Isobaric tags for relative and absolute quantitation (iTRAQ) is an

isobaric labeling Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics. Peptides or proteins are labeled with chemical groups that have (at least nominally) identical mass (isobaric), but vary in terms of distribution of heavy isotopes ...

method used in

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 ...

tandem mass spectrometry Tandem mass spectrometry, also known as MS/MS or MS2, is a technique in instrumental analysis where two or more mass analyzers are coupled together using an additional reaction step to increase their abilities to analyse chemical samples. A com ...

to determine the amount of

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...

s from different sources in a single experiment. It uses stable isotope labeled molecules that can be covalent bonded to the N-terminus and

side chain In organic chemistry and biochemistry, a side chain is a chemical group that is attached to a core part of the molecule called the "main chain" or backbone. The side chain is a hydrocarbon branching element of a molecule that is attached to a ...

amine In chemistry, amines (, ) are compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are formally derivatives of ammonia (), wherein one or more hydrogen Hydrogen is the chemical element wi ...

s of proteins.

Procedure

The ITRAQ method is based on the covalent labeling of the N-terminus and

s of

peptide Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. ...

s from protein digestions with tags of varying mass. There are currently two mainly used reagents: 4-plex and 8-plex, which can be used to label all peptides from different samples/treatments. These samples are then pooled and usually

fractionated Fractionation is a separation process in which a certain quantity of a mixture (of gases, solids, liquids, enzymes, or isotopes, or a suspension) is divided during a phase transition, into a number of smaller quantities ( fractions) in which t ...

liquid chromatography In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it through a system (a ...

and analyzed by tandem mass spectrometry (MS/MS). A database search is then performed using the fragmentation data to identify the labeled peptides and hence the corresponding proteins. The fragmentation of the attached tag generates a low molecular mass reporter ion that can be used to relatively quantify the peptides and the proteins from which they originated.

Data evaluation

At the peptide level, the signals of the reporter ions of each MS/MS spectrum allow for calculating the relative abundance (ratio) of the peptide(s) identified by this spectrum. The abundance of the reporter ions may consist of more than one single signal in the MS/MS data and the signals have to be integrated in some way from the histogram spectrum. At the protein level, the combined ratios a proteins' peptides represent the relative quantification of that protein. The MS/MS spectra can be analyzed using software that is freely available: i-Tracker and jTraqX protein-ms - Browse /jTraqX at SourceForge.net
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Procedure

Data evaluation

References

Further reading