Proteomics - Biomedical Informatics in Translational Research - page 92

ples. This type of profiling requires running each sample separately on one gel for
comparative analysis. It is technically very difficult to duplicate the gel running con-
ditions between samples, which drastically limits the superimposition of images
across the samples and limits the utility of this type of analysis.
The introduction of cyanine fluorescent dyes (Cy3 and Cy5) for labeling pro-
teins made it possible to run a pair of samples within the same gel under identical
conditions and to measure the relative quantities of proteins between the samples
[21]. The application of the differential ingel electrophoresis (DIGE) technique was
further improved by Amersham Inc., which developed a 2D DIGE platform to deter-
mine the statistically significant protein expression alterations that correlate with
the disease or experimentally induced changes in cells [22]. A third dye (Cy2), which
was introduced by Amersham, enables an internal standard (which is a pooled sam-
ple comprised of equal amounts of protein from each sample involved in the DIGE
study) labeled with Cy2 to be included on each gel and allows multiple gels to be
linked for accurate quantitative protein expression analysis between the samples.
The DIGE technique improved the sensitivity (0.025 ng /protein spot) and dynamic
range (~4 orders of magnitude) over the conventional 2DGE technique and the abil-
ity to multiplex the samples in the DIGE design reduced the number of gels that
needed to be run.
The DIGE gels are scanned on a Typhoon variable-mode imager to acquire the
images for the protein sample labeled with each dye, and the images are registered
for protein quantitative analysis across the samples to discern the altered expression
of proteins between the healthy and diseased states. These fluorescent dyes do not
interfere with protein identification by MS because only one lysine per protein mole-
cule is labeled (minimal labeling dyes) and most commonly an unlabeled protein
sample is also loaded along with the labeled protein and the gel is poststained with
Coomassie blue or Syproruby for picking the spots. The differentially expressed
spots whose coordinates are mapped to the coordinates of preparative gel or
poststained DIGE gel spots are used to generate a pick list and the spots are excised
from the gel to identify the proteins of interest. The identification of protein spots
from the DIGE workflows utilizes a robotic workstation to prepare the samples for
MS analysis by MALDI- TOFMS or liquid chromatography with tandem mass
spectrometry (LC/MS/MS).
The ability of the 2DGE technique to fractionate proteins to discrete spots in a
2D gel made it possible to apply a multitude of analyses on the separated proteins to
derive the information pertaining to protein isoforms and post-translational modifi-
cations (mainly phosphorylation and glycosylation) of proteins. This also provides a
means to probe for the presence of antigens associated with the diseased specimens
using the antibodies. For example, the proteins separated on a 2D gel can be sequen-
tially stained with various fluorescent dyes [23] to detect phosphoproteins (Pro-Q
diamond), glycoproteins (Pro-Q Emerald), and total proteins in a biological sample
(Sypro). This parallel analysis using multiple stains will allow determination of the
global functional protein expression differences between the clinical samples. This
global knowledge will aid in designing downstream analyses for a more targeted
approach to identify protein interactions and pathways involved in the diseased
state. Another advantage of fractionating intact proteins in a 2D gel is that they are
available for probing with the antibodies/autoantibodies for antigen identification.