Biology Reference
In-Depth Information
Fig.
2
Protein profi les of seven
B. cinerea
wild-type strains (B05.10, T4, 2100, 2850, 2996, 20518, and BOCL)
that differ both in host and virulence from (
a
) mycelium and (
b
) secreted proteins. This approach allows the
assessment of differences in protein band patterns among strains
Two-DE is the dominant platform in fungal proteomics. The
2-DE consists of a tandem pair of electrophoretic separations: in
the fi rst dimension, proteins are resolved according to their iso-
electric points (pIs), normally using IEF, while in the second
dimension, the proteins are separated according to their approxi-
mate molecular weight using SDS-PAGE. Excellent reviews
describing and discussing the features and protocols of electropho-
retic separations in proteomics strategies have been published [
23
,
48
]. Two main advantages of 2-DE can be emphasized: (a) its high
protein separation capacity, and (b) the possibility of making large-
scale protein-profi ling experiments. Nevertheless, the reproduc-
ibility and resolution of this technique are still remaining challenges.
This method was reported to under-represent proteins with
extreme physicochemical properties (size, isoelectric point, trans-
membrane domains), as well as those with a low abundance [
49
].
After separating proteins, they can be detected using different
staining techniques [
23
,
48
], namely, (a) organic dyes, like colloi-
dal Coomassie Blue staining, (b) zinc-imidazole staining, (c) silver
staining, and (d) fl uorescence-based detection, like Sypro Ruby.
The criteria used to choose the staining method are the level of
sensibility and its compatibility with MS. Gels are digitized, and
bands or spots are studied by specifi c image analysis software (i.e.,
Quantity-One, PD-Quest, BioRad). Bands or spots are excised
from gels and prepared for MS analysis.
The limitations of gel-based analysis have led to the more
recent development of techniques based on LC separation of pro-
teins or peptides, including two-dimensional liquid-phase
chromatography 2-D LC-MS/MS (based on a high-performance
chromatofocusing in the fi rst dimension followed by high-
resolution reversed-phase chromatography in the second) [
50
],
and 1-DE-nanoscale capillary LC-MS/MS, like GeLC-MS/MS
