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].