Isolation and identification of phosphorylated lysine peptides by retention time difference combining dimethyl labeling strategy
Isolation and identification of phosphorylated lysine peptides by retention time difference combining dimethyl labeling strategy
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DOI：
10.1007/s11426-018-9433-3
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发表时间：
2019-06-01
影响因子：
9.6
通讯作者：
Zhang, Yukui
中科院分区：
文献类型：
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作者：
Hu, Yechen;Weng, Yejing;Zhang, Yukui
蛋白质磷酸化在各种生物过程中起着重要作用。尽管O-磷酸蛋白质组学的富集策略已经成熟，但N-P磷酰胺键（组氨酸、精氨酸和赖氨酸的磷酸化）固有的酸不稳定性阻碍了N-磷酸蛋白质组学的进展。在此，我们报告了保留时间差结合二甲基标记（ReDD）策略的磷酸化赖氨酸（pLys）肽的分离和鉴定。通过这种方法，可以在非磷酸化肽的100000倍干扰下分离pLys肽。此外，还应用ReDD策略对大肠杆菌pLys位点进行了定位。大肠杆菌样品中，共鉴定出11个pLys位点，其中K26 p来源于自主性甘氨酰自由基辅因子，经质谱和HPLC共洗脱实验验证。此外，在HeLa细胞中，从100个蛋白质中鉴定出112个pLys位点。所有这些结果表明，ReDD可以提供对Lys磷酸化的第一次一瞥，并且可能是朝着蛋白质磷酸化的全球视角迈出的重要一步。
Protein phosphorylation plays essential roles in various biological procedures. Despite the well-established enrichment strategies for O-phosphoproteomics, the intrinsic acid lability of N-P phosphoramidate bond (phosphorylation of histidine, arginine and lysine) has impaired the progress of N-phosphoproteomics. Herein, we reported a retention time difference combining dimethyl labeling (ReDD) strategy for the isolation and identification of phosphorylated lysine (pLys) peptides. By such a method, pLys peptide could be isolated under 100000-fold interference of non-phosphorylated peptides. Furthermore, ReDD strategy was applied to map pLys sites from E. coli samples, leading to the identification of 11 pLys sites, among which K26p that originating from autonomous glycyl radical cofactor was validated both in mass spectrometry and HPLC co-elution experiments. Furthermore, 112 pLys sites from 100 proteins were identified in HeLa cells. All these results demonstrate that ReDD could provide a first glimpse into Lys phosphorylation, and could be an important step toward the global perspective on protein phosphorylation.