Biology Reference
In-Depth Information
Here, the term proteogenomics refers to differential proteomic
analysis based on the complete genome sequence of the study
organism in contrast to the general defi nition of proteomics-
enabled improvement of genome annotation. At present, about
1,000 prokaryotic complete genome sequences have been deter-
mined, and more than 3,400 genome projects are ongoing (
26
).
In addition, the Genomic Encyclopedia of Bacteria and Archaea
(GEBA) aims at a “phylogenetically balanced genomic representa-
tion of the microbial tree of life” (
27
). Considering these recent
genomic developments as well as the well-established proteomic
technologies (
28
), it can be expected that any prokaryotic isolate
representing a promising model system will be genome-sequenced
and thus applicable for subsequent functional proteomic studies.
2. Establishing 2D
DIGE for Studying
Environmental
Bacteria
Proteomic analyses of genome-sequenced environmental bacteria
provide unprecedented insights at the molecular level. However,
the overall picture of an organism's physiology cannot be derived
by a single comprehensive proteomic measurement. The actual
challenge is to precisely capture distinct subproteomes (proteome
signatures), each refl ecting the organism's response to a specifi c
carbon source, redox state, nutrient limitation, host interaction, or
other environmental condition. Thus, quantitative differential pro-
tein profi ling as it is possible with two-dimensional difference gel
electrophoresis (2D DIGE) plays the central role for physiological
proteomics of environmental bacteria.
The cyanine dye-based 2D DIGE was originally described
by Ünlü et al. (
29
). 2D DIGE, based on three cyanine dyes, the
3-mode Typhoon 9400 scanner, and the DeCyder software, was
evaluated for the fi rst time using a marine bacterium (
30
). 2D
DIGE-based studies from our group with environmental bacteria
are summarized in Table
1
and described in more detail in the
following sections.
2.1. 2D DIGE Method
The 2D DIGE procedures (general experimental setup, labeling
reaction, 2DE separation, image acquisition, and image analysis)
optimized in our laboratory over the last decade for environmental
model organisms have recently been summarized (
31
). Design and
workfl ow of a typical 2D DIGE experiment is divided into four
major steps: fl uorescence labeling, preparation of mixtures of labeled
protein extracts, protein separation by 2DE, image acquisition,
and image analysis (Fig.
3
).
Each protein extract is derived from an individual culture to
account for biological variation. In the following, the typical
2D DIGE experiment is based on 12 parallel gels for reason of
2.1.1. Fluorescence
Labeling
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