Stable isotope labeling by amino acids in cell culture (SILAC) technology is an in vivo metabolic labeling technology that uses stable nuclides to label and quantify intracellular proteins according to mass spectrometry abundance ratios. Combined with different protein affinity enrichment methods, protein quantitative information with higher sensitivity and accuracy can be obtained. In recent years, SILAC has been mainly used in hot research fields such as quantitative proteomics, protein-small molecule interactions, and protein post-translational modifications.
During the experiment, it is necessary to add essential amino acids containing heavy isotopes (eg. 13C,15N) or light isotopes(12C,14N) to the cell culture medium of the experimental group and the control, respectively. Commonly used heavy amino acids are 2H3-leucine, 13C6-arginine, 13C615N4-arginine, 13C6-lysine, etc. The experimental group and the control group cells or cell proteins are mixed in equal proportions after the cell labeling is completed, and the target protein is separated and then enzymatically hydrolyzed. The relative quantification of proteins can be accomplished by comparing the abundance ratio of light and heavy stable isotopes of the same amino acid sequence.
SILAC is a high-throughput method that can identify and quantify hundreds of proteins simultaneously. SILAC significantly reduces quantification errors caused by sample processing, therefore, the method has high quantitative accuracy. Moreover, this method has a wide linear quantitative range, high sensitivity, and low protein consumption (usually only tens of micrograms of protein per sample), and is especially applicable to very low expression levels or post-translational modifications of proteins. The use of intracellular labeling facilitates the analysis of target proteins close to physiological states.
SILAC is an excellent tool for studies of post-translational modification process of proteins. SILAC labeling technology combined with LC-MS can present a global landscape of cell signaling dependent on serine/threonine/tyrosine phosphorylation, enabling the analysis of diverse proteomes and phosphorylation site-specific changes. SILAC combined with LC-MS can better distinguish mono-methylation, di-, trimethylation and other methylation forms according to the quality and retention time of peptides. Glycosylation of proteins can be systematically studied by combination of SILAC and MS or LC-MS. Moreover, SILAC technology can identify biomarkers according to the protein expression in cells or the degree of phosphorylation, methylation and glycosylation, and promote the early diagnosis and treatment of diseases.
SILAC technology offers unique advantages in identifying DNA site-specific binding proteins. For example, combined with DNA affinity enrichment technology to study functional DNA elements such as transcription factor binding sites, single nucleotide polymorphisms, and methylated CpG islands. In addition, SILAC can be used to study protein-proteasome, RNA, and small-molecule drug interactions.
SILAC technology can be utilized for macro proteome analysis, to determine the changes of the whole proteome caused by experimental conditions, and to determine the mechanism of action and binding sites of small molecules. This method is especially suitable for the analysis of multi-target drugs, aiming to find synergy or antagonism between various targets, and promote drug development and mechanism research.