Isotope-coded affinity tag
An Isotope-coded affinity tag (ICAT) is an isotopic labeling method used for quantitative proteomics by mass spectrometry that uses chemical labeling reagents. These chemical probes consist of three elements: a reactive group for labeling an 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.
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 liquid chromatography due to the deuterium interacting with the stationary phase of the column.
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.
- Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R (October 1999). "Quantitative analysis of complex protein mixtures using isotope-coded affinity tags". Nature Biotechnology 17 (10): 994–9. doi:10.1038/13690. PMID 10504701.
- US 6670194 "Rapid quantitative analysis of proteins or protein function in complex mixtures," Rudolf Hans Aebersold et al.
- Yi, E.C et al. (2005). "Increased quantitative proteome coverage with (13)C/(12)C-based, acid-cleavable isotope-coded affinity tag reagent and modified data acquisition scheme". Proteomics 5 (2): 380–7. doi:10.1002/pmic.200400970. PMID 15648049.