Relative Quantification: iCAT™

Isotope-Coded Affinity Tag (iCAT™) strategy. An iCAT™ reagent with specificity toward sulfhydryl groups, an eightfold deuterated linker, and a biotin affinity tag is shown in the Figure . The iCAT™ method includes the following sequential steps:

  1. The side chains of cysteinyl residues in a reduced protein sample representing one cell state are derivatized with the isotopically light form of the iCAT™ reagent. The equivalent groups in a sample representing a second cell state are derivatized with the isotopically heavy reagent.
  2. The two samples are combined and enzymatically cleaved to generate peptide fragments, some of which are tagged.
  3. The tagged (cysteine- containing) peptides are isolated by avidin affinity chromatography.
  4. Finally, the isolated peptides are separated and analyzed by LC-MS/MS.

For Protein quantification the parent peptide masses of iCAT™ labeled peptides are used to quantify the proteins.