Biology Reference
In-Depth Information
Chapter 12
Differential Plant Proteome Analysis by Isobaric Tags
for Relative and Absolute Quantitation (iTRAQ)
María J. Martínez-Esteso , Juan Casado-Vela , Susana Sellés-Marchart ,
María A. Pedreño , and Roque Bru-Martínez
Abstract
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. Briefl y, 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 proce-
dures typically using 4-plex labeling, including tips leading to successful application of iTRAQ technology
for the analysis of plant protein mixtures.
Key words
iTRAQ, Isobaric tag, Liquid chromatography, Mass spectrometry, Plant proteomics,
Quantitation, Strong cation exchange
Abbreviations
ACN Acetonitrile
BCA Bicinchoninic acid
DIGE Differential in-gel electrophoresis
FDR False-positive rate
FWHM Full-wide half-maximum
cICAT Cleavable isotope-coded affi nity tags
LIT Linear ion trap
LOPIT Localization of organelle proteins by isotope tagging
MMTS Methyl methanethiosulfonate
