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amounts of proteins (e.g., 50 mg), a recent report
demonstrated that it is possible to use small
volumes of dilute biological
underexpressed. The most upregulated polypep-
tide was a protein autoimmune regulator. In
spite of the fact that the number of patients
was limited to six, the level of overexpression
for this protein was 14-fold higher in tumor
tissues, suggesting a probable potential interest
in hepatocellular carcinoma diagnostics.
Cerebrospinal
fluids such as cere-
fluid.38
38
When using small samples,
the elution could be avoided; in this case, a tryp-
sination of the beads is made releasing peptides
that are directly analyzed by LC-MS/MS. This
procedure was evaluated under comparative
manner against depletion by Zhi et al.
39
brospinal
fluid has also been investigated
by means of 2-DE differential spot analysis with
the aim of
in
human serum in the search of
finding markers
for diabetes type 1. Beaded peptide libraries
showed a very clear advantage over immunode-
pletion with a signi
finding protein markers related to the
mechanism of neuromyelitis optica.
41
Here also
severals proteins differently expressed were
found. All of them were con
cantly larger number of
gene products found (1,037 versus 497), thus
advocating the use of CPLL followed by direct
analysis for protein biomarker discovery.
rmed in their
differential expression by immunochemical
quantitation using ELISA assays. In a third
example, sputum protein composition analysis
was performed in order to prepare a better
management of obstructive pulmonary dis-
ease.
42
Sputum samples from patients with
different severity of disease associated to
controls were analyzed and the
THE DISCOVERY OF PROTEIN
MARKERS WITH 2-DE
AND ITS ASSOCIATION
WITH LOW-ABUNDANCE
PROTEIN ENRICHMENT
findings were
quite impressive. About 40 spots were differen-
tially expressed; they represented 15 different
proteins, and 2 of them (lipocalin-1 and apolipo-
protein A1) were validated as being potential
speci
As reported above, 2-DE is a convenient
method to multiplex the protein expression
difference with possible applications in diagnos-
tics. However, the dynamic range of protein
extracts frequently prevents the detection of
very dilute protein species.
c markers well correlated with the severity
for the disease.
The above-mentioned examples, selected
randomly, demonstrate the capability of 2-DE
in the discovery of protein markers when the
apparent differences in spot intensity are of at
least two- to threefold.
With the advent of 2D-DIGE, the direct
comparison between two to three samples on
the same plate made a signi
In spite of
this,
2-DE is currently used for the identi
cation of
misexpressed proteins in which it is irreplaceable
for the determination of protein isoform
proportions
and the presence of protein
fragments.
Examples of protein marker discovery by
2-DE are numerous. Within the context of this
review, few illustrative examples are given. A
study of human hepatocellular carcinoma,
40
for
instance, showed that after an appropriate tissue
sample treatment and 2-DE separation, spots
with relevant differences (two folds) were evi-
denced that were excised, digested, and identi-
cant improvement.
Many reports describe the use of 2D-DIGE to
evidence
expression differences
related to
de
ned pathological situations. Such is the
case for protein markers related to prostate
cancer from blood serum
43
after having high-
abundance proteins removed by immunodeple-
tion.
fluorostaining
approach allowed identifying protein markers
of viral hepatitis E.
44
In another study, protein
markers have been found in noninvasive breast
Similarly,
the double
fied by mass spectrometry. Ten proteins were
found to be overexpressed and ten others
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