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学科主题: 物理化学
题名: Application of Separation Technology and Methodology for Shut-Gun Proteome Analysis
作者: Zou HF(邹汉法) ;  Jin WH(靳文海) ;  Xie CH(谢传辉) ;  Feng S(封顺) ;  Ye ML(叶明亮)
会议名称: The 4th Japan-China Joint Seminar Separation Sciences
会议日期: 2005-11-7
出版日期: 2005-11-07
会议地点: 日本
其他题名: 分离技术与方法在代谢组学中的应用
通讯作者: 邹汉法
ISSN: 1342-8284
部门归属: 十八室
主办者: 日本色谱学会
摘要: Proteins from human plasma were prefractionated by online sequential strong cation exchange chromatography and reversed-phase chromatography. The resulting 30 fractions were individually digested by trypsin, and further analyzed by capillary RP-HPLC coupled with linearion trap mass spectrometry. With stringent criteria, a total of 1292 distinct proteins were successfully identified. Considering our strategy allows high-throughput of protein identification in plasma, the prefractionation of proteins before LC-MS analysis is a simple and effective approach to facilitate human plasma proteome analysis. A capillary microreactor with immobilized trypsin monolithic matrix was prepared and used for rapid digestion of complex protein mixture for shotgun proteome analysis. It was found that the digestion of proteins by the capillary microreactor is very efficient due to the presence of high concentration of trypsin in the prepared microreactor, and the immobilized trypsin has higher tolerance with denaturing agent like urea. This capillary microreactor was matched with capillary column LC-MS to analysis of the total cell lysate of Saccharomyces cerevisiae. After sequest database search, a total of 1578 unique peptides corresponding to 541 proteins were identified when 590 ng yeast proteins was digested by the microreactor with incubation time of only 1 min. Finally, an improved strategy for preparation of monolithic silica ODS capillary column with high homogeneity was proposed. The design for constructing an integrated nanoelectrospray emitter on the monolithic silica ODS capillary column was introduced. In comparison with the separated configuration where the emitter is connected to monolithic capillary column by the aid of a zero-dead volume union, the integrated capillary column has inherent advantage of the minimized extra-column volume thus providing improved separation quality. The high separation performance with a 30-cm column allows highly confident identification of 662 distinct proteins through assignment of 1933 unique peptides by analysis of tryptic digest of 500 ng S. cerevisiae proteins. The higher separation efficiency by a 60-cm monolithic capillary column increase the proteome coverage with identification of 1323 proteins through assignment of 5501 unique peptides over 400 min gradient elution.
语种: 中文
内容类型: 会议论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/111908
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,Jin WH,Xie CH,et al. Application of Separation Technology and Methodology for Shut-Gun Proteome Analysis[C]. 见:The 4th Japan-China Joint Seminar Separation Sciences. 日本. 2005-11-7.
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