Biology Reference
In-Depth Information
especially recommended when the analysis of proteins is based on
two-dimensional (2D) electrophoresis, but it depends on the plant
organ from where the protein extraction is intended. Also recom-
mended for two-dimensional electrophoresis is the one described
by Faurobert et al. [
11
] involving a combination of phenol extrac-
tion followed by a precipitation by ammonium acetate in metha-
nol. Another less popular method is the protein precipitation using
a chloroform-methanol mixture with water in 1-4-3 proportions
[
12
]. Several variants to these methods are also described to pre-
cipitate proteins [
13
]; however, the protein solubilization protocol
to be followed is not always easy and frequently necessitates the
presence of zwitterionic surfactants and chaotropes.
All these preliminary operations do not only remove undesired
materials but also contribute to concentrate proteins that are pres-
ent in very low amounts but in a quite large number.
Indeed while the number of native proteins corresponds to the
number of genes, the numerous variants due to extensive post-
translational modifi cations render the reality much different.
Nonetheless it is not the number of proteins that constitutes the
most important issue in their detection and identifi cation, but
rather the level of expression of genes, building up proteomes
where the individual component concentration difference spans
over several orders of magnitude. Among largely expressed pro-
teins there are species that are present only in few copies. The latter
are consequently very diffi cult to detect. In this context enrich-
ment procedures have been devised to improve the situation. Like
in animal proteome investigations, precipitation, fractionation,
depletion, and enrichment are the major approaches.
The published papers on the treatments of proteins from plant
extract samples to try reaching low-abundance species are not as
numerous as described for animal proteins. However, the same
concept has here been applied for the same purpose. Special meth-
ods of precipitations of
Glycine max
leaf extracts to remove
RuBisCO have been used for the detection of rare proteins such as
many phosphorylated proteins [
14
]. Ion-exchange chromatogra-
phy fractionation has also been used as for instance to enrich pro-
tein involved in plant defense from
Arabidopsis thaliana
evidencing
more than 30 misregulated proteins [
15
].
More elegantly immunoaffi nity chromatography has been used
for the selective removal of RuBisCO [
16
] in leaf extracts. Besides
antibodies, other affi nity-based solid-phase techniques have been
used for the removal of nucleotide-binding proteins from leaf
extracts using PolyA [
17
] and PolyU [
18
] as immobilized ligands.
Another affi nity-based selective separation method for the sep-
aration of phosphoproteome is based on the replacement of the
phosphate groups on serine and threonine residues by a biotin tag.
Biotinylated species are then separated using avidin-affi nity chro-
matography [
19
].
