Biology Reference
In-Depth Information
of various plant constituents (polyphenols, polysaccharides) that
strongly interfere with various sample manipulations, such as pro-
tein capture via various chromatographic means and analyses via
different electrophoretic methodologies. The present chapter deals
with such aspects and in particular offers guidelines in one method-
ology that is emerging as perhaps the most powerful in detection of
low- to very-low-abundance proteins, namely, the combinatorial
peptide ligand library (CPLL) technique.
The protein content of plant tissues is much smaller compared to
animal tissues [
4
]. Depending on the organelle, the protein con-
tent is about 20 times less since thick cell walls largely contribute
to the total biomass. In addition large vacuoles present in the cells
also contribute to decrease the average protein mass per biomass
unit. Overall when investigating the plant proteomes the amount
of tissue to start with must be signifi cantly larger than from ani-
mals. The consequence of this situation is that a lot of useless mate-
rial is to be eliminated during protein extraction with the risk of
losing associated proteins. Plant proteins are largely glycosylated
and combined to polysaccharides rendering the extraction chal-
lenging. For example it is known that some proteins are covalently
associated to cell walls [
5
] such as arabinogalactan proteins. Many
of these proteins such as hydroxyproline-rich proteins are also
largely glycosylated with a behavior unrelated to animal glycopro-
teins and thus current technologies cannot directly be applied. For
instance their extraction may require calcium-containing buffers or
other salts [
6
]. In some instances, to succeed in protein extraction,
a deglycosylation is necessary to release proteins that are strongly
involved in cell wall constructs.
In spite of low protein content, the expression of proteins in
plant results in a large dynamic concentration range precluding the
access to low-abundance species. Well-known massively present pro-
teins in plants, depending on the organ, are RuBisCo [
7
] in leaves
and endosperm storage proteins [
8
]. To resolve this question several
approaches have been proposed: (1) extensive fractionation, (2)
removal of dominant species, and (3) compression of concentration
difference between low-abundance and high-abundance proteins.
1.1 Proteins: A Minor
Component of Plant
Extracts
1.2 Necessary
Sample Pretreatment
to Eliminate
Interfering Material
Since interfering substances are present, special treatments are nec-
essary to polish the initial plant crude extract [
9
]. Extraction and
precipitation are the two most popular approaches. TCA associ-
ated with acetone with reducing agents allows precipitating most
proteins leaving in the supernatant a lot of undesired materials
[
10
]. Pellets containing proteins are then separated by centrifuga-
tion and redissolved. Alternatively or in addition to TCA/acetone
precipitation other interfering substances are eliminated by using a
Tris-HCl solution saturated with phenol followed by a precipita-
tion with ammonium acetate or ammonium sulfate. This option is
