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Protein relative quantitation is one of the main targets in many proteomic experiments. Among the range of techniques available for both top-down and bottom-up approaches,
i
sobaric
t
ags for
r
elative and
a
bsolute
q
uantitation (iTRAQ) have gained positions within the top-rank techniques used for this purpose in the recent years. Briefly, each iTRAQ reagent consists of three different components: a reporter group (with a variable mass in the range of 114–117 amu), a balance group, and an amino-reactive group. The isobaric nature of iTRAQ-labeled peptides adds a signal to every peptide in the sample which is detectable in both MS and MS/MS spectra, thus enhancing the sensitivity of detection. During MS/MS, the reporter groups are released as singly charged ions with
m
/
z
ratios ranking from 114 to 117 amu, visible in the low mass region of MS/MS spectra. The iTRAQ technology can be used to analyze up to four different samples using the 4-plex kit (reporter groups 114–115 amu) or can be scaled up to eight different samples using the 8-plex kit (reporter groups 113–121 amu). In this chapter, we focus on the experimental procedures typically using 4-plex labeling, including tips leading to successful application of iTRAQ technology for the analysis of plant protein mixtures.
