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学科主题: 物理化学
题名: A hydrophilic immobilized trypsin reactor with N-vinyl-2-pyrrolidinone modified polymer microparticles as matrix for highly efficient protein digestion with low peptide residue
作者: Jiang, Hao1, 2;  Yuan, Huiming1;  Liang, Yu1;  Xia, Simin1, 2;  Zhao, Qun1, 2;  Wu, Qi1, 2;  Zhang, Lihua1;  Liang, Zhen1;  Zhang, Yukui1
通讯作者: 张丽华
关键词: Immobilized trypsin reactors ;  N-vinyl-2-pyrrolidinone ;  Hydrophilicity ;  Nano-RPLC-ESI-MS/MS ;  Proteomics
刊名: JOURNAL OF CHROMATOGRAPHY A
发表日期: 2012-07-13
DOI: 10.1016/j.chroma.2012.03.014
卷: 1246, 页:111-116
收录类别: SCI ;  ISTP
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: In this work, a novel kind of N-vinyl-2-pyrrolidinone (NVP) modified poly acrylic ester microspheres was prepared, followed by trypsin immobilization to prepare a hydrophilic immobilized enzyme reactor (IMER), to achieve highly efficient protein digestion with low peptide residue. The nonspecific adsorption of peptides on such an IMER was evaluated by the in sequence digestion of bovine serum albumin (BSA) and myoglobin. Without NVP modification, both proteins could be identified after digestion by a 5 cm-length IMER, but 18 peptides of BSA were found in the digests of myoglobin caused by the nonspecific adsorption of the matrix. With NVP modification, the hydrophilicity of IMER was greatly improved, resulting in not only the sequence coverage of myoglobin increased from 63% to 73%, but also no residual peptides from BSA observed in myoglobin digests. Although the sequence coverages of proteins obtained by the IMER were comparable to those obtained by in-solution digestion, the digestion time was shortened from 24 h to 1 min. By such an IMER, a protein mixture, containing BSA, myoglobin, and cytochrome c (100, 1 and 0.01 mu g/mL, respectively), was digested, and all proteins were unambiguously identified with improved sequence coverages than that achieved by in-solution digestion. Furthermore, the hydrophilic IMER was also off-line coupled to nano-RPLC-ESI-MS/MS for the analysis of proteins extracted from yeast. After 1.5 min digestion, 271 protein groups with at least 2 distinct peptides were identified, much more than those obtained by 24 h in-solution digestion (192 protein groups), indicating the great potential of such an IMER for proteome analysis. (C) 2012 Elsevier B.V. All rights reserved.
关键词[WOS]: TANDEM MASS-SPECTROMETRY ;  ENZYME REACTORS ;  CAPILLARY ;  IDENTIFICATION ;  SYSTEM ;  MICROREACTOR ;  PROTEOMICS ;  MONOLITH ;  ONLINE ;  PROTEOLYSIS
语种: 英语
WOS记录号: WOS:000306246000018
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/118205
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
2.Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China

Recommended Citation:
Jiang, Hao,Yuan, Huiming,Liang, Yu,et al. A hydrophilic immobilized trypsin reactor with N-vinyl-2-pyrrolidinone modified polymer microparticles as matrix for highly efficient protein digestion with low peptide residue[J]. JOURNAL OF CHROMATOGRAPHY A,2012,1246:111-116.
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