Biology Reference
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between proteins, changes, and a certain disease
requires accurate and precise proteomic quantita-
tionof a statisticallymeaningful number of human
samples.
Methods and reagents are readily available in
the quantitative proteomic toolbox and several
particular methods have been gaining popu-
larity for discovering potential new protein
biomarkers over the last decade (
Figure 1
).
3
Some methods use differential stable isotope
labeling for the improved quantitation accuracy
and precision while others are label-free for the
simplicity of sample preparation. Recent
advances in proteomic informatics further
make it possible for quantifying changes in
mass spectral signals based on both the label-
based and label-free algorithms.
and fast incorporation of the oxygen stable isotope
labels to peptides; furthermore, water is added at
a very high molar concentration. Water as
a reagent is the cleanest in terms of sample
cleanup and results in no additional sample prep-
aration and separation steps compared to the
label-free methods. Therefore, the enzymatic
18
O-labelingmethodmaintains the limits of quan-
titation (LOQ), an advantage of label-free
approaches.
9
The practical utility of the method
for quantitative pro
ling of clinical samples also
bene
ts from the continuous improvements in
optimizing experimental procedures at individual
laboratories to obtain ef
cient labeling and
prevent the label loss via back-exchange.
10
e
12
Recent advances in data analysis,
13
e
19
as well as
the decreased cost of H
18
O, further facilitate the
method applications. Like all global stable isotope
labeling methods,
20
the enzymatic
18
O-labeling
method enables the protein quantitation based
on multiple peptides.
4
e
6
As a consequence,
protein isomers due to co- and post-translational
modi
Common Techniques
Enzymatic
18
O-Labeling
For clinical human samples, the enzymatic
18
O-labeling of peptides
4,5
is highly applicable
and sensitive. This method utilizes proteases for
labeling peptides that are generated by digesting
proteins in the samples. The differential labeling
of samples is implemented by incubation of
peptides with proteases in buffers that are made
of H
18
OorH
16
O.
6
Counterpart peptides in the
two samples are differentially labeled with two
atoms of oxygen stable isotope at the peptide
C-termini. Typically, the enzymatic
18
O-labeling
allows only the binary relative quantitation of
a diseased sample and a controlled one, using
proteases such as trypsin and Glu-C.
7
A pooled
control can be used as the master control for
quantitation normalization; the
18
O-labeled pro-
teome digest has been used as the universal refer-
ence for biomarker studies.
7,8
Quantitative MS of
the combined two samples allows for the relative
quantitation of peptide (and by implication the
precursor protein) amounts.
The
18
O-labeling method is simple and cost-
effective because it uses ef
cations
17,21
e
23
can be also identi
ed and
quanti
ed for correlation with the disease status.
Recent applications of the enzymatic
18
O-labeling method use samples from human
microdissected specimens,
24
plasma,
8
and sera.
25
Chemical Tagging of Stable Isotope Labels
The biological heterogeneity demands statisti-
cally meaningful numbers of
control and
diseased clinical
samples
to be accurately
ed.
26
e
29
Chemicals for tagging several
proteome samples for simultaneous analysis
can be designed and prepared to incorporate
stable isotope labels differentially. Conse-
quently, these versatile tagging reagents increase
both the accuracy and the sample throughput of
proteomic quantitation. Many chemical reagents
have been investigated for concurrent quantita-
tive proteomic analysis of several samples,
but only a few sets of reagents become widely
used. Two common types of chemicals for
tagging stable isotopes are (1) isobaric
30
e
32
and
(2) mass-difference
33
e
42
tagging reagents.
quanti
cient enzyme catalysis.
The simplest reagent, water, ensures quantitative
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