Technology

The principle of SILAC

The principle of SILAC. Cells differentially labeled by growing them in light medium with normal arginine (Arg-0, blue colour) or medium with heavy arginine (Arg-6, red colour).

High performance liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) can rapidly identify many proteins in complex biological samples. Recently, quantitative methods have been developed which make it possible to obtain precise functional information and to monitor temporal changes in the proteome by MS. In one approach, named SILAC (for stable-isotope labeling with amino acids in cell culture), cells are differentially labeled by cultivating them in the presence of either normal or a heavy isotope–substituted amino acid, such as 13C-labeled arginine. Metabolic incorporation of the amino acids into the proteins results in a mass shift of the corresponding peptides. Due to their mass difference, pairs of chemically identical peptides of different stable-isotope composition can be distinguished in a mass spectrometer. The ratio of intensities for such peptide pairs accurately reflects the abundance ratio for the two proteins. Quantitative proteomics with SILAC has emerged as a very powerful approach to investigate signaling processes. We are using this technology as our central tool to address several challenging questions in cell signaling.