iTRAQ

iTRAQ (isobaric tags for relative and absolute quantitation) is a chemical labeling technology used in tandem mass spectrometry-based proteomics to compare protein expression across multiple samples in a single experiment. After proteins are extracted, digested into peptides, and labeled with isobaric tags that attach to peptide N-termini and lysine side chains, samples are combined and analyzed by LC-MS/MS. Because the tags are isobaric, labeled peptides from different samples co-elute and appear as a single peak at the MS1 level, increasing throughput and reducing run-to-run variability. Upon MS/MS fragmentation, the reporter groups of the tags are released as low-mass reporter ions whose intensities reflect the relative abundance of the corresponding peptide in each sample. Peptide identifications from the MS/MS spectrum are then combined to yield protein-level quantitation.

iTRAQ supports multiplexing of multiple samples; early kits offered 4-plex, and modern kits enable up to eight samples per experiment. The method is widely used for comparative proteomics, biomarker discovery, and pathway analysis, and it does not require metabolic labeling, unlike some other approaches such as SILAC.

Limitations include ratio compression caused by co-isolation of impurity or co-fragmenting peptides, which can underestimate fold changes. Successful application depends on labeling efficiency, sample quality, and high-resolution MS instrumentation with appropriate software for reporter-ion analysis. Despite limitations, iTRAQ remains a foundational multiplexed proteomics approach and has influenced the development of subsequent tagging methods such as TMT.