SILAC allows for the pooling of up to 3 samples together just after culture, ensuring high-quality quantitation by reducing experimental variation.
In order to attain high accuracy in quantitative proteomics, it is important to reduce the number of variables between samples as slight differences in processing can have an impact on peptide identification, making quantitation difficult to resolve. SILAC provides a method in which samples can be metabolically labeled at the very beginning of the experiment while remaining isotopically distinguishable in the mass spectrometer, without the need for chemical reactions or the inclusion of reference peptide sequences.
SILAC involves culturing samples in different media. A “light” media contains amino acids with natural isotopes and the “heavy” media contains amino acids with stable heavy isotopes. These amino acids are metabolically taken up into the cell and over the course of several divisions will be completely incorporated into the proteome of the sample. Once an isotopic difference has been established, the cultures can then be used in the experiments of interest. For example, the effects of an inhibitor on the heavy sample compared to a control on the light sample.
The heavy and light samples can then be pooled and processed for MS analysis simultaneously, with the isotopic differences between the peptides visible as a shift in the mass to charge ratio. By measuring the difference in abundance between the heavy and light peaks, it is possible to get a quantitative difference for each protein within the proteome between both sets of cultures.
The heavy media will contain arginine of lysine amino acids with either 13C or 15N isotopes. This is ideal for most MS protocols that utilise trypsin for digestion, which cleaves proteins at the C-terminus of both lysine and arginine residues. This ensurse that each peptide will possess the isotope following digestion.
It is also possible to use different combinations of isotopes to produce a “medium” media in conjunction with the heavy and light media, essentially allowing for up to 3-plex quantitation.
A core advantage SILAC offers compared to other quantification methods is that it is relatively straightforward to incorporate the stable isotope into the proteome by cell culture.
• Enables MS quantitative tagging of samples at the start of the experiment
• Allows good labelling efficiency of the proteome
• Reduces chances of error during sample preparation
• Can only be applied to samples that can be passaged in cell culture
• Not usable for larger experiments requiring multiple biological triplicates
• Not ideal for experiments involving serum starvation
• For experiments involving dialysed serum, further controls are required