Identifying and quantifying in vivo methylation sites by heavy methyl SILAC

Nat Methods. 2004 Nov;1(2):119-26. doi: 10.1038/nmeth715. Epub 2004 Oct 21.

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 [(13)CD(3)]methionine to the sole biological methyl donor, [(13)CD(3)]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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Algorithms*
  • Binding Sites
  • Carbon Isotopes
  • Chromatography, Liquid / methods*
  • Gene Expression Profiling / methods
  • HeLa Cells
  • Humans
  • Isotope Labeling / methods*
  • Mass Spectrometry / methods*
  • Methionine / analysis
  • Methionine / metabolism*
  • Methylation
  • Neoplasm Proteins / analysis
  • Neoplasm Proteins / metabolism*
  • Protein Binding
  • Proteome / analysis
  • Proteome / metabolism*

Substances

  • Carbon Isotopes
  • Neoplasm Proteins
  • Proteome
  • Methionine