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Isotope Coded Affinity Tagging (ICAT)
Isotope coded affinity tags (ICAT) are a comparative protein quantification method based on chemical tagging. ICATs are compounds consisting of a reactive group, isotope coded linker, and a tag for purification. Usually the reactive group is iodoacetamide, which reacts with cysteine, and the purification tag is biotin. The isotope coded linker can be heavy (deuterium labeled) or light (hydrogen labeled). For ICAT quantification, proteins are collected from the two samples to be compared and one is labeled with the light tag and the other with the heavy tag. The samples are then mixed, digested with a protease, and affinity purified. The purified peptides are analyzed by mass spectrometry and the light- to heavy-ratios for each peptide indicate the relative amount of protein in each sample. ICAT is not a true global technique since proteins that do not contain cysteine will not be labeled. Examples of ICAT applications include the comparison of protein expression in normal versus cancer cells and studies of yeast metabolism. (Credit: Brooke Anderson-White)
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