Quantitative Phosphoproteomic Analysis Using iTRAQ Method

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


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 


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

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

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