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Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed-phase liquid chromatography with mass spectrometry for online protein profiling
Wang, Tingting1,2; Ma, Junfeng1,2; Wu, Shuaibin1,2; Yuan, Huiming1; Zhang, Lihua1; Liang, Zhen1; Zhang, Yukui1
关键词Capillary Isoelectric Focusing Immobilized Enzyme Microreactor Integrated Platform Nanoreversed-phase Liquid Chromatography Trypsin
刊名ELECTROPHORESIS
2011-10-01
DOI10.1002/elps.201100030
32期:20页:2848-2856
收录类别SCI
文章类型Article
WOS标题词Science & Technology ; Life Sciences & Biomedicine ; Physical Sciences
类目[WOS]Biochemical Research Methods ; Chemistry, Analytical
研究领域[WOS]Biochemistry & Molecular Biology ; Chemistry
关键词[WOS]ZONE-ELECTROPHORESIS ; PH GRADIENT ; SEPARATION ; SYSTEM ; PRECONCENTRATION ; MONOLITHS ; DIGESTION ; REACTORS ; CE
英文摘要An integrated platform with the combination of protein and peptide separation was established via online protein digestion, by which proteins were first separated by CIEF, online digested by a trypsin immobilized enzyme microreactor, trapped and desalted by two parallel trap columns, separated by nanoreversed-phase and finally identified by MS. In such a platform, two hollow fiber membrane interfaces were used. One was applied to supply catholyte and electric contact, and another to supply adjustment buffer to improve the compatibility of protein separation and tryptic digestion. A poly(octadecyl acrylate-co-ethylene dimethacrylate) monolithic column served as the trap column to capture sample and to remove the ampholytes from CIEF. A hybrid silica monolith-based immobilized trypsin microreactor was used for online protein digestion. To evaluate the performance of such a platform, a 4-protein mixture with a loading amount of only 0.29 mu g, was analyzed, and sequence coverages for BSA, myoglobin, beta-lactoglobulin and ribonuclease A were 8, 26, 10 and 54%, respectively. Furthermore, such an integrated platform was successfully applied for the analysis of proteins extracted from Escherichia coli, and 101 proteins were positively identified. We anticipate that the integrated platform developed herein will provide a promising tool for low-abundance protein identification with the combination of top-down and bottom-up approaches.
语种英语
WOS记录号WOS:000297424500011
引用统计
文献类型期刊论文
条目标识符http://cas-ir.dicp.ac.cn/handle/321008/142719
专题中国科学院大连化学物理研究所
作者单位1.Chinese Acad Sci, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Natl Chromatog Res & Anal Ctr, Dalian 116023, Peoples R China
2.Chinese Acad Sci, Grad Univ, Beijing, Peoples R China
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Wang, Tingting,Ma, Junfeng,Wu, Shuaibin,et al. Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed-phase liquid chromatography with mass spectrometry for online protein profiling[J]. ELECTROPHORESIS,2011,32(20):2848-2856.
APA Wang, Tingting.,Ma, Junfeng.,Wu, Shuaibin.,Yuan, Huiming.,Zhang, Lihua.,...&Zhang, Yukui.(2011).Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed-phase liquid chromatography with mass spectrometry for online protein profiling.ELECTROPHORESIS,32(20),2848-2856.
MLA Wang, Tingting,et al."Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed-phase liquid chromatography with mass spectrometry for online protein profiling".ELECTROPHORESIS 32.20(2011):2848-2856.
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