Isotop-coded affinity tagging (ICAT)

ICAT is a duplex protein labelling technique to differentially compare relative protein abundances of two samples directly with the mass spectrometer. The ICAT chemistry labels the cysteine residues of proteins. Each sample is labelled either with a light or a heavy label that differ in mass by 9 Da. The mass difference of light and heavy label is due to a different carbon isotope composition of both tags and therefore does not alter the chemical properties of the labelled proteins. After labelling both protein samples are pooled, digested with trypsin and further processed for mass spectrometric analysis.

The relative abundances of differentially labelled proteins can be calculated by the relative peak intensities of tryptic peptides labelled with light versus heavy tag that differentially appear in the mass spectrum with an increment of 9 Da.

The ICAT tag also contains a biotin tag for reducing sample complexity by affinity purification of labelled peptides. ICAT is therefore particularly suitable for the quantification of proteins in very complex protein mixtures.