title: Identification of phosphopeptides with unknown cleavage specificity by a de novo sequencing assisted database search strategy.
abstract: 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.
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China; University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Mingming
authors.initials: M
authors.lastname: Dong
authors.forename: Mingliang
authors.initials: M
authors.lastname: Ye
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.forename: Jing
authors.initials: J
authors.lastname: Dong
authors.forename: Jun
authors.initials: J
authors.lastname: Zhu
authors.forename: Hongqiang
authors.initials: H
authors.lastname: Qin
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Bian
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D030562:Databases, Protein; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D010748:Phosphopeptides; D019286:Endopeptidase K
keywords: De novo sequencing; Endogenous phosphopeptide; Phosphopeptide identification; Phosphoproteomics; Technology