Improved SILAC method for double labeling of bacterial proteome

Stable isotope labeling with amino acids in cell culture (SILAC) is a robust proteomics method with advantages such as reproducibility and easy handling. This method is popular for the analysis of mammalian cells. However, amino acid conversion in bacteria decreases the labeling efficiency and quantification accuracy, limiting the application of SILAC in bacterial proteomics to auxotrophic bacteria or to single labeling with lysine. In this study, we found that adding high concentrations of isotope-labeled (heavy) and natural (light) amino acids into SILAC minimal medium can efficiently inhibit the complicated amino acid conversions. This simple and straightforward strategy facilitated complete incorporation of amino acids into the bacterial proteome with good accuracy. High labeling efficiency can be achieved in different bacteria by slightly modifying the supplementation of amino acids in culture media, promoting the widespread application of SILAC technique in bacterial proteomics. SIGNIFICANCE: Amino acid conversion in bacteria decreases labeling efficiency, limiting the application of Stable isotope labeling with amino acids in cell culture (SILAC) in bacterial proteomics to auxotrophic bacteria or single labeling with lysine. In this study, we found that high concentrations of isotope-labeled (heavy) and natural (light) amino acids facilitate full incorporation of amino acids into the bacterial proteome with good reproducibility. This improved double labeling SILAC technique using medium supplemented with high concentrations of amino acids is suitable for quantitative proteomics research on both gram-positive and -negative bacteria, facilitating the broad application of quantitative proteomics in bacterial studies.