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Isolation and identification of phosphorylated lysine peptides by retention time difference combining dimethyl labeling strategy

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Abstract

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.

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (2017YFA0505003, 2016YFA0501401), the National Natural Science Foundation of China (21505133, 21725506, 91543201), the CAS Key Project in Frontier Science (QYZDY-SSW-SLH017), and Innovation Program from DICP, Chinese Academy of Sciences (DICP TMSR201601).

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  1. These authors contributed equally to this work.

Authors and Affiliations

  1. CAS Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China

    Yechen Hu, Yejing Weng, Bo Jiang, Xiao Li, Xiaodan Zhang, Baofeng Zhao, Qiong Wu, Zhen Liang, Lihua Zhang & Yukui Zhang

  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    Yechen Hu, Yejing Weng & Qiong Wu

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  1. Yechen Hu
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  2. Yejing Weng
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  9. Lihua Zhang
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Correspondence to Lihua Zhang.

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Hu, Y., Weng, Y., Jiang, B. et al. Isolation and identification of phosphorylated lysine peptides by retention time difference combining dimethyl labeling strategy. Sci. China Chem. 62, 708–712 (2019). https://doi.org/10.1007/s11426-018-9433-3

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