Document Detail

Enhancement of the efficiency of phosphoproteomic identification by removing phosphates after phosphopeptide enrichment.
MedLine Citation:
PMID:  17330947     Owner:  NLM     Status:  MEDLINE    
Immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO2) chromatography are simple, widely used, and cost-effective methods to enrich phosphopeptides, but the sample loading buffer composition, desalting procedure, and control of loading amount are critical to avoid nonspecific interactions and to achieve efficient phosphopeptide enrichment. Although the combination of MS3 analysis and high-resolution mass spectrometry (MS) is helpful to identify phosphopeptides, the quality of many MS/MS spectra having a neutral loss peak of phosphate is still too poor to allow sequence identification, and this results in many false-negative as well as false-positive identifications. Here, we present a novel strategy, which is based on the use of alkaline phosphatase to remove phosphates and analysis of phospho/dephosphopeptide retention times to increase the reliability of identification. The use of phospho/dephosphopeptide retention time ratios allows the identification of phosphopeptides with high confidence with the aid of a focused database of dephosphopeptides. This approach was very effective to identify multiple phophorylations in tryptic peptides. A 'true' phosphorylation data set should contain about 90% phospho-Ser and a few percent phospho-Tyr, and this ratio can be used as a quality criterion for evaluation of data sets. By applying this efficient approach, we were able to identify more than one thousand phosphopeptides.
Yasushi Ishihama; Fan-Yan Wei; Ken Aoshima; Toshitaka Sato; Junro Kuromitsu; Yoshiya Oda
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of proteome research     Volume:  6     ISSN:  1535-3893     ISO Abbreviation:  J. Proteome Res.     Publication Date:  2007 Mar 
Date Detail:
Created Date:  2007-03-02     Completed Date:  2007-06-06     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101128775     Medline TA:  J Proteome Res     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1139-44     Citation Subset:  IM    
Laboratory of Seeds Finding Technology, Eisai Company, Ltd. Tokodai 5-1-3, Tsukuba, Ibaraki 300-2635, Japan.
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MeSH Terms
Alkaline Phosphatase / metabolism
Cell Line, Tumor
Cells, Cultured
Neuroblastoma / chemistry,  pathology
Phosphates / metabolism
Phosphopeptides / analysis*
Phosphoproteins / analysis*
Prosencephalon / chemistry,  cytology
Proteomics / methods*,  standards
Reg. No./Substance:
0/Phosphates; 0/Phosphopeptides; 0/Phosphoproteins; 55520-40-6/Tyrosine; 56-45-1/Serine; EC Phosphatase; EC

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