[1][2][3] 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 samples are combined and then separated through chromatography, then sent through a mass spectrometer to determine the mass-to-charge ratio between the proteins.
[4] 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.
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.
The ratios of signal intensities of differentially mass-tagged peptide pairs are quantified to determine the relative levels of proteins in the two samples.