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学科主题: 物理化学
题名: Development of Technology and Method for Phosphoproteome and Glycoproteome 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 1st China-USA Symposium on Separation Sciences
会议日期: 2009-10-17
出版日期: 2009-10-17
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. Furthermore, a new approach was established by integration of the enrichment of phosphopeptides with Ti4+-IMAC and peptide fractionation with strong cation-exchange (SCX) column for large-scale phosphoproteome analysis of human liver, and about 7000 phosphorylation sites were detected. 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. Phosphopeptides enriched from tryptic digests of phosphoproteins were analyzed by nano-LC-MS2-MS3, and the acquired MS2 and MS3 spectra in valid MS2/MS3 pairs were searched against the composite database separately. This methodology combined with multi-protease digestion approach was applied to analyze the phosphorylation of the standards phosphoproteins and cyclic AMP dependent protein kinases. In silico digestion of proteins in human proteome database by trypsin indicates that a significant percentage of tryptic N-glycopeptides is not in the preferred detection mass range of shotgun proteomics approach, that is, from 800 to 3500 Da. And the quite big size of glycan groups may block trypsin to access the K, R residues near N-glycosites for digestion, which will result in generation of big glycopeptides. Thus many N-glycosites could not be localized if only trypsin was used to digest proteins. We developed a comprehensive way to analyze the N-glycoproteome of human liver tissue by combination of hydrazide chemistry method and multiple enzyme digestion, which leads to great improvement in coverage of N-glycosites identified.
语种: 中文
内容类型: 会议论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/113820
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 Method for Phosphoproteome and Glycoproteome Analysis[C]. 见:The 1st China-USA Symposium on Separation Sciences. 中国. 2009-10-17.
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