Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Identifying and quantifying in vivo methylation sites by heavy methyl SILAC

Nature Methods volume 1, pages 119–126 (2004)Cite this article

Abstract

Protein methylation is a stable post-translational modification (PTM) with important biological functions. It occurs predominantly on arginine and lysine residues with varying numbers of methyl groups, such as mono-, di- or trimethyl lysine. Existing methods for identifying methylation sites are laborious, require large amounts of sample and cannot be applied to complex mixtures. We have previously described stable isotope labeling by amino acids in cell culture (SILAC) for quantitative comparison of proteomes. In heavy methyl SILAC, cells metabolically convert [13CD3]methionine to the sole biological methyl donor, [13CD3]S-adenosyl methionine. Heavy methyl groups are fully incorporated into in vivo methylation sites, directly labeling the PTM. This provides markedly increased confidence in identification and relative quantitation of protein methylation by mass spectrometry. Using antibodies targeted to methylated residues and analysis by liquid chromatography–tandem mass spectrometry, we identified 59 methylation sites, including previously unknown sites, considerably extending the number of in vivo methylation sites described in the literature.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The heavy methyl SILAC strategy.
Figure 2: Full incorporation of heavy methyls.
Figure 3: Accurate quantification with heavy methyl SILAC determined from lysate mixing experiments.
Figure 4: (CH2)n substitution matrix for amino acids.
Figure 5: Heavy methyl SILAC encodes methylated peptide pairs with a modification-specific mass tag.
Figure 6: Informative MS/MS spectra and characteristic fragment losses in heavy methyl SILAC.

Similar content being viewed by others

References

  1. Jaenisch, R. & Bird, A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33 (Suppl.), 245–254 (2003).

    Article  CAS  Google Scholar 

  2. Ambler, R.P. & Rees, M.W. Epsilon-N-methyl-lysine in bacterial flagellar protein. Nature 184, 56–57 (1959).

    Article  CAS  Google Scholar 

  3. Bedford, M.T. et al. Arginine methylation inhibits the binding of proline-rich ligands to Src homology 3, but not WW, domains. J. Biol. Chem. 275, 16030–16036 (2000).

    Article  CAS  Google Scholar 

  4. Cote, J., Boisvert, F.M., Boulanger, M.C., Bedford, M.T. & Richard, S. Sam68 RNA binding protein is an in vivo substrate for protein arginine N-methyltransferase 1. Mol. Biol. Cell 14, 274–287 (2003).

    Article  CAS  Google Scholar 

  5. Liu, Q. & Dreyfuss, G. In vivo and in vitro arginine methylation of RNA-binding proteins. Mol. Cell. Biol. 15, 2800–2808 (1995).

    Article  CAS  Google Scholar 

  6. McBride, A.E. & Silver, P.A. State of the arg: protein methylation at arginine comes of age. Cell 106, 5–8 (2001).

    Article  CAS  Google Scholar 

  7. Paik, W.K. & Kim, S. (eds.). Protein Methylation (CRC Press, Boca Raton, Florida, USA, 1990).

    Google Scholar 

  8. Giner, J.L. & Rando, R.R. Novel methyltransferase activity modifying the carboxy terminal bis(geranylgeranyl)-Cys-Ala-Cys structure of small GTP-binding proteins. Biochemistry 33, 15116–15123 (1994).

    Article  CAS  Google Scholar 

  9. Chiu, V.K. et al. Carboxyl methylation of Ras regulates membrane targeting and effector engagement. J. Biol. Chem. 279, 7346–7352 (2004).

    Article  CAS  Google Scholar 

  10. Raman, B. et al. Nω-arginine dimethylation modulates the interaction between a Gly/Arg-rich peptide from human nucleolin and nucleic acids. Nucleic Acids Res. 29, 3377–3384 (2001).

    Article  CAS  Google Scholar 

  11. Fontecave, M., Atta, M. & Mulliez, E. S-adenosylmethionine: nothing goes to waste. Trends Biochem. Sci. 29, 243–249 (2004).

    Article  CAS  Google Scholar 

  12. Katz, J.E., Dlakic, M. & Clarke, S. Automated identification of putative methyltransferases from genomic open reading frames. Mol. Cell. Proteomics 2, 525–540 (2003).

    Article  CAS  Google Scholar 

  13. Mowen, K.A. & David, M. Analysis of protein arginine methylation and protein arginine-methyltransferase activity. Sci. STKE 2001, PL1 (2001).

    CAS  PubMed  Google Scholar 

  14. Mann, M. & Jensen, O.N. Proteomic analysis of post-translational modifications. Nat. Biotechnol. 21, 255–261 (2003).

    Article  CAS  Google Scholar 

  15. Rappsilber, J., Friesen, W.J., Paushkin, S., Dreyfuss, G. & Mann, M. Detection of arginine dimethylated peptides by parallel precursor ion scanning mass spectrometry in positive ion mode. Anal. Chem. 75, 3107–3114 (2003).

    Article  CAS  Google Scholar 

  16. Brame, C.J., Moran, M.F. & McBroom-Cerajewski, L.D. A mass spectrometry based method for distinguishing between symmetrically and asymmetrically dimethylated arginine residues. Rapid Commun. Mass Spectrom. 18, 877–881 (2004).

    Article  CAS  Google Scholar 

  17. Gehrig, P.M., Hunziker, P.E., Zahariev, S. & Pongor, S. Fragmentation pathways of NG-methylated and unmodified arginine residues in peptides studied by ESI-MS/MS and MALDI-MS. J. Am. Soc. Mass Spectrom. 15, 142–149 (2004).

    Article  CAS  Google Scholar 

  18. Ong, S.E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002).

    Article  CAS  Google Scholar 

  19. Zhu, H., Pan, S., Gu, S., Bradbury, E.M. & Chen, X. Amino acid residue-specific stable isotope labeling for quantitative proteomics. Rapid Commun. Mass Spectrom. 16, 2115–2123 (2002).

    Article  CAS  Google Scholar 

  20. Jiang, H. & English, A.M. Quantitative analysis of the yeast proteome by incorporation of isotopically labeled leucine. J. Proteome Res. 1, 345–350 (2002).

    Article  CAS  Google Scholar 

  21. Ong, S.E., Kratchmarova, I. & Mann, M. Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC). J. Proteome Res. 2, 173–181 (2003).

    Article  CAS  Google Scholar 

  22. Siomi, H., Siomi, M.C., Nussbaum, R.L. & Dreyfuss, G. The protein product of the fragile X gene, FMR1, has characteristics of an RNA-binding protein. Cell 74, 291–298 (1993).

    Article  CAS  Google Scholar 

  23. Lee, J. & Bedford, M.T. PABP1 identified as an arginine methyltransferase substrate using high-density protein arrays. EMBO Rep. 3, 268–273 (2002).

    Article  CAS  Google Scholar 

  24. Tang, J., Kao, P.N. & Herschman, H.R. Protein-arginine methyltransferase I, the predominant protein-arginine methyltransferase in cells, interacts with and is regulated by interleukin enhancer-binding factor 3. J. Biol. Chem. 275, 19866–19876 (2000).

    Article  CAS  Google Scholar 

  25. Brahms, H., Meheus, L., de Brabandere, V., Fischer, U. & Luhrmann, R. Symmetrical dimethylation of arginine residues in spliceosomal Sm protein B/B′ and the Sm-like protein LSm4, and their interaction with the SMN protein. RNA 7, 1531–1542 (2001).

    Article  CAS  Google Scholar 

  26. Boisvert, F.M., Cote, J., Boulanger, M.C. & Richard, S. A proteomic analysis of arginine-methylated protein complexes. Mol. Cell. Proteomics 2, 1319–1330 (2003).

    Article  CAS  Google Scholar 

  27. Oda, Y., Huang, K., Cross, F.R., Cowburn, D. & Chait, B.T. Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. USA 96, 6591–6596 (1999).

    Article  CAS  Google Scholar 

  28. Ficarro, S.B. et al. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301–305 (2002).

    Article  CAS  Google Scholar 

  29. Pesavento, J.J., Kim, Y.B., Taylor, G.K. & Kelleher, N.L. Shotgun annotation of histone modifications: a new approach for streamlined characterization of proteins by top down mass spectrometry. J. Am. Chem. Soc. 126, 3386–3387 (2004).

    Article  CAS  Google Scholar 

  30. Harlow, E. & Lane, D. Using Antibodies—A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 1999).

    Google Scholar 

  31. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).

    Article  CAS  Google Scholar 

  32. Olsen, J.V., Ong, S.E. & Mann, M. Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol. Cell. Proteomics 3, 608–614 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank other members of our laboratory for help and fruitful discussions, and J.V. Olsen for critical reading of the manuscript. Work in the Center for Experimental BioInformatics (CEBI) is supported by a generous grant from the Danish National Research Foundation and “Interaction Proteome,” a European Union 6th framework grant.

Author information

Authors and Affiliations

  1. Center for Experimental BioInformatics, University of Southern Denmark, Odense, M 5230, Denmark

    Shao-En Ong, Gerhard Mittler & Matthias Mann

Authors
  1. Shao-En Ong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gerhard Mittler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthias Mann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Gerhard Mittler or Matthias Mann.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Accuracy of methylated peptide ratios from 1-to-1 mixing experiments. (PDF 21 kb)

Supplementary Table 2

Improved confidence in assignment of methylated peptides with Heavy Methyl SILAC. (PDF 23 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ong, SE., Mittler, G. & Mann, M. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC. Nat Methods 1, 119–126 (2004). https://doi.org/10.1038/nmeth715

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth715

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing