SILAC Quantitative Proteomics

Technology
SILAC-based quantitative proteomics is a novel and exciting technology used in high throughput quantitative analysis of large protein complexes and protein-protein interactions. This approach provides a quantitative and 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 cell lines, Dundee Cell Products Ltd is developing strategies and procedures for using SILAQ on primary cells and animal models.

SILAC Methodology: 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 reagents, the proteins become substituted with the heavy forms of arginine and lysine.

Subsequent MS analysis 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 analysed 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.

 

Dundee Cell Products offers a custom SILAQ service that includes consultancy, experimental design, sample processing, mass spectrometry, data analysis and interpretation of results.

 

Key Applications for the technology include:

1. High throughput protein expression profiling

2. Drug discovery and development e.g.  

          - Systematic evaluation of the effects of compounds on proteins including

             localization and interaction partners in proteome wide unbiased screens in cells

           - Improved characterization of chemical compound mechanisms and

             their specificity

           - Identification of potential side effects of novel hit and lead compounds prior to 

             initiating clinical trials

3. Biomarker discovery

4. Functional characterization of protein components in cell-based assays

Benefits
Apart from the use of quantitative proteomics in basic investigative research, SILAQ has benefits in other areas for example;

1. Significant cost savings can be realized by removing compounds from the drug discovery and development process that are non-specific or may cause side effects prior to initiating clinical trials
2. Reliable identification of protein interaction partners in cell extracts by eliminating  non-specific interaction signals, hence faster and cheaper determination of novel protein function(s) in the cell
3. High throughput quantitative protein expression profiling
4. Faster identification of drug targets from phenotypic screen
5. Faster and better characterisation of fusion proteins used in cell-based assays
6. Better and faster hit-target profiling in mammalian cells hence better target validation.
7. Investigative toxicology: faster determination of mechanism of action of xenobiotics in human cells
8. Faster and more efficient novel biomarker identification                                                

See other proteomic services