title: Quantitative phosphoproteomic analysis using iTRAQ method.
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.
authors.affiliation: Division of Functional Genomics, Advanced Science Research Center, Kanazawa University, 920-0934, Kanazawa, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Asano
authors.forename: Takumi
authors.initials: T
authors.lastname: Nishiuchi
mesh_terms: D000478:Alkylation; D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry; D007202:Indicators and Reagents / Q000737:chemistry; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013194:Staining and Labeling; D014357:Trypsin / Q000378:metabolism
chemical_list: D007202:Indicators and Reagents; D010750:Phosphoproteins; D020928:Mitogen-Activated Protein Kinases; D014357:Trypsin; D003545:Cysteine