Current Gel Electrophoresis Approaches to Low-Abundance Protein Marker Discovery - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 181

Excision of
relevant spots

Immunoblot

2-DE
separation
Sample 1

Excision of
differentially
expressed
spots

Spot extraction
and
trypsination

Protein
identification
by LC-MS/MS

2-DE
separation
Sample 2

FIGURE 5 General scheme for 2-DE applications. The main route for biomarker discovery (bold boxes) is the comparison
of two groups of biological extracts. The alternative route (thin boxes) is used when attempting to discover special proteins
such as allergens (immunoblots) or other interacting proteins with speci c targets. Both routes can be advantageously

preceded by enrichment of low-abundance proteins by CPLL treatment.

CONCLUSIONS

a biological sample is compared to another,
only spots that are differentially expressed are
analyzed, thus simplifying the enormous amount
of data that are generally related to a global mass
spectrometry analysis approach. By the same
token, protein isoforms can be individually char-
acterized not only for their identity, but also for
their post-translational modi

Two-dimensional electrophoresis has been
frequently considered as an essential technique
in proteomics investigations; however, limita-
tions are known when dealing with proteomic
extracts with large dynamic concentration range.
2-DE in association with sample treatment tech-
nologies such as CPLL is capable in evidencing
expression differences of low- and very low-
abundance proteins.
Today, 2-DE is frequently considered as a
competitor of mass spectrometry, but in reality
it is fully complementary. It allows sepa-
rating protein isoforms resulting from post-
translational modi

cation that consti-
tutes by itself a signature of metabolic misregula-
tion. 2-DE produces interesting global views of
protein patterns in which hundreds of protein
spots are detectable, rendering this method
very informative and of easy interpretation. In
reality, it is currently the most rapid method for
detecting protein expression differences. The
fact remains that 2-D mapping is not the solution
to all proteomics problems, especially when
dealing with insoluble proteins or tissues for
which only the direct proteolysis followed by
mass spectrometry analysis of peptides is
workable. Nonetheless, even in this domain,
solubilization of proteins is making constant
progress and advances are expected, thus
opening the possibility of a full top-down anal-
ysis for most proteins.

cations that are not directly
evidenced by mass spectrometry. It also allows
immunoblotting to distinguish proteins of
similar category or similar functions. In this
domain of molecular recognition, a number of
possibilities are open that are unachievable by
using only mass spectrometry.
2-DE has a connotation of labor intensity
and slow turnaround, but advantages over
other approaches are numerous. In fact, when