Stable isotope labeling by amino acids in cell culture

SILAC ( stable isotope labeling by an abbreviation for / with amino acids in cell culture ) is a mass spectrometric method for the quantification by isotopic labeling. SILAC used in proteomics.

Principle

Two initially identical cell cultures are grown with different nutrient media. One of the cell cultures in the medium receives only amino acids that carry a heavy isotope. For example, the medium may contain arginine with six 13 C atoms, instead of the usual 12C. In the protein, the proteins are marked. Thereby, as a result all peptides arginine -containing six daltons heavier than the non- labeled peptides of the other cell culture. Alternatively, the two cell cultures can also be labeled with 13C or 15N. To analyze the proteins of both cell cultures are combined and measured together. Based on the different materials of the labeled peptides can be identified. The ratios of the signal intensities correspond to the ratios of the peptides.

Alternatives to SILAC are eg ICAT, the Isobarenmarkierung, the Tandem Mass Tags ( TMT), iTRAQ and label-free quantification (English Label - free quantification ).

Applications

A SILAC - based approach was used to study the signal transduction, for example, for the determination of the substrates of receptor tyrosine kinases, post-translational modifications such as phosphorylation, protein-protein interactions and to examine the regulation of gene expression.

Pulsed SILAC

Pulsed SILAC ( pSILAC ) is a variant of SILAC, in which the labeled amino acids are only given a limited time to the cell cultures, whereby newly formed proteins are measured and their synthesis rate is derived.