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题名: Comprehensive and Reliable Phosphorylation Site Mapping of Individual Phosphoproteins by Combination of Multiple Stage Mass Spectrometric Analysis with a Target-Decoy Database Search
作者: Han, Guanghui1;  Ye, Mingliang1;  Jiang, Xinning1;  Chen, Rui1;  Ren, Jian2, 3;  Xue, Yu2, 3;  Wang, Fangjun1;  Song, Chunxia1;  Yao, Xuebiao2, 3;  Zou, Hanfa1
通讯作者: 邹汉法
刊名: ANALYTICAL CHEMISTRY
发表日期: 2009-07-15
DOI: 10.1021/ac900702g
卷: 81, 期:14, 页:5794-5805
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 ;  Physical Sciences
类目[WOS]: Chemistry, Analytical
研究领域[WOS]: Chemistry
英文摘要: Since the emergence of proteomics, much attention has been paid to the development of new technologies for phosphoproteomcis analysis. Compared with large scale phosphorylation analysis at the proteome level, comprehensive and reliable phosphorylation site mapping of individual phosphoprotein is equally important. Here, we present a modified target-decoy database search strategy for confident phosphorylation site analysis of individual phosphoproteins without manual interpretation of spectra. Instead of using all protein sequences in a proteome database of an organism for the construction of a target-decoy database for phosphoproteome analysis, the composite database constructed for phosphorylation site analysis of individual phosphoproteins only included the sequences of the individual target proteins and a decoy version of a small inhomogeneous protein database. It was found that the confidence of phosphopeptide identifications could be effectively controlled when the acquired MS(2) and MS(3) spectra were searched against the above composite database followed with data processing. Because of the small size of the composite database, the computation time for the database search is very short, which allows the adoption of low-specificity proteases for protein digestion to increase the coverage of phosphorylation site mapping. The sensitivity and comprehensive phosphorylation site mapping of this approach was demonstrated by using two standard phosphoprotein samples of alpha-casein and beta-casein, and this approach was further applied to analyze the phosphorylation of the cyclic AMP-dependent protein kinase (PKA), which resulted in the identification of 17 phosphorylation sites, including five novel sites on four PKA subunits.
关键词[WOS]: PROTEIN-PHOSPHORYLATION ;  IN-VIVO ;  PHOSPHOPEPTIDE IDENTIFICATIONS ;  AFFINITY-CHROMATOGRAPHY ;  AUTOMATIC VALIDATION ;  SIGNALING NETWORKS ;  ENRICHMENT ;  PROTEOMICS ;  STRATEGY ;  FRAGMENTATION
语种: 英语
原文出处: 查看原文
WOS记录号: WOS:000268135000028
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/102971
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 R&A Ctr, Dalian 116023, Peoples R China
2.Univ Sci & Technol China, Hefei Natl Lab Phys Sci Microscale, Hefei 230027, Peoples R China
3.Univ Sci & Technol China, Sch Life Sci, Hefei 230027, Peoples R China

Recommended Citation:
Han, Guanghui,Ye, Mingliang,Jiang, Xinning,et al. Comprehensive and Reliable Phosphorylation Site Mapping of Individual Phosphoproteins by Combination of Multiple Stage Mass Spectrometric Analysis with a Target-Decoy Database Search[J]. ANALYTICAL CHEMISTRY,2009,81(14):5794-5805.
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