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Protein quantitation by chemical incorporation of isotopes

Absolute quantitation: determine the absolute concentration of proteins/peptides in a selected fluid/cell/tissue: can apply to a series of samples.
Relative quantitation: determine the concentration of proteins/peptides by comparison to an internal standard and/or similar fluid/cell/tissue at a different physiological stage.

Labeling

Biological incorporation

Pre-harvesting. Labeling of peptide/protein is achieved by growing cell in media enriched in stable isotope containing amino acids. SILAC.

Chemical incorporation

Post-harvesting. Derivatisation reagent for chemical modification of proteins in a site-specific manner after harvest of the proteins.

Enzymatic incorporation

Labeling is achieved during enzymatic cleavage where proteolysis incorporates an O from the solvent (H2O) into the C-terminus.

ICAT – Isotope-coded affinity tagging

In MS, protein quantitation is often based on the use of stable isotopes. Gel-based methods use proteins for quantitation while MS uses peptides for quantitation. The general approach is to label alternative samples with equivalent reagents, one of which contains a light isotope. The samples are mixed, separated into fractions, and analyzed by MS. The ratio of 2 isotopic variants can be determined from the heights of the peaks in the mass spectra and used to identify proteins with differential abundance. MS methods are more reproducible and sensitive than gel-based methods for protein quantitation.

ICAT uses stable isotope labeling to perform quantitative analysis of paired protein samples, followed by separation and identification of proteins within these complex mixtures by LC-MS. The isotopic tags bind covalently to Cys within a protein. The tags are almost identical, possessing the same structure and chemical properties, but exist in two isotopic forms:

• Light - possessing eight hydrogens
• Heavy - possessing eight deutriums

When bound to the same peptide, a concrete mass change of exactly 8 mass units will be evident when analyzed by MS.

The tag has three functional elements:

1. a biotin tag, used during affinity capture
2. isotopically encoded linker chain
3. reactive group which will bind to and modify Cys residues

Using ICAT is a 4 step process:

1. free cysteines in a protein are reacted with a special affinity tag
2. labeled proteins are enzymatically digested
3. labeled peptides are separated from bulk using LC prior to MS
4. MS detects the mass differences in the same peptides

Two samples are separately treated with affinity tags. One with light ICAT and the other with heavy ICAT. The samples are mixed, digested and passed through MS.

The strength of this technique lies in its ability to allow quantification and identification within a single analysis. It also can be applied to samples from any source as it does not require metabolic labeling. The advantage over 2D gel is its speed and ease of automation.

Weaknesses of this method include the frequent need for extensive sample fractionation before MS/MS analysis. Since the procedure targets Cys residues, proteins that do not contain Cys cannot be quantified. This represents about 10% of the proteins.

Heavy Oxygen Labeling

An alternative to ICAT labeling of proteins that is not selective for cysteine-containing peptides is to label the peptides after digestion. When trypsin cleaves a protein and generates a peptide with a new C-terminus, it introduces an oxygen atom derived from a molecule of water into the carboxyl group of the peptide. This can be exploited for the identification of y-series ions in fragment ion spectra but it can also be used to differentially label peptides derived from alternative protein samples if normal water is used in one buffer and water substituted with heavy oxygen O-18 is used in the other. The abundance of the peptides can then be compared, since they will appear as doublets separated by two mass units.