Biology Reference
In-Depth Information
Peptides derivatized with the so-called isobaric
chemicals have the same nominal mass and
produce differentiable ions only after gas-phase
fragmentation. Therefore, concurrent analysis of
multiple samples does not have the increased
spectrometric complexity during the
dissociation in the gas phase. Other gas-phase
dissociation methods can also produce unique
reporter ions for quantifying the tagged peptides.
Recently, 8-plex iTRAQ was reported to produce
more consistent ratios than 4-plex iTRAQ with
comparable detectability, allowing for reliable
quanti
rstMS anal-
ysis during a tandem (MS/MS) experiment. Only
so-called reporter ions are separated during
a second MS analysis, thus with decreased chem-
ical interference. In addition, sequence ions from
different samples are additive. This advantage is
very important because it ensures that pooling
multiple samples for a single analysis does not
result in the signal dilution of peptide sequence
ions. Thus, this chemical tagging of stable isotopes
has minimal effect on peptide sequencing and
identi
cation of the increased number of
samples.
43
TMT and iTRAQ applications have
been expanded in complex biological samples
and proteome dynamics, including the subcel-
lular proteome
46,47
and synaptosomes.
48
Contin-
uous efforts to increase the isobaric reagent
repository are being made, such as synthesis
of DiART,
49
e
51
MBIT,
52
SPIMT,
53
and DiLeu,
54
in order to use less expensive deuterium labels
or not use isotopes for reduced analysis cost.
2
Co-selection of precursor ions with small mass
differences is a major interfering factor in quanti-
tative proteomics using isobaric mass tagging. All
tagged peptides generate the same quantitation
reporter ions, increasing the background signal
for a peptide of interest.
55
Such an interference
results in decreased ratios for changes in the
peptide concentration. Contribution of the back-
ground signal is more signi
cation. Tagging of the N-termini and Lys
side chain residues by isobaric tag for relative
and absolute quantitation (iTRAQ
) and tandem
mass tag (TMT
) reagents is speci
c and occurs
ciently without major side reactions.
43
However, there always are some unavoidable
increases in the sample complexity due to incom-
plete reactions and minor side reactions, espe-
cially when the sample has a large dynamic
range. Sample loss due to additional sample
cleanup steps is also unavoidable. Both are intrin-
sically associated with peptide chemical derivati-
zations. Because the tagging step is performed in
the later part of sample preparation, prior experi-
mental variability is also an issue.
44
Therefore,
compromised limits of detection (LOD) and
LOQ can be observed when chemicals are used
for multiplexed quantitation of clinical samples
with minute amounts. In contrast, isobaric chem-
ical labels have been reported to be more precise
and reproducible than metabolic labeling in
arecentstudy.
45
Three sets of isobaric reagents are commer-
cially available: 4-plex iTRAQ,
31
6-plex TMT,
32
and 8-plex iTRAQ
27
reagents, together with
new reagents being developed. Peptides derivat-
ized with these reagents produce reporter ions at
the regions of
m/z
113
e
116, 126
e
131, and
113
e
119 and 121 upon collisional-induced
ef
cant for peptides
with large concentration changes.
56,57
The inter-
ference also increases its magnitude with the
increase in sample complexity, and it is a signifi-
-
cant issue for complex proteome samples like
human plasma.
55
Therefore, fractionation and
separation of proteins and peptides for reducing
the sample complexity for a particular MS anal-
ysis is very important for quantitative proteomics
of human samples. From the protein biomarker
development aspect, it is less of an issue,
however. Proteins with concentration changes
are only candidates for protein biomarkers, which
are further quanti
ed in a later stage of the
biomarker development, using targeted MS in
which the interference of co-selected precursor
ions can be reduced if not eliminated.
The other type of chemical mass tagging uses
the so-called mass-difference tagging reagents
for incorporating peptides (and proteins) with
stable
labels.
38
e
42
isotope
The
reductive
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