5.3 Isobaric tag for relative and absolute quantitation
iTRAQ is a mass spectrometry-based method using isobaric tags (8-plex or 4-plex) to label proteins for quantification. Labeling targets N-termini and amine groups on side chains. Liquid chromatography separates labeled proteins, which are then analyzed by mass spectrometry. iTRAQ allows simultaneous protein identification and quantification [59].
Shotgun proteomics involves extracting and digesting protein mixtures from diverse samples. Multiple iTRAQ reaction mixtures, capable of labeling up to 8 samples, differentially label the proteins. After labeling, the samples are combined in a 1:1 ratio for multiplexing. Subsequently, they undergo high-performance liquid chromatography separation and mass spectrometry analysis. The 8-plexed sample can be separated using one of three methods: (1) IEF separation on gel followed by MALDI mass spectrometry (MS and MS/MS of the spots), (2) Gel-C-MS separation followed by high-resolution mass spectrometry, or (3) High-performance liquid chromatography separation coupled with high-resolution mass spectrometry [60].