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SILAC is used to quantify protein expression differences in up to 2 or 3 samples from cells. Cells are grown in identical conditions, with one cell line incorporating heavy isotopic amino acids (usually Arginine [U-13C6, 15N4] and Lys [U-13C6]) thereby taking advantage of the normal metabolic machinery of the cell to label the proteins. Metabolic labeling avoids problems associated with missing labels that can occur when using chemical labeling techniques.
When the labeled analog of an amino acid is supplied to cells in culture instead of the natural amino acid, it is incorporated into all newly synthesized proteins. After a number of cell divisions, each instance of this particular amino acid will be replaced by its isotope labeled analog.
A small percentage of the sample is removed early on to check incorporation of the heavy amino acids and a growth of 8-10 doubling times is recommended for a high incorporation.
At this point the 2 or 3 samples are then mixed immediately after the cells are grown. (As a result, downstream processes such as protein extraction, fractionation, and digestion are all subjected to the same magnitude of experimental variations, reducing the error on the final intensity ratios of the samples analyzed, and the obtained quantification will be more accurate and reliable.) Then their proteins are subjected to extracted, digested with a protease. The light and heavy proteins or peptides from digestion are chemically identical and thus co-elute in SDS PAGE separation and strong cation exchange fractionation.
Then each HPLC fraction is subjected to LC-MS/MS analysis (on the LTQ Orbitrap XL or other mass spectrometer) to identify the peptide sequences. Since the peptides originating from the 2 or 3 different samples are identical in all regards with the exception of their masses, their masses are used to quantify these peptides in the sample using the Matrix Science Quantitation Tool Box.
1. Instrument
2. Workflow
3.
Key Applications for the technology include:
a. 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
b. Faster identification of drug targets from phenotypic screen
c. Characterization of fusion proteins used in cell-based assays
d. Better and faster hit-target profiling in mammalian cells hence better target validation
e. Investigative toxicology: faster determination of mechanism of action of xenobiotics in human cells
f. Novel biomarker identification
g. 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 leads compounds prior to initiating clinical trials
h.
Functional characterization of
protein components in cell-based
assays
4. Reference: details of SILAC labeling technique can be found in following publication:
Blagoev, B., Kratchmarova, I., Ong, S. E., Nielsen, M., Foster, L. J. and Mann, M, A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. (2003). Nature Biotechnology. 21, 315-318.
5. Sample Amount
1. Recommended sample amount is 50 to 100μg if the CEX fractionation is performed
2. 0.5 to 5μg is recommended for a single LC-MS/MS run
*SILAC Labeling is performed in the investigators lab
6. Sample preparation
If 2-D PAGE is chosen for separation, some hints will be given. Sample preparation and solubilization are crucial factors for the overall performance of the 2-D PAGE technique. Protein complexes and aggregates should be completely broken up in order to avoid the appearance of new spots due to a partial protein solubilization. Thus, samples have to be sent to the Creative Proteomics either in a fresh solution containing 8 M urea, 4% (w/v) CHAPS, Tris base 40 mM, DTE 65 mM and a trace of bromophenol blue (see below) or as a dried powder. The minimum amount of protein has to be around 100 µg. The sample may contain salt in amounts lower than 100 mM. Put your samples in an Eppendorf tube, freeze them at -20 degree Celsius and put them into dry ice for shipment.
7. Time delay
Allow six weeks for delivery of the results.
Creative Proteomics offers a custom SILAQ service that includes advice on sample processing, MS/MS analysis, data analysis and interpretation of results.
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SILAC