y. W. and A. L. B.). Z. D. was partially supported by funds from Zhejiang Academy of Agricultural Sciences.


  1. 1.
    Hunter T (2007) The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol Cell 28:730–738PubMedCrossRefGoogle Scholar
  2. 2.
    Kersten B, Agrawal GK, Durek P, Neigenfind J, Schulze W, Walther D, Rakwal R (2009) Plant phosphoproteomics: an update. Proteomics 9:964–988PubMedCrossRefGoogle Scholar
  3. 3.
    Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154–1169PubMedCrossRefGoogle Scholar
  4. 4.
    Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Stable isotope labeling by amino acids in cell culture. SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386PubMedCrossRefGoogle Scholar
  5. 5.
    Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 17:994–999PubMedCrossRefGoogle Scholar
  6. 6.
    Macek B, Mann M, Olsen JV (2009) Global and site-specific quantitative phosphoproteomics: principles and applications. Annu Rev Pharmacol Toxicol 49:199–221PubMedCrossRefGoogle Scholar
  7. 7.
    Nuhse TS, Bottrill AR, Jones AM, Peck SC (2007) Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses. Plant J 51:931–940PubMedCrossRefGoogle Scholar
  8. 8.
    O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021PubMedGoogle Scholar
  9. 9.
    Unlu M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18:2071–2077PubMedCrossRefGoogle Scholar
  10. 10.
    Tonge R, Shaw J, Middleton B, Rowlinson R, Rayner S, Young J, Pognan F, Hawkins E, Currie I, Davison M (2001) Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics 1:377–396PubMedCrossRefGoogle Scholar
  11. 11.
    Tan HT, Tan S, Lin Q, Lim TK, Hew CL, Chung MC (2008) Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells. Mol Cell Proteomics 7:1174–1185PubMedCrossRefGoogle Scholar
  12. 12.
    Thon JN, Schubert P, Duguay M, Serrano K, Lin S, Kast J, Devine DV (2008) Comprehensive proteomic analysis of protein changes during platelet storage requires complementary proteomic approaches. Transfusion 48:425–435PubMedCrossRefGoogle Scholar
  13. 13.
    Wu WW, Wang G, Baek SJ, Shen RF (2006) Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/TOF. J Proteome Res 5:651–658PubMedCrossRefGoogle Scholar
  14. 14.
    Thelen JJ, Peck SC (2007) Quantitative proteomics in plants: choices in abundance. Plant Cell 19:3339–3346PubMedCrossRefGoogle Scholar
  15. 15.
    Tang W, Deng Z, Oses-Prieto JA, Suzuki N, Zhu S, Zhang X, Burlingame AL, Wang ZY (2008) Proteomics studies of brassinosteroid signal transduction using prefractionation and two-dimensional DIGE. Mol Cell Proteomics 7:728–738PubMedGoogle Scholar
  16. 16.
    Gu Y, Deng Z, Paredez AR, DeBolt S, Wang ZY, Somerville C (2008) Prefoldin 6 is required for normal microtubule dynamics and organization in Arabidopsis. Proc Natl Acad Sci USA 105:18064–18069PubMedCrossRefGoogle Scholar
  17. 17.
    Deng Z, Zhang X, Tang W, Oses-Prieto JA, Suzuki N, Gendron JM, Chen H, Guan S, Chalkley RJ, Peterman TK, Burlingame AL, Wang ZY (2007) A proteomics study of brassinosteroid response in Arabidopsis. Mol Cell Proteomics 6:2058–2071PubMedCrossRefGoogle Scholar
  18. 18.
    He JX, Gendron JM, Yang Y, Li J, Wang ZY (2002) The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid signaling pathway in Arabidopsis. Proc Natl Acad Sci USA 99:10185–10190PubMedCrossRefGoogle Scholar
  19. 19.
    Tang W, Kim T-W, Oses-Prieto JA, Sun Y, Deng Z, Zhu S, Wang R, Burlingame AL, Wang Z-Y (2008) BSKs mediate signal transduction from the receptor kinase BRI1 in Arabidopsis. Science 321:557–560PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Plant BiologyCarnegie Institution for ScienceStanfordUSA
  2. 2.Institute of Virology and BiotechnologyZhejiang Academy of Agricultural ScienceHangzhouChina

Personalised recommendations