Subject Area物理化学
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)
Conference Name35th International Symposium on High Performance Liquid Phase Separations and Related Techniques
Conference Date2010-6-19
Conference Place美国
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.
Document Type会议论文
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],2010:0/2.
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