Chapter title |
Quantitative Phosphoproteomic Analysis Using iTRAQ Method.
|
---|---|
Chapter number | 19 |
Book title |
Plant MAP Kinases
|
Published in |
Methods in molecular biology, May 2014
|
DOI | 10.1007/978-1-4939-0922-3_19 |
Pubmed ID | |
Book ISBNs |
978-1-4939-0921-6, 978-1-4939-0922-3
|
Authors |
Asano T, Nishiuchi T, Tomoya Asano, Takumi Nishiuchi |
Editors |
George Komis, Jozef Šamaj |
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. |
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