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学科主题: 物理化学
题名: High efficient large scale proteome analysis strategies with serially coupled long microcolumn
作者: Tao DY(陶定银) ;  Zhang LH(张丽华) ;  Liang Z(梁振) ;  Zhang YK(张玉奎)
会议名称: The 24th MSB 2009
会议日期: 2009-10-19
出版日期: 2009-10-19
会议地点: 中国
通讯作者: 张丽华
部门归属: 十八室
主办者: 大连化物所
摘要: Comprehensive analysis of whole proteomes is an extraordinary challenge. LC/MS/MS is commonly used to separate peptides digested from proteomes extracted from cells or tissues. To increase protein identification capacity, improvement on separation efficiency and peak capacity of HPLC and sample fractionation to reduce sample complexity are two effective ways. Increasing column length is one of the effective solutions to improve the separation capacity of RPLC. However, it was hard to prepare a long microcolumn due to high backpressure generated during packing procedure. In our recent work, through connecting microcolumns of 5, 10 and 15 cm length via unions with minimal dead volume, long microcolumns with lengths up to 30 cm were obtained, and applied for analyzing protein digests. With a 30 cm long microcolumn (300 m i.d.), 318 proteins extracted from E. Coli were identified by RPLC-ESI MS/MS (LCQ), compared with 70 proteins by a 5 cm long microcolumn. In addition, such a column was applied in 2-D SCX-RPLC/MS/MS for peptides separation and protein identification. Compared to that obtained with normal 10 cm-long RPLC column, higher peak capacity (2365 vs 2015) and more identified proteins or protein groups (1836 vs 1358) with FDR<1% were obtained within shorter time (26.7 h vs 39 h). Furthermore, protein pre-fractionation by preparative microscale solution isoelectric focusing (PMSIEF), peptide separation by RPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS, were combined, and applied for high resolution separation and high sensitive detection of proteins extracted from E. coli. By comparison with that identified only by RPLC-MS/MS with serially coupled long microcolumn, the identified protein number was increased by 3.53 times (835/236), and the Mws and pIs ranges were extended (2563.0 Da to 219155.7 Da vs 2563.0 Da to 163837.0, 3.98 to 11.81 vs 4.01 to 11.42). In addition, a larger scale proteome analysis by combining PMSIEF fractionation technology with 2-D SCX-RPLC/MS/MS with a serially coupled long RPLC column was performed, and more proteins were identified.
语种: 中文
内容类型: 会议论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/113612
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:
Tao DY,Zhang LH,Liang Z,et al. High efficient large scale proteome analysis strategies with serially coupled long microcolumn[C]. 见:The 24th MSB 2009. 中国. 2009-10-19.
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