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题名: Preparation of high efficiency and low carry-over immobilized enzymatic reactor with methacrylic acid-silica hybrid monolith as matrix for on-line protein digestion
作者: Yuan, Huiming1;  Zhang, Lihua1;  Zhang, Yukui1
关键词: Immobilized trypsin reactor ;  Organic-inorganic hybrid monolith ;  One-pot strategy ;  Low carry-over protein digestion ;  Proteomics
刊名: JOURNAL OF CHROMATOGRAPHY A
发表日期: 2014-12-05
DOI: 10.1016/j.chroma.2014.10.067
卷: 1371, 页:48-57
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: In this work, a novel kind of organic-silica hybrid monolith based immobilized enzymatic reactor (IMER) was developed. The monolithic support was prepared by a single step "one-pot" strategy via the polycondensation of tetramethoxysilane and vinyltrimethoxysilane and in situ copolymerization of methacrylic acid and vinyl group on the precondensed siloxanes with ammonium persulfate as the thermal initiator. Subsequently, the monolith was activated by N-(3-dimethylaminopropyl) - N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), followed by the modification of branched polyethylenimine (PEI) to improve the hydrophilicity. Finally, after activated by EDC and NHS, trypsin was covalently immobilized onto the monolithic support. The performance of such a microreactor was evaluated by the in sequence digestion of bovine serum albumin (BSA) and myoglobin, followed by MALDI-TOF-MS analysis. Compared to those obtained by traditional in-solution digestion, not only higher sequence coverages for BSA (74 +/- 1.4% vs. 59.5 +/- 2.7%, n = 6) and myoglobin (93 +/- 3% vs. 81 +/- 4.5%, n = 6) were obtained, but also the digestion time was shortened from 24 h to 2.5 min, demonstrating the high digestion efficiency of such an IMER. The carry-over of these two proteins on the IMER was investigated, and peptides from BSA could not be found in mass spectrum of myoglobin digests, attributed to the good hydrophilicity of our developed monolithic support. Moreover, the dynamic concentration range for protein digestion was proved to be four orders of magnitude, and the IMER could endure at least 7-day consecutive usage. Furthermore, such an IMER was coupled with nano-RPLC-ESI/MS/MS for the analysis of extracted proteins from Escherichia coli. Compared to formerly reported silica hybrid monolith based IMER and the traditional in-solution counterpart, by our developed IMER, although the identified protein number was similar, the identified distinct peptide number was improved by 7% and 25% respectively, beneficial to improve the reliability of protein identification. The IMER was further online integrated with two-dimensional nano-HPLC-MS/MS system for the analysis of protein extracts from hepatocellular carcinoma (HCC) cells with low metastasis rate, and more than 3000 protein groups were identified, with only 46 proteins identified from the residues of the IMER. All these results demonstrated that such a hybrid monolith based IMER would be of great promise in the high throughput and high confidence proteome analysis. (C) 2014 Elsevier B.V. All rights reserved.
关键词[WOS]: CAPILLARY LIQUID-CHROMATOGRAPHY ;  TANDEM MASS-SPECTROMETRY ;  PROTEOMIC ANALYSIS ;  TRYPSIN REACTOR ;  MICROREACTOR ;  PEPTIDE ;  COLUMN ;  IDENTIFICATION ;  SEPARATION ;  SYSTEM
语种: 英语
WOS记录号: WOS:000345810200006
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/145666
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 Res & Anal Ctr, Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China

Recommended Citation:
Yuan, Huiming,Zhang, Lihua,Zhang, Yukui. Preparation of high efficiency and low carry-over immobilized enzymatic reactor with methacrylic acid-silica hybrid monolith as matrix for on-line protein digestion[J]. JOURNAL OF CHROMATOGRAPHY A,2014,1371:48-57.
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