Isotope-coded affinity tag
Encyclopedia
Isotope-coded affinity tags (ICATs) 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 (e.g., iodoacetamide
to modify cysteine
residues), an isotopically coded linker, and a tag (e.g., biotin
) for the affinity isolation of labeled proteins/peptides. For the quantitative comparison of two proteomes, one sample is labeled with the isotopically light (d0) probe and the other with the isotopically heavy (d8) version. To minimize error, both samples are then combined, digested with a protease (i.e., trypsin), and subjected to avidin
affinity chromatography
to isolate peptides labeled with isotope-coded tagging reagents. These peptides are then analyzed by liquid chromatography-mass spectrometry
(LC-MS). The ratios of signal intensities of differentially mass-tagged peptide pairs are quantified to determine the relative levels of proteins in the two samples.
The original tags were developed using deuterium, but later the same group redesigned the tags using 13C instead to circumvent issues of peak separation during LC due to the deuterium interacting with the stationary phase of the column.
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...
that relies on chemical labeling reagents. These chemical probes consist of three general elements: a reactive group capable of labeling a defined amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
side chain (e.g., iodoacetamide
Iodoacetamide
2-Iodoacetamide is an alkylating agent used for peptide mapping purposes. Its actions are similar to those of iodoacetate. It is commonly used to bind covalently with the thiol group of cysteine so the protein cannot form disulfide bonds. Also used in ubiquitin studies as an inhibitor of...
to modify cysteine
Cysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...
residues), an isotopically coded linker, and a tag (e.g., biotin
Biotin
Biotin, also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin discovered by Bateman in 1916. It is composed of a ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring...
) for the affinity isolation of labeled proteins/peptides. For the quantitative comparison of two proteomes, one sample is labeled with the isotopically light (d0) probe and the other with the isotopically heavy (d8) version. To minimize error, both samples are then combined, digested with a protease (i.e., trypsin), and subjected to avidin
Avidin
Avidin is a tetrameric biotin-binding protein produced in the oviducts of birds, reptiles and amphibians deposited in the whites of their eggs. In chicken egg white, avidin makes up approximately 0.05% of total protein...
affinity chromatography
Affinity chromatography
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...
to isolate peptides labeled with isotope-coded tagging reagents. These peptides are then analyzed by liquid chromatography-mass spectrometry
Liquid chromatography-mass spectrometry
Liquid chromatography–mass spectrometry is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high...
(LC-MS). The ratios of signal intensities of differentially mass-tagged peptide pairs are quantified to determine the relative levels of proteins in the two samples.
The original tags were developed using deuterium, but later the same group redesigned the tags using 13C instead to circumvent issues of peak separation during LC due to the deuterium interacting with the stationary phase of the column.
See also
- ProteomicsProteomicsProteomics 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...
- Mass spectrometryMass spectrometryMass 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...
- 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...
- Tandem mass tags (TMT)Tandem 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...
- Isobaric tags for relative and absolute quantitation (iTRAQ)ITRAQIsobaric tags for relative and absolute quantitation are a non-gel-based technique used to quantify proteins from different sources in a single experiment. It uses isotope-coded covalent tags...
- Tandem mass tags (TMT)
- Label-free quantificationLabel-free quantificationLabel-free quantification is a method in mass spectrometry that aims to determine the differential expression level of proteins in two or more biological samples...
- Stable isotope labeling by/with amino acids in cell culture (SILAC)SilacSILAC 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