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

Abstract

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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References

  1. 1.

    Adermann, K., John, H., Standker, L., Forssmann, W.G.: Exploiting natural peptide diversity: novel research tools and drug leads. Curr. Opin. Biotechnol. 15, 599–606 (2004)

  2. 2.

    Slavoff, S.A., Mitchell, A.J., Schwaid, A.G., Cabili, M.N., Ma, J., Levin, J.Z., Karger, A.D., Budnik, B.A., Rinn, J.L., Saghatelian, A.: Peptidomic discovery of short open reading frame-encoded peptides in human cells. Nat. Chem. Biol. 9, 59–64 (2013)

  3. 3.

    Fricker, L.D.: Neuropeptides and other bioactive peptides: from discovery to function. Colloquium Series on Neuropeptides. Morgan Claypool Life Sci. 1(2), 1–122 (2012)

  4. 4.

    Richards, A.L., Hebert, A.S., Ulbrich, A., Bailey, D.J., Coughlin, E.E., Westphall, M.S., Coon, J.J.: One-hour proteome analysis in yeast. Nat. Protoc. 10, 701–714 (2015)

  5. 5.

    Wang, Y., Chen, J., Chen, L., Zheng, P., Xu, H.B., Lu, J., Zhong, J., Lei, Y., Zhou, C., Ma, Q., Li, Y., Xie, P.: Urinary peptidomics identifies potential biomarkers for major depressive disorder. Psychiatry Res. 217, 25–33 (2014)

  6. 6.

    de Araujo, C.B., Russo, L.C., Castro, L.M., Forti, F.L., do Monte, E.R., Rioli, V., Gozzo, F.C., Colquhoun, A., Ferro, E.S.: A novel intracellular peptide derived from g1/s cyclin d2 induces cell death. J. Biol. Chem. 289, 16711–16726 (2014)

  7. 7.

    Salzman, N.H., Hung, K., Haribhai, D., Chu, H., Karlsson-Sjoberg, J., Amir, E., Teggatz, P., Barman, M., Hayward, M., Eastwood, D., Stoel, M., Zhou, Y., Sodergren, E., Weinstock, G.M., Bevins, C.L., Williams, C.B., Bos, N.A.: Enteric defensins are essential regulators of intestinal microbial ecology. Nat. Immunol. 11, 76–83 (2010)

  8. 8.

    Schrader, M., Schulz-Knappe, P.: Peptidomics technologies for human body fluids. Trends Biotechnol. 19, S55–S60 (2001)

  9. 9.

    Greening, D.W., Kapp, E.A., Ji, H., Speed, T.P., Simpson, R.J.: Colon tumour secretopeptidome: insights into endogenous proteolytic cleavage events in the colon tumour microenvironment. Biochim. Biophys. Acta. 1834, 2396–2407 (2013)

  10. 10.

    Hu, L., Li, X., Jiang, X., Zhou, H., Jiang, X., Kong, L., Ye, M., Zou, H.: Comprehensive peptidome analysis of mouse livers by size exclusion chromatography prefractionation and nanoLC-MS/MS identification. J. Proteome Res. 6, 801–808 (2007)

  11. 11.

    Fan, J., Deng, X., Gallagher, J.W., Huang, H., Huang, Y., Wen, J., Ferrari, M., Shen, H., Hu, Y.: Monitoring the progression of metastatic breast cancer on nanoporous silica chips. Philos Transact. Ser A, Math, Phys Eng Sci. 370, 2433–2447 (2012)

  12. 12.

    Terracciano, R., Gaspari, M., Testa, F., Pasqua, L., Tagliaferri, P., Cheng, M.M., Nijdam, A.J., Petricoin, E.F., Liotta, L.A., Cuda, G., Ferrari, M., Venuta, S.: Selective binding and enrichment for low-molecular weight biomarker molecules in human plasma after exposure to nanoporous silica particles. Proteomics. 6, 3243–3250 (2006)

  13. 13.

    Chertov, O., Biragyn, A., Kwak, L.W., Simpson, J.T., Boronina, T., Hoang, V.M., Prieto, D.A., Conrads, T.P., Veenstra, T.D., Fisher, R.J.: Organic solvent extraction of proteins and peptides from serum as an effective sample preparation for detection and identification of biomarkers by mass spectrometry. Proteomics. (4), 1195–1203 (2004)

  14. 14.

    Jiang, L., He, L., Fountoulakis, M.: Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. J. Chromatogr. A. 1023, 317–320 (2004)

  15. 15.

    Chen, I.H., Xue, L., Hsu, C.C., Paez, J.S., Pan, L., Andaluz, H., Wendt, M.K., Iliuk, A.B., Zhu, J.K., Tao, W.A.: Phosphoproteins in extracellular vesicles as candidate markers for breast cancer. Proc. Natl. Acad. Sci. U. S. A. 114, 3175–3180 (2017)

  16. 16.

    Schunter, A.J., Yue, X., Hummon, A.B.: Phosphoproteomics of colon cancer metastasis: comparative mass spectrometric analysis of the isogenic primary and metastatic cell lines SW480 and SW620. Anal. Bioanal. Chem. 409, 1749–1763 (2017)

  17. 17.

    Hu, Y., Shah, P., Clark, D.J., Ao, M., Zhang, H.: Reanalysis of global proteomic and phosphoproteomic data identified a large number of glycopeptides. Anal. Chem. 90, 8065–8071 (2018)

  18. 18.

    Wang, Q., Wang, K., Ye, M.: Strategies for large-scale analysis of non-histone protein methylation by LC-MS/MS. Analyst. 142, 3536–3548 (2017)

  19. 19.

    Uhlmann, T., Geoghegan, V.L., Thomas, B., Ridlova, G., Trudgian, D.C., Acuto, O.: A method for large-scale identification of protein arginine methylation. Mol Cell Proteomics : MCP. 11, 1489–1499 (2012)

  20. 20.

    Ma, M., Zhao, X., Chen, S., Zhao, Y., Yang, L., Feng, Y., Qin, W., Li, L., Jia, C.: Strategy based on deglycosylation, multiprotease, and hydrophilic interaction chromatography for large-scale profiling of protein methylation. Anal. Chem. 89, 12909–12917 (2017)

  21. 21.

    Fricker, L.D., Gelman, J.S., Castro, L.M., Gozzo, F.C., Ferro, E.S.: Peptidomic analysis of HEK293T cells: effect of the proteasome inhibitor epoxomicin on intracellular peptides. J. Proteome Res. 11(1981), (2012)

  22. 22.

    Zhang, J., Xin, L., Shan, B., Chen, W., Xie, M., Yuen, D., Zhang, W., Zhang, Z., Lajoie, G.A., Ma, B.: PEAKS DB: de novo sequencing assisted database search for sensitive and accurate peptide identification. Mol Cell Proteomics : MCP. 11, M111 010587 (2012)

  23. 23.

    Colaert, N., Helsens, K., Martens, L., Vandekerckhove, J., Gevaert, K.: Improved visualization of protein consensus sequences by iceLogo. Nat. Methods. 6, 786–787 (2009)

  24. 24.

    da Huang, W., Sherman, B.T., Lempicki, R.A.: Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009)

  25. 25.

    da Huang , W., Sherman, B.T., Lempicki, R.A.: Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009)

  26. 26.

    DeLaney, K., Buchberger, A., Li, L.: Identification, quantitation, and imaging of the crustacean peptidome. Methods Mol. Biol. 1719, 247–269 (2018)

  27. 27.

    Zhang, X., Petruzziello, F., Zani, F., Fouillen, L., Andren, P.E., Solinas, G., Rainer, G.: High identification rates of endogenous neuropeptides from mouse brain. J. Proteome Res. 11, 2819–2827 (2012)

  28. 28.

    Gelman, J.S., Sironi, J., Castro, L.M., Ferro, E.S., Fricker, L.D.: Peptidomic analysis of human cell lines. J. Proteome Res. 10, 1583–1592 (2015)

  29. 29.

    Jung, S.Y., Li, Y., Wang, Y., Chen, Y., Zhao, Y., Qin, J.: Complications in the assignment of 14 and 28 Da mass shift detected by mass spectrometry as in vivo methylation from endogenous proteins. Anal. Chem. 80, 1721–1729 (2008)

  30. 30.

    Hornbeck, P.V., Zhang, B., Murray, B., Kornhauser, J.M., Latham, V., Skrzypek, E.: PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res. 43, D512–D520 (2015)

  31. 31.

    Lachner, M., O'Carroll, D., Rea, S., Mechtler, K., Jenuwein, T.: Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature. 410, 116–120 (2001)

  32. 32.

    Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Tempst, P., Reinberg, D.: Histone methyltransferase activity associated with a human multiprotein complex containing the enhancer of Zeste protein. Genes Dev. 16, 2893–2905 (2002)

  33. 33.

    Garcia, B.A., Hake, S.B., Diaz, R.L., Kauer, M., Morris, S.A., Recht, J., Shabanowitz, J., Mishra, N., Strahl, B.D., Allis, C.D., Hunt, D.F.: Organismal differences in post-translational modifications in histones H3 and H4. J. Biol. Chem. 282, 7641–7655 (2007)

  34. 34.

    Guo, A., Gu, H., Zhou, J., Mulhern, D., Wang, Y., Lee, K.A., Yang, V., Aguiar, M., Kornhauser, J., Jia, X., Ren, J., Beausoleil, S.A., Silva, J.C., Vemulapalli, V., Bedford, M.T., Comb, M.J.: Immunoaffinity enrichment and mass spectrometry analysis of protein methylation. Mol Cell Proteomics : MCP. 13, 372–387 (2014)

  35. 35.

    Zhu, G., Reynolds, L., Crnogorac-Jurcevic, T., Gillett, C.E., Dublin, E.A., Marshall, J.F., Barnes, D., D'Arrigo, C., Van Trappen, P.O., Lemoine, N.R., Hart, I.R.: Combination of microdissection and microarray analysis to identify gene expression changes between differentially located tumour cells in breast cancer. Oncogene. 22, 3742–3748 (2003)

  36. 36.

    Jakobsson, M.E., Malecki, J., Nilges, B.S., Moen, A., Leidel, S.A., Falnes, P.O.: Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation. Nucleic Acids Res. 45, 8239–8254 (2017)

  37. 37.

    Wang, K., Dong, M., Mao, J., Wang, Y., Jin, Y., Ye, M., Zou, H.: Antibody-free approach for the global analysis of protein methylation. Anal. Chem. 88, 11319–11327 (2016)

  38. 38.

    Bremang, M., Cuomo, A., Agresta, A.M., Stugiewicz, M., Spadotto, V., Bonaldi, T.: Mass spectrometry-based identification and characterisation of lysine and arginine methylation in the human proteome. Mol. BioSyst. 9, 2231–2247 (2013)

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Acknowledgements

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.

Funding

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.

Author information

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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Keywords

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