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学科主题: 分析化学
题名: Integrated Microfluidic System Containing Two Dimensional Separation and MS Identification for Proteomics Analysis
作者: Liang Y(梁玉) ;  Zhang LH(张丽华) ;  Zhang YK(张玉奎) ;  Liang Z(梁振) ;  Dai ZP(戴忠鹏) ;  Liang ZC(梁作成)
会议文集: Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy
会议名称: Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy
会议日期: 2011-3-13
出版日期: 2011
会议地点: 亚特兰大
通讯作者: 张丽华
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: 匹兹堡光谱学会
摘要: Microfluidic systems have been paid much attention to achieve high throughput proteomics analysis, with the advantages of improved portablility, low sample and reagent consumption, accelerated analytical speed, and facile integration. In this paper, the microchip containing SCX monolith and C18 tip was developed and integrated with ESI-MS/MS for two dimensional separation and MS identification of peptides. The SCX monolith in the microchannel was prepared by the photopolymerization of ethylene glycol methacrylate phosphate and bisacrylamide in a trinary porogenic solvent, consisting of dimethylsulfoxide, dodecanol, and N,N-dimethylformamide, which exhibits high dynamic binding capacity and high permeability. The C18 tip was prepared by a fused-silica capillary with a pulled 5-m needle tip, packed with C18 particles (5 m, 300 Å), inset and glued to the outlet of microchannel, coupled with ESI-MS/MS directly, used as the separation column and the chip-to-MS interface. 4 g tryptic digests of membrane proteins extracted from rat liver were loaded on SCX monolith by buffer C (0.1% formic acid water). Then a series of stepwise elution with salt concentration of 250, 350, 500, 750 and 1000 mM CH3COONH4 adjusted by buffer C and buffer D (1000 mM CH3COONH4 at pH 3) was used to elute peptides from SCX monolith onto C18 tip, followed by nano-RPLC-ESI-MS/MS analysis. After database searching using MASCOT with the significance threshold p<0.05, Ions score cut-off 20, and bold red required, 295 proteins were successfully identified with 879 peptides, containing 120 transmembrane proteins, which demonstrated the applicability of such a system for proteomics analysis.
英文摘要: Microfluidic systems have been paid much attention to achieve high throughput proteomics analysis, with the advantages of improved portablility, low sample and reagent consumption, accelerated analytical speed, and facile integration. In this paper, the microchip containing SCX monolith and C18 tip was developed and integrated with ESI-MS/MS for two dimensional separation and MS identification of peptides. The SCX monolith in the microchannel was prepared by the photopolymerization of ethylene glycol methacrylate phosphate and bisacrylamide in a trinary porogenic solvent, consisting of dimethylsulfoxide, dodecanol, and N,N-dimethylformamide, which exhibits high dynamic binding capacity and high permeability. The C18 tip was prepared by a fused-silica capillary with a pulled 5-m needle tip, packed with C18 particles (5 m, 300 Å), inset and glued to the outlet of microchannel, coupled with ESI-MS/MS directly, used as the separation column and the chip-to-MS interface. 4 g tryptic digests of membrane proteins extracted from rat liver were loaded on SCX monolith by buffer C (0.1% formic acid water). Then a series of stepwise elution with salt concentration of 250, 350, 500, 750 and 1000 mM CH3COONH4 adjusted by buffer C and buffer D (1000 mM CH3COONH4 at pH 3) was used to elute peptides from SCX monolith onto C18 tip, followed by nano-RPLC-ESI-MS/MS analysis. After database searching using MASCOT with the significance threshold p<0.05, Ions score cut-off 20, and bold red required, 295 proteins were successfully identified with 879 peptides, containing 120 transmembrane proteins, which demonstrated the applicability of such a system for proteomics analysis.
语种: 英语
内容类型: 会议论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/116033
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:
Liang Y,Zhang LH,Zhang YK,et al. Integrated Microfluidic System Containing Two Dimensional Separation and MS Identification for Proteomics Analysis[C]. 见:Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy. 亚特兰大. 2011-3-13.
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