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Comprehensive and Reliable Phosphorylation Site Mapping of Individual Phosphoproteins by Combination of Multiple Stage Mass Spectrometric Analysis with a Target-Decoy Database Search
Han, Guanghui1; Ye, Mingliang1; Jiang, Xinning1; Chen, Rui1; Ren, Jian2,3; Xue, Yu2,3; Wang, Fangjun1; Song, Chunxia1; Yao, Xuebiao2,3; Zou, Hanfa1; Zou HF(邹汉法)
Source PublicationANALYTICAL CHEMISTRY
2009-07-15
DOI10.1021/ac900702g
Volume81Issue:14Pages:5794-5805
Indexed BySCI
SubtypeArticle
Department18
Funding Project1809
Contribution Rank1;1
WOS HeadingsScience & Technology ; Physical Sciences
WOS SubjectChemistry, Analytical
WOS Research AreaChemistry
WOS KeywordPROTEIN-PHOSPHORYLATION ; IN-VIVO ; PHOSPHOPEPTIDE IDENTIFICATIONS ; AFFINITY-CHROMATOGRAPHY ; AUTOMATIC VALIDATION ; SIGNALING NETWORKS ; ENRICHMENT ; PROTEOMICS ; STRATEGY ; FRAGMENTATION
AbstractSince 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.
Language英语
URL查看原文
WOS IDWOS:000268135000028
Citation statistics
Cited Times:21[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://cas-ir.dicp.ac.cn/handle/321008/102971
Collection中国科学院大连化学物理研究所
Corresponding AuthorZou HF(邹汉法)
Affiliation1.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
GB/T 7714
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.
APA Han, Guanghui.,Ye, Mingliang.,Jiang, Xinning.,Chen, Rui.,Ren, Jian.,...&邹汉法.(2009).Comprehensive and Reliable Phosphorylation Site Mapping of Individual Phosphoproteins by Combination of Multiple Stage Mass Spectrometric Analysis with a Target-Decoy Database Search.ANALYTICAL CHEMISTRY,81(14),5794-5805.
MLA Han, Guanghui,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".ANALYTICAL CHEMISTRY 81.14(2009):5794-5805.
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