Asano Tomoya, Nishiuchi Takumi
Division of Functional Genomics, Advanced Science Research Center, Kanazawa University, 920-0934, Kanazawa, Japan.
Methods Mol Biol. 2014;1171:251-8. doi: 10.1007/978-1-4939-0922-3_19.
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.
丝裂原活化蛋白激酶（MAPK）级联反应在植物感知发育和环境信号并做出反应的过程中发挥着重要作用。磷酸化蛋白质组学有助于识别受MAPK依赖信号通路调控的靶蛋白。在此，我们介绍一种使用化学标记方法的定量磷酸化蛋白质组学分析。相对和绝对定量等压标签（iTRAQ）方法是一种基于质谱的技术，可在一次实验中对多达八个不同样品中的蛋白质表达进行定量。在该技术中，肽段用一些稳定同位素编码的共价标签进行标记。我们在植物毒素处理后，对拟南芥野生型和应激反应mapkk突变体进行了定量磷酸化蛋白质组学比较。为了全面鉴定MAPKK的下游磷酸化蛋白，从经植物毒素处理的野生型和mapkk突变体植物中提取总蛋白。磷酸化蛋白通过Pro-Q® Diamond磷酸化蛋白富集试剂盒进行纯化，并用胰蛋白酶消化。所得肽段用iTRAQ试剂进行标记，然后通过MALDI TOF/TOF分析仪进行定量和鉴定。我们鉴定出许多在mapkk突变体中与野生型相比含量降低的磷酸化蛋白。
