Biology Reference
In-Depth Information
electrophoresis (2-DE) has assumed the role of
prima donna. In spite of the enormous develop-
ment of mass spectrometry, 2-DE represents
a unique analytical method. It separates proteins
from complex extracts on the basis of two very
important intrinsic properties, namely the isoelec-
tric point
many very low-abundance proteins becomes
extremely dif
cult, if not impossible. It is in
this context that sample treatment techniques
have been devised: some are based on sample
fractionation, others on removal of high-
abundance proteins (depletion or subtraction),
and others on drastic reduction of the dynamic
concentration range, thus allowing the enrich-
ment of many low-abundance proteins.
This chapter is essentially focused on the
enrichment of protein samples associated with
2-DE analysis, a tandem technology considered
as a powerful tool in proteomics investigations.
first dimension (dependent
essentially on the amino acid composition) and
the mass for the second dimension (dependent
essentially on the number of amino acids
composing the polypeptide chain). This tech-
nology is well documented (see technical details
and protocols in reference).
1
2-DE offers a high degree of resolution and is
uniquely able to resolve protein isoforms and
truncated forms that are both extremely impor-
tant information to make a clear distinction
between two or more samples. Protein spots
after staining have different intensities in rela-
tion to their initial concentration within the
sample. With this property, it is easy to under-
stand how the use of this technique can
contribute to discover differences in protein
expression. Once these differences are high-
lighted, the relevant spots are excised and
treated with trypsin and the resulting peptides
analyzed by tandem mass spectrometry for the
formal identi
for the
THE EVOLUTION OF 2-DE
TOWARD PROTEOMICS
APPLICATIONS
Historically, two-dimensional electrophoresis
was developed with the objective of improving
the resolution of one-dimensional electropho-
resis. Two different separation parameters were
thus crossed over. However, the
rst attempts
in this adventure were not signi
cantly better
than single-dimensional separations.
3
More
sophisticated protocols involving electrophoretic
mobility as the
cation of the proteins of interest.
Because a biological sample contains a large
number of proteins that appear as spots that
are very close to each other and even superim-
posed, narrow pH gradients can be used to
resolve the question.
Many published studies report the immense
potential of 2-DE and a large number of
first dimension followed by
molecular sieving were used for the separation
of ribosomes with ef
cient resolution of small
and large ribosomal units.
4
A major break-
through in the domain (
Figure 1
) came with the
association of two orthogonal separation princi-
ples, combining isoelectric focusing and mass
fractionation in the presence of SDS.
5
This tech-
nology allowed for the
findings
in the domain of differential expression investi-
gations with proposals for protein markers
speci
first time the resolution
of more than 1,000 distinct proteins on a single
plate while giving indications of the isoelectric
point and the molecular mass values. Initially,
the
c for a given disease.
2
Nonetheless, the concentration differences
among proteins in biological extracts are huge,
reaching in serum up to at least 12 orders of
magnitude, where frequently only few proteins
represent 95 to 99% of the total protein content.
In the presence of massive amounts of
first dimension was operated using free
Ampholines
lled
glass tube in which proteins migrated according
to their isoelectric point. After extrusion, the gel
was placed at the edge of a plate of polyacryl-
amide for electrophoresis migration in the
in a polyacrylamide gel
e
few
high-abundance proteins,
the detection of
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