A genetic engineering solution to the "arginine conversion problem" in stable isotope labeling by amino acids in cell culture (SILAC)

Mol Cell Proteomics. 2010 Jul;9(7):1567-77. doi: 10.1074/mcp.M110.000208. Epub 2010 May 10.

Abstract

Stable isotope labeling by amino acids in cell culture (SILAC) provides a straightforward tool for quantitation in proteomics. However, one problem associated with SILAC is the in vivo conversion of labeled arginine to other amino acids, typically proline. We found that arginine conversion in the fission yeast Schizosaccharomyces pombe occurred at extremely high levels, such that labeling cells with heavy arginine led to undesired incorporation of label into essentially all of the proline pool as well as a substantial portion of glutamate, glutamine, and lysine pools. We found that this can be prevented by deleting genes involved in arginine catabolism using methods that are highly robust yet simple to implement. Deletion of both fission yeast arginase genes or of the single ornithine transaminase gene, together with a small modification to growth medium that improves arginine uptake in mutant strains, was sufficient to abolish essentially all arginine conversion. We demonstrated the usefulness of our approach in a large scale quantitative analysis of proteins before and after cell division; both up- and down-regulated proteins, including a novel protein involved in septation, were successfully identified. This strategy for addressing the "arginine conversion problem" may be more broadly applicable to organisms amenable to genetic manipulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / chemistry
  • Amino Acids / metabolism
  • Arginine / chemistry
  • Arginine / metabolism*
  • Cell Culture Techniques*
  • Cells, Cultured
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Genetic Engineering / methods*
  • Isotope Labeling / methods*
  • Mass Spectrometry / methods
  • Molecular Sequence Data
  • Proteomics / instrumentation
  • Proteomics / methods*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Schizosaccharomyces / metabolism

Substances

  • Amino Acids
  • Fungal Proteins
  • Recombinant Fusion Proteins
  • Arginine