ITRAQ
Encyclopedia
Isobaric tags
for relative and absolute quantitation
(iTRAQ) are a non-gel-based technique used to quantify protein
s from different sources in a single experiment. It uses isotope
-coded covalent tags. iTRAQ is used in proteomics
to study quantitative changes in the proteome.
s of peptide
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
by nano liquid chromatography 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.
Isobaric labeling
Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics. Peptides or proteins are labeled with various chemical groups that are isobaric, or the same in mass, but which fragment during tandem mass spectrometry to yield reporter ions of different mass...
for relative and absolute quantitation
Quantitative proteomics
The aim of quantitative proteomics is to obtain quantitative information about all proteins in a sample. Rather than just providing lists of proteins identified in a certain sample, quantitative proteomics yields information about differences between samples. For example, this approach can be used...
(iTRAQ) are a non-gel-based technique used to quantify protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s from different sources in a single experiment. It uses isotope
Isotope
Isotopes are variants of atoms of a particular chemical element, which have differing numbers of neutrons. Atoms of a particular element by definition must contain the same number of protons but may have a distinct number of neutrons which differs from atom to atom, without changing the designation...
-coded covalent tags. iTRAQ is used in proteomics
Proteomics
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with...
to study quantitative changes in the proteome.
Procedure
The method is based on the covalent labeling of the N-terminus and sidechain amineAmine
Amines are organic compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are derivatives of ammonia, wherein one or more hydrogen atoms have been replaced by a substituent such as an alkyl or aryl group. Important amines include amino acids, biogenic amines,...
s of peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
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
See also: Fractionated spacecraftFractionation is a separation process in which a certain quantity of a mixture is divided up in a number of smaller quantities in which the composition changes according to a gradient. Fractions are collected based on differences in a specific property of the...
by nano liquid chromatography and analyzed by tandem mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
(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.
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.Protein level
The combined ratios a proteins' peptides represent the relative quantification of that protein.Software
The data of the MS/MS spectra can be analyzed using software that is freely available- i-Tracker
- jTraqX
See also
- Quantitative proteomicsQuantitative proteomicsThe aim of quantitative proteomics is to obtain quantitative information about all proteins in a sample. Rather than just providing lists of proteins identified in a certain sample, quantitative proteomics yields information about differences between samples. For example, this approach can be used...
- Isobaric labelingIsobaric labelingIsobaric labeling is a mass spectrometry strategy used in quantitative proteomics. Peptides or proteins are labeled with various chemical groups that are isobaric, or the same in mass, but which fragment during tandem mass spectrometry to yield reporter ions of different mass...
- ICATIsotope-coded affinity tagIsotope-coded affinity tags are a gel-free method for quantitative proteomics that relies on chemical labeling reagents. These chemical probes consist of three general elements: a reactive group capable of labeling a defined amino acid side chain , an isotopically coded linker, and a tag for the...
- Tandem mass tagsTandem mass tagsTandem mass tags are chemical labels used for mass spectrometry -based quantification and identification of biological macromolecules such as proteins, peptides and nucleic acids. TMT belongs to a family of reagents referred to as isobaric mass tags...
- Label-free quantitation
- SILACSilacSILAC is a technique based on mass spectrometry that detects differences in protein abundance among samples using non-radioactive isotopic labeling. It is a popular method for quantitative proteomics.-Procedure:Two populations of cells are cultivated in cell culture...
- Isobaric labeling