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

  • Yechen Hu
  • Yejing Weng
  • Bo Jiang
  • Xiao Li
  • Xiaodan Zhang
  • Baofeng Zhao
  • Qiong Wu
  • Zhen Liang
  • Lihua ZhangEmail author
  • Yukui Zhang
Communications
  • 13 Downloads

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.

Keywords

phosphorylated lysine retention time difference dimethyl labeling N-phosphorylation proteome LC-MS/MS 

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Notes

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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Copyright information

© Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Yechen Hu
    • 1
    • 2
  • Yejing Weng
    • 1
    • 2
  • Bo Jiang
    • 1
  • Xiao Li
    • 1
  • Xiaodan Zhang
    • 1
  • Baofeng Zhao
    • 1
  • Qiong Wu
    • 1
    • 2
  • Zhen Liang
    • 1
  • Lihua Zhang
    • 1
    Email author
  • Yukui Zhang
    • 1
  1. 1.CAS Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalianChina
  2. 2.University of Chinese Academy of SciencesBeijingChina

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