DIGE: Past and Future - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 7

software dedicated to DIGE proteomics. These reagents and tools
now make DIGE one of the most versatile and sensitive comparative
proteomics methods currently available. This volume is a testament
to the broad applicability and robustness of DIGE.
3. The Future
The goal of comparative proteomics is to discover protein changes
between cells and tissues under a variety of conditions and circum-
stances. Implicit in this goal is the desire to detect all protein
species within the proteome. Given the chemical complexity of the
proteome and its large number of different protein species, sepa-
rating the proteome into discrete entities is a virtually impossible
task, but we are obligated to do as best we can.
Historically, conventional 2DE has had diffi culty in resolving
proteins that are very large (>250,000 Da) or very basic (>9.5 p I ,
many of which are ribosomal proteins). Mass spectrometry-centric
methods also have diffi culties with proteins outside these ranges.
Fortunately, these proteins represent a very minor component of
the proteome. 2DE also has diffi culty resolving integral membrane
proteins. This may be due to their hydrophobicity, which causes
protein aggregation, and glycosylation, which causes heterogene-
ity in mass and charge so that membrane proteins do not appear as
discrete spots. Important strides in improving 2DE's capacity to
resolve membrane proteins have been made by methods such as
introducing novel detergents, lipid removal, and deglycosylation
( 2-4 ). Finally, concern has been raised about protein overlap or
comigration on 2DE gels. The development of narrow pH-range
isoelectric focusing gels and very large-format 2DE gels has greatly
improved the resolution of 2DE gels where over 10,000 protein
species are now detectable ( 5, 6 ). These improvements have sig-
nifi cantly increased the resolving power of 2DE. One can expect
even further advances since many laboratories continue to study
new ways to increase the resolution of 2DE. These efforts have
improved resolution relative to protein mass and p I , but compara-
tively little has been done to improve resolution relative to protein
abundance.
Proteins exist in cells over an approximately 10 5 -fold concen-
tration range, while in serum the concentration range is on the
order of tens of millions fold. Currently, DIGE imagers are capable
of detecting proteins over a 20,000-fold concentration range. In
contrast, conventional mass spectrometers have a dynamic range of
about 1,000-fold. There is a clear need to improve the sensitivity
and dynamic range of DIGE fl uorescent-gel imagers. I am heart-
ened by the fact that technology for detecting single-molecule
fl uorescence already exists. Hence, it should be possible to build