Subject Area物理化学
Application of Separation Technology and Methodology for Shut-Gun Proteome Analysis
Zou HF(邹汉法); Jin WH(靳文海); Xie CH(谢传辉); Feng S(封顺); Ye ML(叶明亮)
Conference NameThe 4th Japan-China Joint Seminar Separation Sciences
Conference Date2005-11-7
Conference Place日本
Alternative Title分离技术与方法在代谢组学中的应用
Funding Organization日本色谱学会
AbstractProteins 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.
Document Type会议论文
Corresponding AuthorZou HF(邹汉法)
Recommended Citation
GB/T 7714
Zou HF,Jin WH,Xie CH,et al. Application of Separation Technology and Methodology for Shut-Gun Proteome Analysis[C],2005:127/1.
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