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学科主题: 物理化学
题名: Combination of online enzyme digestion with stable isotope labeling for high-throughput quantitative proteome analysis
作者: Wang, Fangjun1;  Wei, Xiaoluan1;  Zhou, Hu2, 3;  Liu, Jing1;  Figeys, Daniel2;  Zou, Hanfa1
通讯作者: 邹汉法
关键词: Miscleavage ;  Online enzyme digestion ;  Quantitative proteome analysis ;  Stable isotope labeling ;  Technology
刊名: PROTEOMICS
发表日期: 2012-11-01
DOI: 10.1002/pmic.201200162
卷: 12, 期:21, 页:3129-3137
收录类别: SCI
文章类型: Article
部门归属: 18
项目归属: 1809
产权排名: 1, 1
WOS标题词: Science & Technology ;  Life Sciences & Biomedicine
类目[WOS]: Biochemical Research Methods ;  Biochemistry & Molecular Biology
研究领域[WOS]: Biochemistry & Molecular Biology
英文摘要: Various enzyme reactors and online enzyme digestion strategies have been developed in recent years. These reactors greatly enhanced the detection sensitivity and proteome coverage in qualitative proteomics. However, these devices have higher rates of miscleavage in protein digestion. Therefore, we investigated the effect of online enzyme digestion on the quantification accuracy of quantitative proteomics using chemical or metabolic isotope labeling approaches. The incomplete digestion would introduce some unexpected variations in comparative quantification when the samples are digested and then chemically isotope labeled in different aliquots. Even when identical protein aliquots are processed on these devices using post-digestion chemical isotope labeling and the CVs of the ratios controlled to less than 50% in replicate analyses, about 10% of the quantified proteins have a ratio greater than two-fold, whereas in theory the ratio is 1:1. Interestingly, the incomplete digestion with enzyme reactor is not a problem when metabolic isotope labeling samples were processed because the proteins are isotopically labeled in vivo prior to their simultaneous digestion within the reactor. Our results also demonstrated that both high quantification accuracy and high proteome coverage can be achieved in comparative proteome quantification using online enzyme digestion even when a limited amount of metabolic isotope labeling samples is used (1683 proteins comparatively quantified from 105 Hela cells).
关键词[WOS]: TANDEM MASS-SPECTROMETRY ;  PHASE LIQUID-CHROMATOGRAPHY ;  TRYPSIN REACTOR ;  COUPLED ONLINE ;  CELL-CULTURE ;  AMINO-ACIDS ;  IDENTIFICATION ;  PROTEOLYSIS ;  CAPILLARY ;  SILAC
语种: 英语
WOS记录号: WOS:000310564100002
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/118443
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, CAS Key Lab Separat Sci Analyt Chem, Natl Chromatog Res & Anal Ctr, Dalian 116023, Peoples R China
2.Univ Ottawa, Ottawa Inst Syst Biol, Ottawa, ON, Canada
3.Chinese Acad Sci, Shanghai Inst Mat Med, Shanghai 200031, Peoples R China

Recommended Citation:
Wang, Fangjun,Wei, Xiaoluan,Zhou, Hu,et al. Combination of online enzyme digestion with stable isotope labeling for high-throughput quantitative proteome analysis[J]. PROTEOMICS,2012,12(21):3129-3137.
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