Current Gel Electrophoresis Approaches to Low-Abundance Protein Marker Discovery - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 176

cancer from 90 patients with preclinical
biomarkers identi

A number of real-life examples have been
described with good results. One of them is
the identi

ed. 45 More examples can be
found in the scienti

c and medical literature.
Considering the practical advantages of
reducing the dynamic concentration range
compared to the depletion of high-abundance
proteins, the association of CPLL technology
with 2-DE opens up, as already anticipated by
Sihlbom et al., 46 several interesting possibilities
in protein discovery with speci

cation of novel allergens. CPLL
enrichment recently demonstrated that trace
allergens could be enhanced and detected in
extracts where the presence of high-abundance
proteins normally complicates the task. After
CPLL treatment, 2-DE immunoblots with sera
from various patients revealed the presence of
many more spots than expected with very
different topologies. Some are isoforms of the
same protein; others are allergens of low-
abundance never described before. The scat-
tered allergic response related to patient
behavior indicated that active allergens are not
thesamefromoneindividualtoanother,sug-
gesting possible targeted personalized therapy.
Such was the case with Hevea brasiliensis latex
proteome 50 and more recently for the cypress
pollen proteome. 51
Due to the recent introduction of CPLL tech-
nology, it may be too early for popular adoption;
however, each time CPLLs were associated with
two-dimensional electrophoresis, very compel-
ling results have been reported. For instance,
when investigating the potential of the CPLL
process to

c emphasis on
protein marker detection. Such integration
supposes some efforts of compatibility. For
instance, if CPLL-treated proteins are recovered
by Laemmli buffer elution, a direct 2-DE analysis
cannot be performed because the sample compo-
sition is incompatible with the

first dimension of
migration unless treated to eliminate the deter-
gent. Thus special attention must be paid to the
selection of appropriate elution methods, always
keeping in mind that the desorption process effi-

-
ciency must approach the total recovery. In this
respect, several reports have been published on
the elution methods associated with subsequent
analytical methods. 47 Protein elution from
peptide libraries using TUC solution (2M
thiourea-7M urea-4% CHAPS) containing cysteic
acid is a good option as it ensures almost protein
desorption without distortion of protein isoelec-
tric migration. 48 The CPLL-treated sample is
thus directly loadable on the

find biomarkers of clinical interest,
Beseme et al. 52 found that it was possible to
access a large number of novel proteins by
two-dimensional electrophoresis. In a restricted
acidic pH range, 557 spots were found compared
to untreated serum in which a large number of
them did not belong to high-abundance species.
In recent work dealing with follicular

first dimension of
protein separation without preliminary treat-
ment, preventing thus the risk of protein losses.
If the CPLL-treated sample is very rich in protein
diversity that precludes a reasonable spot resolu-
tion, it is advised to fractionate the protein
mixture either directly operated from the same
CPLL beads by fractionated elution as already
described. 49 Table 1 summarizes the options
that are offered to the practitioner of 2-DE
when dealing with various elution methods
following CPLL sample treatment.
Low-abundance protein detection from bio-
logical extract comprising dominating species
was well demonstrated with human serum and
red blood cell lysates ( Figure 2 ).

uid
proteins, CPLLs demonstrated their ability to
signi

cantly improve the detection of many
protein spots that were normally masked by
major proteins, 53 thus opening the way to the
detection of markers associated with speci c

diseases. By using the same biological fluid, Mar-

tinkova et al. 54 find biomarkers of

ovarian hyperstimulation syndrome. First they
accessed low-abundance proteins by means of
CPLLs and analyzed the resulting samples by
various methods

tried to

including two-dimensional