Proteomic and informatic analyses of signal transducing network

为了从蛋白质组学和生物信息学两方面对信号转导系统进行大规模的分析，本课题进行了几个方面的研究。建立了蛋白质组学研究设施。2. 该设备用于对包括枯草芽孢杆菌在内的各种物种进行蛋白质组学分析。3. 建立分析方法，进行差异蛋白表达分析，并用于蓝藻的光诱导变化。4 . .提出了一种利用抗体结合磁珠分离细胞器的新方法。该方法用于分离过氧化物酶体，并对纯化的细胞器进行蛋白质组学分析。在几种新发现的蛋白质中，发现了一种新的Lon蛋白酶异构体是过氧化物酶体特异性的。5. 我们建立了一种用前驱体扫描模式运行的三重四极杆质谱仪检测磷酸肽的新方法。该方法用于蛋白激酶C δ同工酶磷酸化位点的鉴定。在鉴定的六个磷酸化位点中，发现三个酪氨酸残基在氧化应激下被磷酸化。6. 为了分析EGF受体下游的信号通路，分离酪氨酸磷酸化蛋白并进行蛋白质组学分析。在鉴定出的150种蛋白质中，三分之一是新蛋白质。7. 将包含色氨酸肽序列数据的大规模蛋白质组学数据直接映射到原始基因组序列上，并比较标注的基因组数据与蛋白质组学数据的差异。除了原始核苷酸序列错误和基因注释错误外，还发现了几个新基因。8. 解决了几种信号蛋白和蛋白质复合物的三维结构，并阐明了修饰调节的蛋白质-蛋白质相互作用的详细机制。

To conduct large-scale analyses of signal transduction system both by proteomics and bioinformatics, several themes were pursued.1.A facility for proteomics was established. 2. The facility was used to conduct proteomic analyses of various species including B. subtilis. 3. Analytical methodology was established to conduct differential protein expression analysis, and used for the light-induced change of cyanobacteria. 4.. A new methodology was developed for the isolation of cellular organelles using antibody-bound magnetic beads. The method was used to isolate peroxisomes, and the purified organelle was subjected to proteomic analysis. Among several novel proteins identified, a novel isoform of Lon protease was found to be peroxisome specific. 5. We have established a new method to detect phosphopeptides using triple quadrupole mass spectrometer running in precursor scanning mode. The method was applied for the identification of phosphorylation sites on protein kinase C δ isozyme. Of six phosphorylation sites identified, three tyrosine residues were found to be phosphorylated upon oxidative stress. 6. To analyze the signaling pathways downstream of EGF receptor, tyrosine-phosphorylated proteins were isolated and analyzed by proteomic analysis. Of 150 proteins thus identified, one-third were novel proteins. 7. A large-scale proteomic data containing the sequence data of tryptic peptides were directly mapped onto the raw genome sequence, and the discrepancy between the annotated genome data and proteomic date was compared. Several novel genes in addition to the errors in the raw nucleotide sequence, and wrong annotations of genes were discovered. 8. The 3D structures of several signaling protein and protein complexes were solved, and the detailed mechanisms of modification-regulated protein-protein interaction were elucidated.