Isobaric labeling
Encyclopedia
Isobaric labeling is a mass spectrometry
strategy used in quantitative proteomics
. Peptides or proteins are labeled with various chemical groups that are (at least nominally) isobaric, or the same in mass, but which fragment during tandem mass spectrometry
to yield reporter ions of different mass. In a typical bottom-up proteomics
workflow, proteins are enzymatically digested by a protease
to produce peptides, which are then labeled with different isobaric tags. The samples are mixed in equal ratios. During a liquid chromatography-mass spectrometry
analysis, the peptides are fragmented to produce sequence-specific product ions, which help to determine the peptide sequence, as well as the reporter tags, whose abundances reflect the relative ratio of the peptide in the samples that were combined.
and isobaric tags for relative and absolute quantitation (iTRAQ)
. TMT is available in duplex and 6-plex forms, while iTRAQ is available in 4-plex and 8-plex forms.
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...
strategy used in quantitative proteomics
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...
. Peptides or proteins are labeled with various chemical groups that are (at least nominally) isobaric, or the same in mass, but which fragment during tandem mass spectrometry
Tandem mass spectrometry
Tandem mass spectrometry, also known as MS/MS or MS2, involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages.-Tandem MS instruments:...
to yield reporter ions of different mass. In a typical bottom-up proteomics
Bottom-up proteomics
Bottom-up proteomics is a common method to identify proteins and characterize their amino acid sequences and post-translational modifications by proteolytic digestion of proteins prior to analysis by mass spectrometry. The proteins may first be purified by a method such as gel electrophoresis...
workflow, proteins are enzymatically digested by a protease
Protease
A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein....
to produce peptides, which are then labeled with different isobaric tags. The samples are mixed in equal ratios. During a 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...
analysis, the peptides are fragmented to produce sequence-specific product ions, which help to determine the peptide sequence, as well as the reporter tags, whose abundances reflect the relative ratio of the peptide in the samples that were combined.
Availability
There are two types of isobaric tags commercially available: tandem mass tags (TMT)Tandem mass tags
Tandem 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...
and isobaric tags for relative and absolute quantitation (iTRAQ)
ITRAQ
Isobaric 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...
. TMT is available in duplex and 6-plex forms, while iTRAQ is available in 4-plex and 8-plex forms.
See also
- Isotope-coded affinity tag (ICAT)Isotope-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...
- Stable isotope labeling 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...
- Label-free quantitation