Biology Reference
In-Depth Information
Most of these techniques have as essential objective to get rid
of RuBisCO except in the case of Widjaja et al. [
15
] where the
authors tried to combine depletion and enrichment. Unfortunately
the approach proposed is a combination of various fractionation
techniques that are all together labor intensive and add the high
risk of losing very low-abundance proteins due to the repeated
manipulations. The only method that concomitantly reduces the
dynamic range of protein concentration is the one involving CPLL
as detailed below. It proved its properties with a number of plant
extracts such as maize seeds [
20
], spinach [
21
],
Arabidopsis t
.
leaves [
22
], rubber plant latex [
23
], and wines [
24
,
25
].
Low-abundance proteins are generally undetectable because of
two distinct phenomena: (1) their signal is covered by the high-
abundance proteins in either 2D electrophoresis and mass spec-
trometry and (2) because their concentration is below the sensitivity
level of the analytical methods. In the past few years a method has
been devised to compress the dynamic concentration range in
order to decrease the concentration of most concentrated species
reducing thus the signal coverage and also the concentration of
rare species. This process is operated by the so-called CPLL. This
approach has been extensively described for a number of biological
extracts including from plants [
26
-
30
].
CPLL is a mixture of small beads (ca. 65
1.3 Protocol
to Enhance Low-
Abundance Proteins
in Plant Proteomics
m diameters) to
which hexapeptides are covalently linked. The number of pep-
tides reaches various millions depending on the number of amino
acids used for the synthesis; however, each bead carries a single
type of peptide in a large number of copies. This is thus a mixed
bed of beads different from each other and individually capable
to capture a protein or a group of them. When a plant protein
extract is exposed to such a CPLL under large overloading con-
ditions, each bead with affi nity to an abundant protein will rap-
idly become saturated and the vast majority of the same protein
will remain unbound. In contrast, trace proteins will not saturate
the corresponding partner beads unless the sample volume is
large enough to provide for increasing amounts of proteins.
Once the excess of unbound proteins is eliminated by fi ltration
or centrifugation, all captured proteins can be collected by elu-
tion at a concentration range that is largely lower than in the
original biological sample. Trace proteins thus become detect-
able by current analytical methods. In theory each bead carrying
a single peptide ligand should interact with proteins that share
the same epitope complementary to the peptide bait. However,
because in a number of cases peptide ligands are very similar and
due to the mass action law (individual protein concentration,
affi nity constants, and conditions of exposure) several proteins
are found on a single bead (or peptide ligand) and a same pro-
tein can be captured by several different beads.
μ
