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].