Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) is a technique based on mass spectrometry that detects differences in protein abundance among samples using non-radioactive

isotopic labeling Isotopic labeling (or isotopic labelling) is a technique used to track the passage of an isotope (an atom with a detectable variation in neutron count) through a reaction, metabolic pathway, or cell. The reactant is 'labeled' by replacing specific ...

. It is a popular method for

quantitative proteomics Quantitative proteomics is an analytical chemistry technique for determining the amount of proteins in a sample. The methods for protein identification are identical to those used in general (i.e. qualitative) proteomics, but include quantificatio ...

Procedure

Two populations of cells are cultivated in

cell culture Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This te ...

. 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

isotope Isotopes are two or more types of atoms that have the same atomic number (number of protons in their nuclei) and position in the periodic table (and hence belong to the same chemical element), and that differ in nucleon numbers (mass numbers) ...

s. For example, the medium can contain arginine labeled with six

carbon-13 Carbon-13 (13C) is a natural, stable isotope of carbon with a nucleus containing six protons and seven neutrons. As one of the environmental isotopes, it makes up about 1.1% of all natural carbon on Earth. Detection by mass spectrometry A mas ...

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. Thereafter, all peptides containing a single arginine are 6 Da heavier than their normal counterparts. Alternatively, uniform labeling with ¹³C or ¹⁵N can be used. Proteins from both cell populations are combined and analyzed together by mass spectrometry as 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 -Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. The word "tyrosine" is from the G ...

labeled with nine

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 In biology, cell signaling (cell signalling in British English) or cell communication is the ability of a cell to receive, process, and transmit signals with its environment and with itself. Cell signaling is a fundamental property of all cellula ...

, post translation modifications such as

phosphorylation In chemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology and could be driven by natural selection. Text was copied from this source, wh ...

protein–protein interaction Protein–protein interactions (PPIs) are physical contacts of high specificity established between two or more protein molecules as a result of biochemical events steered by interactions that include electrostatic forces, hydrogen bonding and th ...

and regulation of gene expression. In addition, SILAC has become an important method in

secretomics Secretomics is a type of proteomics which involves the analysis of the secretome—all the secreted proteins of a cell, tissue or organism. Secreted proteins are involved in a variety of physiological processes, including cell signaling and matrix ...

, the global study of secreted proteins and secretory pathways. It can be used to distinguish between proteins secreted by cells in culture and serum contaminants. Standardized protocols of SILAC for various applications have also been published. While SILAC had been mostly used in studying eukaryotic cells and cell cultures, it had been recently employed in bacteria and its multicellular biofilm in antibiotic tolerance, to differentiate tolerance and sensitive subpopulations.

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. It had also been used to study biofilm tolerance to antibiotics to differentiate tolerant and sensitive subpopulations

NeuCode SILAC

Traditionally the level of multiplexing in SILAC was limited due to the number of SILAC isotopes available. Recently, a new technique called NeuCode (neutron encoding) SILAC, has augmented the level of multiplexing achievable with metabolic labeling (up to 4). The NeuCode amino acid method is similar to SILAC but differs in that the labeling only utilizes heavy amino acids. The use of only heavy amino acids eliminates the need for 100% incorporation of amino acids needed for SILAC. The increased multiplexing capability of NeuCode amino acids is from the use of mass defects from extra neutrons in the stable isotopes. These small mass differences however need to be resolved on high resolution mass spectrometers.

References

External links