Biology Reference
In-Depth Information
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RPB-A
Reverse-phase chromatography buffer A
RPB-B
Reverse-phase chromatography buffer B
RPB-C
Reverse-phase chromatography buffer C
SDS
Sodium dodecyl sulfate
SMPW
Spectrum mill proteomics workbench
TEAB
Triethylammonium bicarbonate
TCEP
Tris(2-carboxyethyl)phosphine
1
Introduction
Isobaric tags for relative and absolute quantitation (iTRAQ) is a
recently developed technique for a mass spectrometric based quan-
titation of proteins. Since its fi rst description by Ross et al. [
1
], in
2004, the amine-specifi c isobaric tag for relative and absolute pro-
tein quantifi cation (iTRAQ) has become a consolidated technique
in quantitative proteomics because large-fold changes of protein
expression within broad dynamic ranges of protein abundance can
be measured quite accurately [
2
]. The iTRAQ method allows for
the multiplexed identifi cation and quantifi cation of proteins in
four different samples (4-plex) and has been recently scaled to
measure protein changes in up to eight different samples (8-plex) [
3
].
These reagents were designed to bear an isobaric skeleton
among the different tags within the experiment. In the case of a
4-plex experiment, these tags are constituted in three parts: a pep-
tide reactive group which is common for all of them, a specifi c
reporter group unique to each tag (114-117 amu), and a neutral
balance group (31-28 amu) to maintain a total mass of 145 amu
(Fig.
1a
). MS/MS fragmentation splits the four unique reporters
away from their corresponding tags giving rise to a strong reporter
ion signals in the low mass area of any given spectra. As displayed
in Fig.
1b
, the intensities detected correspond to the addition of
the intensities of the four peptide populations, each of them bear-
ing a different tag. Concomitant peptide sequencing and protein
quantitation are allowed by combining −
b
and −
y
fragment ions
together with reporter ions in a single MS/MS spectrum (Fig.
1c
).
iTRAQ reagents were designed to react through the peptide reac-
tive group (NHS) with all primary amines found in tryptic pep-
tides, including N-termini,
-amino groups of Lys side chains, and,
possibly, Tyr side chains. Labeling all the peptides within the four
samples and pooling them prior to the mass spectrometric analyses
imply enhancing the peptide coverage for any protein. The general
workfl ow is depicted in Fig.
1
(panels B and C), showing the
typical appearance of a 4-plex iTRAQ kit (Life technologies). Each
sample is separately reduced, alkylated, digested, and labeled with
an iTRAQ tag (114-117 Da) and then pooled together. The
iTRAQ technique is currently a popular approach for quantitative
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