期刊
PROTEOMICS
卷 14, 期 21-22, 页码 2410-2416出版社
WILEY
DOI: 10.1002/pmic.201400268
关键词
De novo sequencing; Endogenous phosphopeptide; Phosphoproteomics; Phosphopeptide identification; Technology
资金
- China State Key Basic Research Program Grant [2013CB911202, 2012CB910101, 2012CB910604]
- Creative Research Group Project of NSFC [21321064]
- National Natural Science Foundation of China [21275142, 21235006, 81161120540, 81361128015]
- National Key Special Program on Infection diseases [2012ZX10002009-011]
- Analytical Method Innovation Program of MOST [2012IM030900]
In theory, proteases with broad cleavage specificity could be applied to digest protein samples to improve the phosphoproteomic analysis coverage. However, in practice this approach is seldom employed. This is because the identification of phosphopeptides without enzyme specificity by conventional database search strategy is extremely difficult due to the huge search space. In this study, we investigated the performance of a de novo sequencing assisted database search strategy for the identification of such phosphopeptides. Firstly, we compared the performance of conventional database search strategy and the de novo sequencing assisted database search strategy for the identification of peptides and phosphopeptides without stetting enzyme specificity. It was found that the identification sensitivity dropped significantly for the conventional one while it was only slightly decreased for the new approach. Then, this new search strategy was applied to identify phosphopeptides generated by Proteinase K digestion, which resulted in the identification of 717 phosphopeptides. Finally, this strategy was utilized for the identification of serum endogenous phosphopeptides, which were generated in vivo by different kinds of proteases and kinases, and the identification of 68 unique serum endogenous phosphopepitdes was successfully achieved.
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