Biology Reference
In-Depth Information
The fi rst example deals with proteins from leaf extracts from
Spinacia oleracea
[
41
]. Protein extraction (after removal of unde-
sired pigments and polyphenols) was performed in physiological
saline solution containing 5 mM EDTA, 1 mM DTT, and a tablet
of complete protease inhibitor in order to prevent protease action.
The homogenate was clarifi ed by centrifugation at 10,000 ×
g
for
10 min and directly submitted to protein capture with CPLLs, fol-
lowed by elution in 4 % boiling SDS and 20 mM DTT. The proteins
found in the treated sample were compared to untreated extract:
322 unique gene products were found, 114 of which common
between native and CPLL-treated extract. While only 18 unique
proteins were found in the control, 114 new others have been found
thanks to CPLL intervention. They presumably represent low-
abundance species since they were undetectable in the crude native
extract. A number of proteins related to chloroplast category have
been found after CPLL treatment such as inorganic pyrophospha-
tase 1, phosphoglycerate kinase, glutamate decarboxylase 2, glu-
cose-1-phosphate adenylyltransferase, fructose-bisphosphate
aldolase 2, ribulose-phosphate 3-epimerase, and 5-methyltetrahy-
dropteroyltriglutamate-homocysteine methyltransferase. A second
category of enrichment of cellular components was ribosomal pro-
teins such as 30S, 40S, 50S, and 60S.
Figure
3
represents the comparative results between native and
treated extracts.
The proteome of
Hevea brasiliensis
latex has been explored after
treatment with CPLL [
23
]. In this case the fresh plant exudate was
fi rst centrifuged at high speed to remove latex grains and then pro-
teins were precipitated in the cold with ammonium sulfate at 90 %
saturation. The supernatant was discarded, and protein pellets resus-
pended in distilled water containing protease inhibitor cocktail tab-
lets and then dialyzed (MWCO 3,500 membrane) overnight at 4 °C
against PBS. Two successive treatments with CPLL were performed
(ProteoMiner and carboxylated ProteoMiner). Protein elution was
operated sequentially using 1 M NaCl; 2 M thiourea, 7 M urea, 3 %
CHAPS; 9 M urea in 50 mM citric acid, pH 3.3; and hydro-organic
mixture composed of acetonitrile, isopropanol, trifl uoroacetic acid,
and water at fi nal concentrations of, respectively, 16.6, 33.3, 0.5, and
49.5 %. A total of 300 unique gene products have been identifi ed
from a proteome that was largely unknown, most of them thanks to
the CPLL treatment (Fig.
4
). In addition, when searching for aller-
gens by confrontation with the sera of 18 patients, several species
have been identifi ed (2D electrophoresis and immunoblots). Among
them were major known allergens, and others unknown such as heat-
shock protein, proteasome subunit (30 kDa), 8 kDa protease inhibi-
tor, hevamine A, and glyceraldehyde-3-phosphate dehydrogenase.
Proteome investigations have also been operated on cypress
pollen in view of fi nding novel allergens [
42
]. Pollen is a quite pecu-
liar part of plants with proteins that are part of the tissue wall and
