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学科主题: 物理化学
题名: Preparing a metal-ion chelated immobilized enzyme reactor based on the polyacrylamide monolith grafted with polyethylenimine for a facile regeneration and high throughput tryptic digestion in proteomics
作者: Wu, Shuaibin1, 3;  Zhang, Lei;  Yang, Kaiguang1;  Liang, Zhen1;  Zhang, Lihua;  Zhang, Yukui1
通讯作者: 杨开广
关键词: Metal-ion chelation ;  Polyacrylamide monolith ;  Polyethylenimine ;  Tryptic digestion ;  Regenerable immobilized enzyme reactor
刊名: ANALYTICAL AND BIOANALYTICAL CHEMISTRY
发表日期: 2012
DOI: 10.1007/s00216-011-5501-6
卷: 402, 期:2, 页:703-710
收录类别: SCI
文章类型: Article
部门归属: 18
项目归属: 1810
产权排名: 1,1
类目[WOS]: Biochemical Research Methods ;  Chemistry, Analytical
研究领域[WOS]: Biochemistry & Molecular Biology ;  Chemistry
英文摘要: Initially, a poly (glycidyl methacrylate-co-acrylamide-co-methylenebisacrylamide) monolith was prepared in the 100 mu m i.d. capillary, and then was grafted with polyethylenimine (Mw, similar to 25,000) for adsorbing Cu(2+), followed by chelating trypsin. As a result, efficient digestion for BSA (100 ng/mu L) was completed within 50 s via such immobilized enzyme reactor (IMER); yielding 47% sequence coverage by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Compared with the conventional method for preparing the metal-ion chelated IMER, the regeneration of such IMER can be achieved facilely by the respective 30 min desorption and re-adsorption of trypsin, and 51% sequence coverage was obtained for 50 s BSA digestion after regeneration. BSA down to femtomole was also efficiently digested by the prepared regenerable IMER. Meanwhile, after the consecutive digestion of myoglobin and BSA, there was not any mutual interference for both during MALDI-TOF MS identification, indicating the low nonspecific adsorption of such regenerable IMER. To test the applicability of regenerable IMER for complex sample profiling, proteins (150 ng) extracted from Escherichia coli were digested within 80 s by the regenerable IMER and further analyzed by nanoreversed phase liquid chromatography-electrospray ionization-mass spectrometry successfully, showing its practicability for the high throughput analysis of complex samples.
关键词[WOS]: LASER-DESORPTION/IONIZATION-TIME ;  MAGNETIC SILICA MICROSPHERES ;  PHASE LIQUID-CHROMATOGRAPHY ;  EFFICIENT PROTEIN DIGESTION ;  TANDEM MASS-SPECTROMETRY ;  TRYPSIN IMMOBILIZATION ;  IDENTIFICATION ;  ONLINE ;  MICROREACTOR ;  ADSORPTION
语种: 英语
WOS记录号: WOS:000298645300012
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/118276
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, Natl Chromatog R&A Ctr, Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China
2.Chinese Acad Sci, Dalian Inst Chem Phys, State Key Lab Catalysis, Dalian 116023, Peoples R China
3.Chinese Acad Sci, Grad Sch, Beijing 100049, Peoples R China

Recommended Citation:
Wu, Shuaibin,Zhang, Lei,Yang, Kaiguang,et al. Preparing a metal-ion chelated immobilized enzyme reactor based on the polyacrylamide monolith grafted with polyethylenimine for a facile regeneration and high throughput tryptic digestion in proteomics[J]. ANALYTICAL AND BIOANALYTICAL CHEMISTRY,2012,402(2):703-710.
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