A systematic approach to assess amino acid conversions in SILAC experiments

SILAC is a widely accepted approach for quantitative proteomics in which proteins are labeled with stable isotopes during cell culture. A major drawback of this technique is the metabolic conversion of labeled amino acids that may hamper accurate quantification. A paradigmatic example of this phenomenon is the generation of labeled proline from arginine, known to occur in a good number of biological models. We propose a novel methodology to identify and quantitate metabolic conversions as well as to evaluate labeling efficiency in SILAC experiments. In this approach, labeled proteins are reduced to amino acids by acid hydrolysis before LC–MS/MS analysis. Since it is carried out at the amino acid level, tracking the fate of the isotope label is straightforward and can be performed for each amino acid independently. After applying this method to mammalian cells, grown in the presence of heavy arginine and lysine, labeling efficiency and amino acid conversions could be accurately evaluated. Only undesirable labeling of proline was found to occur at a significant extent, varying greatly among cell lines. Finally, increasing proline concentration in the growing medium was shown to be effective at preventing arginine conversion without any noticeable side effect.