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学科主题分析化学
Integrated microfluidic system for proteomic analysis consisting of on-line protein digestion, peptides separation and identification
Liang Y(梁玉); Tao DY(陶定银); Liang Z(梁振); Zhang LH(张丽华); Zhang YK(张玉奎)
会议文集Integrated microfluidic system for proteomic analysis consisting of on-line protein digestion, peptides separation and identification
会议名称35th International Symposium on High Performance Liquid Phase Separations and Related Techniques
会议日期2010-6-19
2011
会议地点Boston
页码0-0
出版者待补充
出版地待补充
合作性质墙报
部门归属1810
主办者CASSS
英文摘要Recently, microfluidic systems have been paid much attention to achieve high throughput proteome analysis. In our recent work, an integrated microfluidic platform, consisting of on-line protein digestion by immobilized enzymatic reactor (IMER) prepared in a microchannel, peptides separation and identification by nanoRPLC-ESI-MS/MS, was developed and successfully applied into the analysis of E. Coli cell lysate. To achieve rapid on-line protein digestion, a novel monolithic hydrophilic polymer-based IMER was prepared within the specified position of a microchannel, by photopolymerization of N-acryloxysuccinimide and poly (ethylene glycol) diacrylate, followed by trypsin immobilization via succinimide functionalities, by which 4μg myoglobin could be digested within 6 min, with sequence coverage as 81.5%. To achieve on-line separation and identification of protein digests, a fused-silica capillary (6-cm length, 75-μm i.d., 190-μm o.d.) with one end pulled to a fine point of ca. 5 μm was packed with C18 particles (5 m, 300Å), glued to the outlet channel of IMER without dead volume, and coupled with ESI-MS/MS directly. Sample could be introduced via a pump and a valve (A) connected to the inlet of IMER. Another microchannel vertical to the outlet of IMER was fabricated, and connected to binary pumps via another valve (B). With valve B blocked, proteins were introduced into IMER, on-line digested, and trapped on C18 tip. Then, with valve A blocked, peptides were separated by the nanoRPLC column and identified by ESI-MS/MS. By such an integrated microfluidic platform, within 85 min, a mixture of BSA, myoglobin and cytochrome C were identified with the average sequence coverage as 51.29%, 60.78% and 57.69%, respectively. In addition, ca. 50 proteins were identified from one 5-min RPLC fraction of E. Coli cell lysate.; Recently, microfluidic systems have been paid much attention to achieve high throughput proteome analysis. In our recent work, an integrated microfluidic platform, consisting of on-line protein digestion by immobilized enzymatic reactor (IMER) prepared in a microchannel, peptides separation and identification by nanoRPLC-ESI-MS/MS, was developed and successfully applied into the analysis of E. Coli cell lysate. To achieve rapid on-line protein digestion, a novel monolithic hydrophilic polymer-based IMER was prepared within the specified position of a microchannel, by photopolymerization of N-acryloxysuccinimide and poly (ethylene glycol) diacrylate, followed by trypsin immobilization via succinimide functionalities, by which 4μg myoglobin could be digested within 6 min, with sequence coverage as 81.5%. To achieve on-line separation and identification of protein digests, a fused-silica capillary (6-cm length, 75-μm i.d., 190-μm o.d.) with one end pulled to a fine point of ca. 5 μm was packed with C18 particles (5 m, 300Å), glued to the outlet channel of IMER without dead volume, and coupled with ESI-MS/MS directly. Sample could be introduced via a pump and a valve (A) connected to the inlet of IMER. Another microchannel vertical to the outlet of IMER was fabricated, and connected to binary pumps via another valve (B). With valve B blocked, proteins were introduced into IMER, on-line digested, and trapped on C18 tip. Then, with valve A blocked, peptides were separated by the nanoRPLC column and identified by ESI-MS/MS. By such an integrated microfluidic platform, within 85 min, a mixture of BSA, myoglobin and cytochrome C were identified with the average sequence coverage as 51.29%, 60.78% and 57.69%, respectively. In addition, ca. 50 proteins were identified from one 5-min RPLC fraction of E. Coli cell lysate.
文献类型会议论文
条目标识符http://cas-ir.dicp.ac.cn/handle/321008/115952
专题中国科学院大连化学物理研究所
通讯作者Zhang LH(张丽华)
推荐引用方式
GB/T 7714
Liang Y,Tao DY,Liang Z,et al. Integrated microfluidic system for proteomic analysis consisting of on-line protein digestion, peptides separation and identification[C]. 待补充:待补充,2011:0-0.
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