Biology Reference
In-Depth Information
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time (min)
Fig. 2 Representative chromatogram corresponding to the fractionation of an iTRAQ-labeled complex mixture
of peptides by strong cation exchange (SCX). Peptide elution was achieved using a gradient of SCX-B buffer
containing 0.5 M KCl at 0.1 mL/min constant fl ow rate. 1-min eluate fractions were collected using an auto-
mated sample collector and, then, redistributed in 23 different fractions (F1 to F23) corresponding to approxi-
mately equal chromatographic areas (mAU × min −1 at 280 nm). The redistribution of peptides according to
previously measured UV areas precludes the injection of samples too concentrated or too diluted and improves
protein identifi cation and quantitation
proteomics but the analysis of complex pools of peptides typically
leads to certain bias such as underestimation of the number of
proteins identifi ed and compression of quantitation ratios. A good
way to circumvent these limitations is the introduction of peptide
fractionation steps prior to mass spectrometric analysis. In this
sense, we chose the use of peptide fractionation by strong cation
exchange (SCX) chromatography (Fig. 2 ). The eluted peptide
fractions were analyzed by LC-MS/MS.
Currently, stable isotopic labeling followed by analysis through
liquid chromatography coupled to tandem mass spectrometry
(LC-MS/MS) is successfully used to characterize and quantify
changes in protein levels in complex biological samples. It is a
widely used technique for the differential expression study of
proteomes. Since 2005, several publications are reported using
iTRAQ technique for the study of plant proteomes [ 4 - 7 ].
The iTRAQ technology is a powerful technique allowing a
wide fl exibility in the experimental design which means a broad
range of applications to address biological questions. iTRAQ has
been extensively used to achieve a temporal quantitation of a bio-
logical process allowing monitoring time-course experiments
[ 8 - 12 , 29 ]. Also, its application to study the effect of a treatment
[ 13 , 14 ], a physiological response [ 15 , 16 ], proteome comparisons
[ 17 ], or functional studies based on mutant proteome differential
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