Category Archives: Protein quantitation

What is SILAC?

SILAC: stable isotope labeling by amino acids in cell culture

SILAC incorporates stable-isotope labeled (or ‘heavy’) amino acids into cellular proteomes through normal metabolic processes. This is achieved by replacing natural (or ‘light’) amino acids in a growth medium with ‘heavy’ amino acids. The presence of heavy amino acids does not affect cell growth or morphology, but when ‘light’ and ‘heavy’ populations are mixed, the differences in protein abundances between the two populations can be determined by MS. SILAC affords the most accurate relative quantification compared with any chemical derivatization methods (e.g. iTRAQ or TMT) because the two (or more) samples are combined upstream of the protein extraction, at the intact cell level. Any cell culture system with defined amino acid sources is amenable to SILAC. Sufficient incorporation of ‘heavy’ amino acids usually requires several cell doublings; and the entire experiment can be completed within a few days.

Your first SILAC-based workflow should include a pilot experiment and a two-phase SILAC experiment. Once you are confident in your skills, your cells are adapted to the SILAC medium and have completely incorporated the ‘heavy’ amino acid(s), you can start with the part 2.2.

1.      Pilot experiment (no labeling)

The purpose of a pilot experiment is to go through the entire procedure from growing cells to analyzing MS results, identify existing and potential problems, and find solutions prior to purchasing the isotopically-labeled amino acids. The pilot experiment is the only way to find out how many cells are needed to get good MS data, whether your enrichment/fractionation procedure is optimal, and whether the proteins of interest are well-represented.

The cost of MS analysis is low compared to the cost of isotopically labeled amino acids and the cost of other supplies and labor, so there is no advantage in shunning the ‘dry run’ experiment and moving on to the SILAC. The outcome of the pilot experiment is the optimized protocol.

2.      SILAC experiment

2.1  Adaptation phase (untreated cells, heavy vs. light)

Adaptation phase experiment allows the cells to adapt to the SILAC medium and evaluates the degree of incorporation of the heavy amino acid over time. An aliquot of cells from only the heavy sample is analyzed. It is unnecessary to perform extensive fractionation of proteins before the analysis at this stage. A mixture of peptides from a gel band is analyzed by LC-MS, several peptides are selected, and the m/z region corresponding to the light form of each peptide is examined. In the final SILAC analysis, the proportion of proteins in the fully labeled ‘heavy’ state must be >98%, i.e. no detectable signal should be present in the m/z region of the ‘light’ form of a peptide.

2.2  Experiment phase (differential treatment, heavy vs. light)

Once the heavy amino acids are fully incorporated, two cell populations (from adaptation phase) can undergo a differential treatment, for example a drug is added to the ‘light’ population and the ‘heavy’ population is left untreated (control). The experiment follows the protocol that has been optimized during the pilot study. The outcome of the SILAC experiment is a record of quantitative and qualitative changes in the cell protein profile in response to the treatment.


A recent example of study that uses SILAC

‘Identifying novel targets of oncogenic EGF receptor signaling in lung cancer through global phosphoproteomics’, Zhang et al., Proteomics, 2015, 15, 340–355; doi: 10.1002/pmic.201400315

A generic SILAC protocol

‘A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC)’, Ong and Mann, Nature Protocols, 2007, 1, 2650 – 2660; doi:10.1038/nprot.2006.427

SILAC methods, recipes, publications, links, etc.

Isobaric ions

Two (or more) ions are isobaric if they have the same nominal mass but different exact mass. For example positive radical ions of N2, CO, and C2H4 all have nominal mass of 28 units (z = 1). However, their exact (monoisotopic) masses are 28.00559, 27.99436, and 28.03075 units respectively.

TMT reagents are called isobaric tags, which can be confusing. These labeling reagents within each set (e.g. 2-plex or 6-plex) have the same nominal mass and the same exact mass, therefore they are not isobars as defined by IUPAC.

What is TMT?

TMT (tandem mass tags) are labeling reagents for comparative mass-spectrometry-based proteomics, a Thermo equivalent of iTRAQ. Most popular TMT are amine reactive, but SH-reactive tags are also available. Each isobaric TMT reagent has an amine-reactive NHS-ester group, a spacer arm, and a tandem MS reporter. Either intact proteins or their tryptic digests can be labeled, and up to 6 experimental conditions can be compared in terms of protein expression differences. You can find more information about TMT reagents here.

Our facility is capable of performing both TMT and iTRAQ experiments.