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Nature Methods
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Identifying and quantifyingin vivo methylation sites by heavy methyl SILAC

Nature Methodsvolume 1, pages119–126 (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 intoin 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 ofin vivo methylation sites described in the literature.

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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.

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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.

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Authors and Affiliations

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

    Shao-En Ong, Gerhard Mittler & Matthias Mann

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  1. Shao-En Ong

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  2. Gerhard Mittler

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  3. Matthias Mann

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Corresponding authors

Correspondence toGerhard Mittler orMatthias Mann.

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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)

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Ong, SE., Mittler, G. & Mann, M. Identifying and quantifyingin vivo methylation sites by heavy methyl SILAC.Nat Methods1, 119–126 (2004). https://doi.org/10.1038/nmeth715

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