5.1 ICAT labeling
Proteins from cancer cells are tagged with isotope-coded affinity tags
(ICAT reagents) and analyzed with a mass spectrometer, efficiently
identifying disease-related proteins. This method allows simultaneous
detection and assessment of various proteins, aiding researchers in
pinpointing disease causes for potential targeted therapies [55].
ICAT, an early mass spectrometry-based labeling method, targets cysteine
residues with three components: a biotin affinity tag, a thiol-reactive
group, and a linker with specific isotopes. It labels cysteine residues
with either 1H or 2H atoms (or 12C and 13C) using ICAT reagents.
Afterward, proteins are purified with avidin affinity chromatography,
digested by trypsin, and analyzed using LC-MS/MS to generate a spectrum.
ICAT specifically tags cysteine residues, making up 3.3% of vertebrate
amino acids, potentially missing proteins without Cys residues and
posing quantitation challenges for those with a single Cys residue
[56].