DICP OpenIR
Subject Area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)
Source PublicationIntegrated microfluidic system for proteomic analysis consisting of on-line protein digestion, peptides separation and identification
Conference Name35th International Symposium on High Performance Liquid Phase Separations and Related Techniques
Conference Date2010-6-19
2011
Conference PlaceBoston
Pages0-0
Publisher待补充
Publication Place待补充
Cooperation Status墙报
Department1810
Funding OrganizationCASSS
AbstractRecently, 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.
Document Type会议论文
Identifierhttp://cas-ir.dicp.ac.cn/handle/321008/115952
Collection中国科学院大连化学物理研究所
Corresponding AuthorZhang LH(张丽华)
Recommended Citation
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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