Biomedical Engineering Reference
In-Depth Information
2.5. IsobaricTagging
(iTRAQ,TMT,CILAT)
Isobaric tagging is a multiplex peptide labelling method that relies
on the introduction of stable isotope tags that are chemically
identical but distinguishable by MS/MS due to their fragmen-
tation into reporter ions of different masses. The most commonly
used method of this type has been the 4-plex iTRAQ reagents,
which are
N
-hydroxysuccinimidyl esters for the labelling of pri-
mary amino groups. A specific reporter group in each tag based
on
N
-methylpiperazine generates ions of 114, 115, 116 and 117
m
/
z
upon CID fragmentation. These appear in an ion-sparse
region of MS/MS spectra and their relative intensities provide
the relative abundance of labelled peptides between the samples.
The reporter groups in each tag are mass-balanced with a linker
group making the tags isobaric. The major advantage of this
MS/MS-dependent strategy is that the multiplex labelling does
not increase the mass complexity of the samples and only pep-
tides subjected to CID fragmentation are quantified. In addition,
higher signal-to-noise ratios can be achieved with MS/MS-based
detection versus MS-mode measurements.
global protein expression of wild-type and mutant yeast strains
defective in the nonsense-mediated and 5
to 3
mRNA decay
pathways using 2D-LC linked to MALDI- and ESI-MS/MS.
Under optimised labelling conditions, there was an estimated 97%
labelling of N termini and lysine -amino groups, with a min-
imal degree of unlabelled or tyrosine-labelled peptides. Lysine-
derivatised peptides were more frequently identified, possibly due
to their higher ionisation efficiency versus arginine-terminated
peptides. Peptide ratios were averaged for each protein and 685
proteins were quantified in all three yeast strains using two or
more significant scoring peptides. A high degree of reproducibil-
ity (CV 15-17%) for individual peptides contributing to any given
protein was reported. This study also determined the absolute lev-
els of a target protein after spiking with a synthetic peptide stan-
dard labelled with one of the isobaric tags. An 8-plex version of
iTRAQ has also recently been commercialised, generating a spec-
trum of eight unique reporter ions at 113, 114, 115, 116, 117,
118, 119 and 121
m
/
z
increasing sample throughput for com-
tocol for iTRAQ labelling of multiple samples with subsequent
2D-LC-MS/MS-based quantification.
Use of the iTRAQ strategy has increased rapidly in the last
few years with numerous applications including the study of
temporal changes in cellular signalling and protein localisation
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