Isotopic labeling for relative quantification using MS. Proteins or peptides derived from 2 different cell states are derivatized with light and heavy isotopes of the same chemical reagent. The samples are then combined and analyzed by MS. The relative abundance levels of the proteins are calculated by comparing the peak heights of the light- and heavy-labeled peptides. The isotopic label can be incorporated in vivo, in vitro, at the protein, or at the peptide level. In cell culture, the incorporation was achieved by using media that included the isototopic labels, such as ¹⁵N or ¹⁴N media, minimal medium, or minimal medium with isotopically labeled leucine (leuD10).^61,62,94,95  The stable isotope-labeling approach has also been used for detecting the numbers of the tagged amino acid residues and, therefore, for unambiguously recognition of the protein.^62,95  A method that gained increased popularity is the isotope-coded affinity tag (ICAT) approach.¹³  The label contains a thiol group (which reacts with cysteine residues), 8 hydrogens (light) or deuteriums (heavy), used for relative quantification, and a biotin group that is selectively recognized during the affinity extraction step by an avidin moiety attached to the chromatographic column. Proteins from 2 cell states are derivatized with either the heavy or the light isotope and the mixture is digested and separated by affinity extraction prior to tandem mass spectrometric analysis. The ICAT analysis with the initial reagent used was complicated by the fact that heavy-labeled peptides elute earlier than the light-labeled peptides during LC separation, the so-called deuterium effect.⁹⁶  Newer cleavable ¹³C-labeled ICAT reagents have significant advantages over the original reagents.⁹⁷