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学科主题: 物理化学
题名: Development of Technology and Methods for Phosphoproteome Analysis
作者: Zou HF(邹汉法) ;  Ye ML(叶明亮) ;  Jiang XN(江新宁) ;  Han GH(韩广辉) ;  Wang FJ(王方军)
会议名称: The First China-Canada Symposium on Systems Biology
会议日期: 2010-10-20
出版日期: 2010-10-20
会议地点: 中国
通讯作者: 邹汉法
部门归属: 十八室
主办者: 大连化学物理研究所
摘要: The elucidation of protein post-translational modifications, such as phosphorylation, remains a challenging analytical task for proteomic studies. Since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric, a prerequisite for their identification is the specific enrichment of phosphopeptide prior to mass spectrometric analysis. A new type of the immobilized metal ion affinity chromatography (IMAC) through the chelating interaction between phosphate groups on the polymer and Zr4+ and Ti4+ (Zr4+- and Ti4+-IMAC) has been developed for enriching phosphopeptides. We also compared Zr4+- and Ti4+-IMAC to other enrichment methods including Fe3+-IMAC, TiO2 and ZrO2, and demonstrate superior selectivity and efficiency of Zr4+- and Ti4+-IMAC for the isolation and enrichment of phosphopeptides. Manual checking is commonly employed to validate the phosphopeptide identifications from database searching of tandem mass spectra. A simple automatic validation approach was developed for phosphopeptide identification by combining consecutive stage mass spectrometry data and the target-decoy database searching strategy. Only phosphopeptides identified from both MS2 and its corresponding MS3 were accepted for further filtering, which greatly improved the reliability in phosphopeptide identification. A classification filtering strategy to improve the phosphopeptide identification and phosphorylation site localization was developed. Phosphopeptide identifications were classified into four classes according to their different characteristics, and then, the identifications from each class of mass spectra were processed and filtered separately using different filtering strategies. A software suite named ArMone was specially designed for the management and analysis of phosphoproteome data. It can readily identify phosphopeptides with high reliability and high sensitivity, and can effectively pinpoint the most probable phosphorylation site. By integration of the enrichment of phosphopeptides with Ti4+-IMAC and data process software, large-scale phosphoproteome analysis of human liver with identification of 10,000 phosphorylation sites were performed. Compared with large scale phosphorylation analysis at proteome level, comprehensive and reliable phosphorylation site mapping of individual phosphoprotein is equally important. A novel method for confident phosphorylation site analysis of individual phosphoproteins was developed without manual interpretation of spectra. This methodology combined with multi-protease digestion approach was applied to analyze the phosphorylation of the standards phosphoproteins and cyclic AMP dependent protein kinases.
语种: 中文
内容类型: 会议论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/114192
Appears in Collections:中国科学院大连化学物理研究所_会议论文

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Recommended Citation:
Zou HF,Ye ML,Jiang XN,et al. Development of Technology and Methods for Phosphoproteome Analysis[C]. 见:The First China-Canada Symposium on Systems Biology. 中国. 2010-10-20.
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