Biology Reference
In-Depth Information
This method was described for the
first time by
Unlu et al. in 1997
14
and then improved progres-
sively in terms of reproducibility and reliability
for differential protein expression.
15
fractionation, depletion of dominant proteins,
and enrichment by the reduction of dynamic
concentration range. The third of these, consid-
ered to be the most powerful approach for
enhancing low-abundance species, is described
in detail here.
LOW-ABUNDANCE PROTEINS
ARE NOT RESOLVED
BY 2-DE ALONE
Proteome Fractionation: A Complex
Procedure with Protein Losses
Preliminary fractionation using mainly clas-
sical chromatography methods has repeatedly
been proposed for the simpli
Although two-dimensional electrophoresis is
a useful technique for the analysis of proteomes,
it suffers from the presence of high-abundance
species such as albumin in serum reducing the
detectability of low-abundance species. Pub-
lished studies suggested the use of narrow-
range pH gradients (covering a few or even less
than 1 pH unit),
16
which would exclude albumin
frommost pH ranges and would allow the detec-
tion of low-abundance proteins, especially with
large sample loadings. Unfortunately, because
protein loading comprises the entire proteome,
massive precipitation outside the pI of migration
can physically entrap species that would not
migrate and would thus escape detection.
A better approach for the detection of low-
abundance proteins is reducing the dynamic
concentration range of the proteome sample.
cation of complex
protein mixtures. Fractions thus obtained contain
a lower number of proteins in each fraction that
could be (1) better separated, (2) more ef
ciently
analyzed by mass spectrometry, and (3) adapted
to detect more species. These methods are
intended to separate groups of proteins based
on their chemical and physical properties. This
goal is obtained by adsorption-elution mecha-
nisms that induce not only fractionations but
also some level of concentration. A number of
chromatography methods have been applied to
proteome fractionation including size exclusion,
ion exchange chromatography,
17
and hydro-
phobic interaction chromatography.
18
When the main question is the separation
of homogeneous groups of proteins, speci
c
methods are adopted, such as af
nity chroma-
tography; few examples are discussed here:
ENHANCING LOW-ABUNDANCE
PROTEINS
a)
Glycoproteins: Lectins are largely used for the
fractionation of glycoproteins. The capture of
glycoproteins is performed in physiological
conditions of pH and ionic strength, as is the
displacement elution with competing sugars,
so that these species are not denatured and
collected in their native state. A strategic
approach of using lectins as a way to capture
categories of glycoproteins is reported by
Dayarathna et al.
19
Concanavalin A, wheat
germ agglutinin, and jacalin were used to
improve conditions for analysis of
subproteomes for speci
The direct and natural approach to increase
the concentration of very dilute proteins is to
concentrate biological samples by lyophilization
or membrane concentration. However, this
approach suffers from at least two, if not three,
dif
culties: (1) the sample would become more
viscous (especially by starting from serum or
plasma), (2) the concentration would apply to
all proteins in the sample; and (3) high-
abundance species would become even more
concentrated with increased detrimental effects.
In this context, scientists tried different strategies:
ed diseases.
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