Advertisement

Quantitative Phosphoproteomic Analysis Using iTRAQ Method

  • Tomoya Asano
  • Takumi NishiuchiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1171)

Abstract

The MAPK (mitogen-activated kinase) cascade plays important roles in plant perception of and reaction to developmental and environmental cues. Phosphoproteomics are useful to identify target proteins regulated by MAPK-dependent signaling pathway. Here, we introduce the quantitative phosphoproteomic analysis using a chemical labeling method. The isobaric tag for relative and absolute quantitation (iTRAQ) method is a MS-based technique to quantify protein expression among up to eight different samples in one experiment. In this technique, peptides were labeled by some stable isotope-coded covalent tags. We perform quantitative phosphoproteomics comparing Arabidopsis wild type and a stress-responsive mapkk mutant after phytotoxin treatment. To comprehensively identify the downstream phosphoproteins of MAPKK, total proteins were extracted from phytotoxin-treated wild-type and mapkk mutant plants. The phosphoproteins were purified by Pro-Q® Diamond Phosphoprotein Enrichment Kit and were digested with trypsin. Resulting peptides were labeled with iTRAQ reagents and were quantified and identified by MALDI TOF/TOF analyzer. We identified many phosphoproteins that were decreased in the mapkk mutant compared with wild type.

Key words

Quantitative proteomics iTRAQ Phosphoproteome Phytopathogen Fusarium Trichothecene MAPK 

References

  1. 1.
    Dong J, Bergmann DC (2010) Stomatal patterning and development. Curr Top Dev Biol 91:267–297PubMedCrossRefGoogle Scholar
  2. 2.
    Mishra NS, Tuteja R, Tuteja N (2006) Signaling through MAP kinase networks in plants. Arch Biochem Biophys 452:55–68PubMedCrossRefGoogle Scholar
  3. 3.
    Samaj J, Baluska F, Hirt H (2004) From signal to cell polarity: mitogen-activated protein kinases as sensors and effectors of cytoskeleton dynamicity. J Exp Bot 55:189–198PubMedCrossRefGoogle Scholar
  4. 4.
    Mao G, Meng X, Liu Y, Zheng Z, Chen Z et al (2011) Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis. Plant Cell 23:1639–1653PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Kline-Jonakin KG, Barrett-Wilt GA, Sussman MR (2011) Quantitative plant phosphoproteomics. Curr Opin Plant Biol 14:507–511PubMedCrossRefGoogle Scholar
  6. 6.
    Jones AME, Bennett MH, Mansfield JW, Grant M (2006) Analysis of the defence phosphoproteome of Arabidopsis thaliana using differential mass tagging. Proteomics 6:4155–4165PubMedCrossRefGoogle Scholar
  7. 7.
    Nespoulous C, Rofidal V, Sommerer N, Hem S, Rossignol M (2012) Phosphoproteomic analysis reveals major default phosphorylation sites outside long intrinsically disordered regions of Arabidopsis plasma membrane proteins. Proteome Sci 10:62PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Jorrín JV, Maldonado AM, Castillejo MA (2007) Plant proteome analysis: a 2006 update. Proteomics 7:2947–2962PubMedCrossRefGoogle Scholar
  9. 9.
    Asano T, Nishiuchi T (2011) Comparative analysis of phosphoprotein expression using 2D-DIGE. Methods Mol Biol 744:225–233PubMedCrossRefGoogle Scholar
  10. 10.
    Colcombet J, Hirt H (2008) Arabidopsis MAPKs: a complex signalling network involved in multiple biological processes. Biochem J 413:217–226PubMedCrossRefGoogle Scholar
  11. 11.
    Meszaros T, Helfer A, Hatzimasoura E, Magyar Z, Serazetdinova L et al (2006) The Arabidopsis MAP kinase kinase MKK1 participates in defence responses to the bacterial elicitor flagellin. Plant J 48:485–498PubMedCrossRefGoogle Scholar
  12. 12.
    Teige M, Scheikl E, Eulgem T, Doczi F, Ichimura K et al (2004) The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis. Mol Cell 15:141–152PubMedCrossRefGoogle Scholar
  13. 13.
    Qiu JL, Zhou L, Yun BW, Nielsen HB, Fiil BK et al (2008) Arabidopsis mitogen-activated protein kinase kinases MKK1 and MKK2 have overlapping functions in defense signaling mediated by MEKK1, MPK4, and MKS1. Plant Physiol 148:212–222PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Gao M, Liu J, Bi D, Zhang Z, Cheng F et al (2008) MEKK1, MKK1/MKK2 and MPK4 function together in a mitogen-activated protein kinase cascade to regulate innate immunity in plants. Cell Res 18:1190–1198PubMedCrossRefGoogle Scholar
  15. 15.
    Xing Y, Jia W, Zhang J (2008) AtMKK1 mediates ABA-induced CAT1 expression and H2O2 production via AtMPK6-coupled signaling in Arabidopsis. Plant J 54:440–451PubMedCrossRefGoogle Scholar
  16. 16.
    Nishiuchi T, Masuda D, Nakashita H, Ichimura K, Shinozaki K et al (2006) Fusarium phytotoxin trichothecenes have an elicitor-like activity in Arabidopsis thaliana, but the activity differed significantly among their molecular species. Mol Plant Microbe Interact 19:512–520PubMedGoogle Scholar
  17. 17.
    Shilov IV, Seymour SL, Patel AA, Loboda A, Tang WH et al (2007) The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra. Mol Cell Proteomics 6:1638–1655PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Division of Functional Genomics, Advanced Science Research CenterKanazawa UniversityKanazawaJapan

Personalised recommendations