Stable Isotopic Labeling by Amino Acids in Cell Culture or SILAC

SILAC (1) 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. The light and heavy amino acids are chemically identical and thus co-elute in SDS PAGE and HPLC separation. But, since the peptides are isotopically distinct, the light and heavy labeled peptides are clearly distinguishable by mass spectrometry. 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. Samples should be mixed immediately after the cells are grown.

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As a result, downstream processes are all subjected to the same magnitude of experimental variations, reducing the error on the final intensity ratios of the samples analyzed. SILAC labeled samples are analyzed by LC-MS/MS (sometimes with prior strong cation exchange fractionation) on the LTQ Orbitrap XL, with quantitation using the Matrix Science Quantitation Tool Box. Results are loaded into YPED. Figure 1 shows the results from a SILAC experiment where endothelial cells were grown in “light” and “heavy” (Lys-6) medium. Light to heavy ratios of 2:1, 4:1, 2:3, and 1:3 were mixed followed by a 1D SDS PAGE (upper panel). The gel lane was sequentially cut, digested with trypsin and analyzed on the LTQ Orbitrap XL. An example of the total ion current (TIC) or 1 peptide pair is shown (bottom panel). The quantitated ratios were in excellent agreement with the expected ratios.

Recommended sample amount is 50 to 100µg if the CEX fractionation is performed
0.5 to 5µg is recommended for a single LC-MS/MS run

*SILAC Labeling is performed in the investigators lab

Ong, S., Blagoev, B., Kratchmarova, I., Kristensen, D., Steen, H., Pandey, A., and Mann, M. (2002) Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics. Molecular & Cellular Proteomics, 1(5):376-386.