Biology Reference
In-Depth Information
precipitate in an appropriate buffer. This method minimizes protein
degradation/modification. Furthermore, it removes interfering
compounds such as polysaccharides, polyphenols, pigment, and lip-
ids, which may cause problems during IEF [
23
], and prevents protease
activities [
24
]. TCA treatment complicates subsequent protein solu-
bilization for IEF, especially with hydrophobic proteins. These prob-
lems have been partially overcome by the use of chaotropes (urea and
thiourea) [
25
], new zwitterionic detergents [
26
-
30
], and a brief
treatment with sodium hydroxide [
24
] that led to an increase in reso-
lution and capacity of 2-DE gels. Other protein extraction methods
have reported an improvement when using an acidic extraction solu-
tion to reduce streaking of fungal samples caused by their cell wall
[
31
], as well as with the use of a phosphate buffer solubilization
before the precipitation [
32
,
33
]. Finally, the combined use of TCA
precipitation and phenol extraction provides a better spot defi nition
due to the fact that it reduces streaking and leads to a higher number
of detected spots [
34
-
36
]. Alternative protocols for protein extrac-
tion from spores of
Aspergillus
ssp. have been optimized, since they
use acidic conditions, step organic gradient, and variable sonication
treatments (ultrasonic homogenizer and sonic water bath) [
20
].
Special protocols are required for secreted proteins due to the
fact that there may be problems like a very low protein concentra-
tion that are sometimes below the detection limit of colorimetric
methods (Bradford, Lowry, or BCA), or the presence of polysaccha-
rides, mucilaginous material, salts, and secreted metabolites (low-
molecular organic acids, fatty acids, phenols, quinones, and other
aromatic compounds). The presence of these extracellular com-
pounds may impair standard methods for protein quantifi cation and
may result in a strong overestimation of total protein number [
37
].
This determination can also be affected by the high concentration of
reagents from the solubilization buffer (i.e., urea, thiourea, or DTT)
that may interfere in the spectrophotometric measurement, produc-
ing an overestimation of the total amount of protein in which,
depending on the method, the differences vary in the order of two
magnitudes [
38
]. Comparisons of different standard methods for
protein precipitation have demonstrated their limited applicability in
analyzing the whole fungal secretome [
39
-
44
].
Electrophoresis is the most employed technique for protein
separation in fungal research. Despite its simplicity, 1-DE remains
as a useful technique that provides relevant information, especially
in the case of comparative proteomics with large number of sam-
ples to analyze. It is possible by using this technique to distinguish
between phenotypes of different wild-type strains of
B. cinerea,
and to identify proteins involved in the pathogenicity mechanisms
(Fig.
2
) [
45
,
46
]. With appropriate software tools, 1-DE is a sim-
ple, reliable technique for fi ngerprinting of crude extracts, and it is
especially useful in the case of hydrophobic and low-molecular-
weight proteins [
47
].