Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/SILAC



SILAC (stable isotope labeling by amino acids in cell culture) is a technique based on mass spectrometry that detects differences in protein abundance among samples using non-radioactive isotopic labeling. It is a popular method for quantitative proteomics.

Procedure
Two populations of cells are cultivated in cell culture. One of the cell populations is fed with growth medium containing normal amino acids. In contrast, the second population is fed with growth medium containing amino acids labeled with stable (non-radioactive) heavy isotopes. For example, the medium can contain arginine labeled with six carbon-13 atoms (13C) instead of the normal carbon-12 (12C). When the cells are growing in this medium, they incorporate the heavy arginine into all of their proteins. Therefore, all of the arginine containing peptides are now 6 Da heavier than their normal counterparts. The trick is that the proteins from both cell populations can be combined and analyzed together by mass spectrometry. Pairs of chemically identical peptides of different stable-isotope composition can be differentiated in a mass spectrometer owing to their mass difference. The ratio of peak intensities in the mass spectrum for such peptide pairs reflects the abundance ratio for the two proteins.

Applications
A SILAC approach involving incorporation of tyrosine labeled with nine carbon-13 atoms (13C) instead of the normal carbon-12 (12C) has been utilized to study tyrosine kinase substrates in signaling pathways. SILAC has emerged as a very powerful method to study cell signaling, post translation modifications such as phosphorylation, protein-protein interaction and regulation of gene expression. Standardized protocols of SILAC for various application have also been published.

Pulsed SILAC
Pulsed SILAC (pSILAC) is a variation of the SILAC method where the labelled amino acids are added to the growth medium for only a short period of time. This allows monitoring differences in de novo protein production rather than raw concentration.