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The SILAC experiment
Advantages and limitations
SILAC was developed in CEBI (Ong et. al. MCP 2002) as a simple and accurate approach for mass spectrometric (MS)-based quantitative proteomics. The method relies on the incorporation of amino acids with substituted stable isotopic nuclei (in this case deuterium 2 H, 13 C, 15 N). In SILAC, two groups of cells are grown in culture media that are identical except in one respect: the first media contains the ‘light' and the other a ‘heavy' form of a particular amino acid (for e.g. L-leucine or deuterated L-leucine). Through the use of essential amino acids (those not synthesizable by the cell-type) the cells are forced to use the particular labeled or unlabeled form. Thus, with each cell doubling the cell population replaces at least half of the original form of the amino acid, eventually incorporating 100% of a given ‘light' or ‘heavy' form of the amino acid.
Incorporation of the labeled amino acid (d3) over five timepoints, showing the eventual replacement of unlabeled d0 form of the peptide by the d3 form after 5 days of adaptation. (from Ong et. al 2002 , M CP ) Asterix (*) marks an unrelated peptide.
SILAC relies on metabolic incorporation of the quantitative label. Cells are encoded with the label as they grow in cell culture. All SILAC labeled proteins are therefore fully labeled, in contrast to chemical modification methods which may not have 100% labeling efficiency. This also means that SILAC-labeling of tissue samples are not possible. Primary culture of cells harvested from tissue in SILAC is possible. Alternatively, the use of the tissue sample as the natural stable isotope abundance sample (LIGHT) is a valid comparison with sample labeled with SILAC (HEAVY).
The SILAC approach neither requires additional purifications to remove excess labeling reagent, nor does it involve multi-step labeling protocols. Hence the labeling process is straightforward and highly efficient – 100% of the sample is available for analysis. Cells are cultivated in labeling media under typical cell culture conditions. Unlabeled and labeled samples can be combined prior to lysis of the cells and treated as a single sample in all subsequent steps. This allows the experimenter to use any method of protein or even peptide purification (after enzymatic digestion) without introducing error into the final quantitative analysis. The amount of labeled protein in SILAC required for quantitative analyses is far less even than in the case of chemical incorporation where a large excess of labeling reagent and sample is required to ensure an unbiased labeling reaction.
The schematic above is intended as a general outline of the various strategies for quantitative labeling of proteins and peptides for the purpose of mass spectrometry-based quantitative proteomics. The white, blue or grey shading is to indicate the point at which "labeled" sample can be differentiated from the "normal" sample in the schema.
The comparison of the various MS-quantitation methods indicates the stage at which the two labeled states exist in the experiment. With labeled cells in SILAC, one can proceed to do sub-cellular purification of organellar structures or multi-protein complexes whilst they exist in their native forms. The two samples can be combined as whole cells and a single subcellular preparation of nuclei, mitochondria etc. be performed. Any sample preparation bias introduced by the comparison of two separate preparation steps (as would be the case in a chemical modification method) would thus be avoided.