Proteomic and informatic analyses of signal transducing network

为实现蛋白质组学和生物信息学对信号转导系统的大规模分析，本研究主要开展了以下几个方面的工作：1.建立了蛋白质组学研究平台。2.该设施用于进行包括B在内的各种物种的蛋白质组学分析。枯草杆菌。3.建立了一套分析方法，对蓝藻进行差异蛋白表达分析，并用于蓝藻光诱导变化的研究。4..开发了一种使用抗体结合的磁珠分离细胞器的新方法。该方法被用来分离过氧化物酶体，纯化的细胞器进行蛋白质组学分析。在几个新的蛋白质鉴定，一个新的异构体的Lon蛋白酶被发现是过氧化物酶体特异性。5.建立了一种三重四极杆质谱仪前体扫描检测磷酸肽的新方法。该方法已用于蛋白激酶C δ同工酶磷酸化位点的鉴定。在鉴定的6个磷酸化位点中，发现3个酪氨酸残基在氧化应激后被磷酸化。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通过蛋白质组学分析进行分析。在150种蛋白质中，有三分之一是新蛋白质。7.将包含胰蛋白酶肽序列的大规模蛋白质组数据直接映射到原始基因组序列上，并比较注释的基因组数据与蛋白质组数据之间的差异。除了原始核苷酸序列中的错误之外，还发现了几个新的基因，以及基因的错误注释。8.解决了几个信号蛋白和蛋白质复合物的三维结构，并阐明了修饰调控蛋白质-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.