5.2 Stable Isotopic Labeling with Amino Acids in Cell Culture (SILAC)
SILAC is a quantitative proteomics method that uses mass spectrometry (MS) for labeling and distinguishing proteins within cells or samples. It employs ”light” and ”heavy” amino acid labels to differentiate proteins in cultured cells. SILAC is valuable for studying post-translational modifications, cell signaling, gene expression, and secretory protein pathways in cell culture [57]. Stable amino acids (like 13C or 15N-labeled arginine or lysine) are integrated into the entire proteome during cell culture with isotope-labeled residues using SILAC. Two cell populations are grown in different media: ”light” with natural amino acids and ”heavy” with stable isotope-labeled amino acids. After multiple cell divisions (usually at least five cycles in mammalian cells), proteins in heavy media are entirely labeled. The labeling efficiency is checked before quantification, and then equal amounts of labeled and unlabeled cells or protein extracts are combined. Peptides are generated from these samples and analyzed using LC-MS/MS. SILAC quantification is based on comparing the ratio of isotope-labeled peptides to unlabeled peptides, allowing for quantitative comparison of light and heavy samples using signal intensities [58].