Biology Reference
In-Depth Information
diffi cult to solubilize. This adds to the large diffi culties of eliminating
any undesired types of materials such as pigments and polyphenols.
Here the authors have fi rst tried to extract the maximum amount
and number of proteins involved in insoluble matrices and then to
eliminate all that would complicate protein fractionation. The fact
that proteins are only a small part of the pollen matrix necessitated
to start from very large amounts of dry pollen. The fi rst extraction
operation was performed in the presence of antiproteases using a
solution of 10 mM phosphate containing 0.2 % of a nonionic deter-
gent—Nonidet NP40—and 3 M urea, followed by a second extrac-
tion with physiological buffered saline. After precipitation with 80 %
saturation ammonium sulfate, the two collected protein solutions
were independently treated with CPLL at three different pHs to
maximize the enrichment in low-abundance species. The analysis of
treated proteins compared to non-treated extracts was performed by
either SDS-PAGE followed by LC-MS/MS or by 2D electrophore-
sis followed by immunoblots against the serum of patients allergic to
cypress pollen, followed by spot excision and identifi cation by mass
spectrometry. In a nut, what was found is an extremely large number
of peptides of which only few could be assigned to identifi ed pro-
teins; the majority of peptides could not be assigned due to the lack
of genome knowledge. As expected however, the number of identifi -
able proteins was signifi cantly increased by CPLL treatment com-
pared to the control and only few were in common. The number of
isoforms was also largely increased after CPLL when comparing 2D
map results. Moreover the number of positive spots to IgE from
various patients allowed identifying many allergens unknown up to
the present investigation and also indicating that allergic profi les are
extremely different from an individual to another.
Products derived from plants such as some drinks (e.g., wines,
bier, colas, and others) have also been analyzed after treatment with
CPLL. Wines were directly loaded on beads for the capture of pos-
sible traces of proteins [
24
,
25
]. The volume of these beverages
compared to the volume of beads was in all cases huge since around
1,000 mL of each drink was loaded on 0.1 mL of beads. Useless to
say that considering the minute amount of protein possibly captured
the elution was performed at once using SDS desorption as described.
In most investigated wines traces of casein were found (on the aver-
age from 20 to 60
g/L),
casein being one of refi ning agents used after fermentation. However,
for other younger untreated wine (e.g., white Recioto, from
Garganega grapes in the Veneto, Italy) CPLLs captured close to 100
unique gene products derived from the grapes [
43
]. As far as beer is
concerned a large number of proteins were found [
41
], several from
barley, two from maize, and more than four dozens from yeast such
as
S. cerevisiae
,
S. pastorianus
,
and S. bayanus
.
μ
g/L, with a detectability of as low as 1
μ
