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Discovery of Missing Methylation Sites on Endogenous Peptides of Human Cell Lines


Methylation of proteins has considerable impacts on physiological processes including signal transduction, DNA damage repair, transcriptional regulation, gene activation, and inhibition of gene expression. However, the traditional proteomics-based approach suffers from limited identification rates of these critical methylation sites on endogenous peptides. In this work, a peptidomics-based workflow was established to discover and characterize the global methylome of endogenous peptides in human cells. The reliability of our strategy was validated by methyl-SILAC labeling, resulting in 83% true-positive identifications in the HeLa cell line. We applied this approach to seven human cell lines, and 700 methylated forms on 646 putative methylation sites were identified in total, with over 61% of the methylation sites being newly identified. This study provides a complementary strategy for a traditional proteomics-based approach that enables identification of missing methylation sites and creates a first methylome draft of endogenous peptides of human cell lines, offering a valuable resource for in-depth studies of biological functions of methylated endogenous peptides.

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This work is supported by the National Key R&D Program of China (No. 2016YFA0501302 to CJ and No. 2017YFA0505702 to CJ), the National Science Foundation of China (No. 21675006 to CJ), and a fund (BWS17J025 to CJ). LL acknowledges funding support from National Institutes of Health grants RF1AG052324 (to LL), R01 DK071801 (to LL), and U01CA231081 (to LL). We thank the mass spectrometry facility of the National Center for Protein Science-Beijing (Phoenix Center) for accessing the instruments. We thank Dr. Min Ma, Qianqian Wang, and Ting Zhao from the National Center for Protein Science-Beijing (Phoenix Center) for helpful discussions and insightful suggestions on this project.


All mass spectrometry data have been deposited to the iProx with the dataset identifier IPX0001467000. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.

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Correspondence to Lingjun Li or Chenxi Jia.

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Yan, X., Li, L. & Jia, C. Discovery of Missing Methylation Sites on Endogenous Peptides of Human Cell Lines. J. Am. Soc. Mass Spectrom. 30, 2537–2547 (2019). https://doi.org/10.1007/s13361-019-02270-y

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  • Endogenous peptides
  • Peptide methylation
  • Mass spectrometry
  • Peptidomics
  • Post-translational modification (PTM)
  • Methylome