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题名: Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed-phase liquid chromatography with mass spectrometry for online protein profiling
作者: 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
发表日期: 2011-10-01
DOI: 10.1002/elps.201100030
卷: 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
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.
关键词[WOS]: ZONE-ELECTROPHORESIS ;  PH GRADIENT ;  SEPARATION ;  SYSTEM ;  PRECONCENTRATION ;  MONOLITHS ;  DIGESTION ;  REACTORS ;  CE
语种: 英语
WOS记录号: WOS:000297424500011
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/142719
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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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

Recommended Citation:
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.
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