PROTEOMICS:
SILAC ANALYSIS
SILAC-based quantitative proteomics is an innovative service used in high throughput quantitative analysis of large protein complexes, chemical biology, biomarker discovery and protein-protein interactions. SILAC is an acronym that stands for Stable Isotope Labeling with Amino acids of Cells in culture. This approach provides an unbiased strategy that can reveal how specifically either inhibitors, or other perturbations, affect the dynamic properties and/or cellular distributions of proteins. It can also be used as a sensitive and effective method to determine the specific interaction partners of proteins under study. Although the technology is currently being used in basic investigative research mainly on continuously growing cell lines, we have developed strategies and procedures for using SILAC on differentiated cells.
SILAC Experimental Design
SILAC studies are performed using cells with the normal isotopes 12C and 14N in which the amino acids arginine and lysine are to be replaced with the heavy isotopes 13C and/or 15N. Heavy isotope substitution is made on the basic amino acids arginine and lysine because these are the sites of trypsin cleavage, thereby conveniently generating a set of tryptic peptides each with increased mass that can readily be detected and quantitated by mass spectrometry. When mammalian cells are grown using SILAC media, the proteins become substituted with the heavy forms of arginine and lysine. Subsequent MS/MS and software analyses resolves and quantitates the relative ratios of each isotopic form of every peptide. This measures whether the level of each protein in the complex has increased, decreased or remained at the same level after the treatment used. The accuracy of the quantitation is enhanced because the measurements are made at the level of individual peptides, while typically multiple (2-10+) peptides are analyzed for each protein. In this way the response of large numbers of endogenous, untagged proteins (many hundreds to thousands) in the cell or a specific complex can be evaluated quantitatively at multiple time points in a single experiment.
Gemini Biosciences offers a custom SILAC analysis service that includes advice on SILAC experimental design, sample processing, MS/MS analysis, data analysis and interpretation of results.
SILAC Workflow
