Biology Reference
In-Depth Information
suppression. As a consequence, immunodeple-
tion does not appear as an ideal technical option
to the discovery of disease markers, as recently
assessed by Tu et al.
33
and by Afkarian et al.
34
they will become saturated as more sample is
offered to the bead library. The consequence of
this last mechanism is the concentration and
hence the enrichment of low-abundance species.
Once the equilibrium of the above-mentioned
phenomena is reached, the excess of proteins is
eliminated by
Enrichment by the Reduction
of Dynamic Range
A few years ago, solid-phase combinatorial
peptide ligand libraries (CPLL) were proposed
for the reduction of proteins dynamic concentra-
tion range.
35
filtration or centrifugation and
the captured proteins released by appropriate
elution. The eluted protein pool comprises all
proteins that are present in the initial protein
sample, but
the proportion of
individual
The principle is based on the
proteins is largely modi
ed. High-abundance
af
nity chromatography operated in a mixed
bed of a multitude of ligands where the protein
sample is added under large overloading condi-
tions. The mixed af
proteins are signi
cantly reduced, whereas
low-abundance ones are enriched. The compres-
sion of dynamic range can reach more than four
orders of magnitude depending on the condi-
tions of work where adsorption and elution are
two distinct contributing phenomena of
nity bed is a ligand library
prepared by a combination of natural amino
acids to form hexapeptides attached covalently
to hydrophilic beads. Each peptide is individu-
ally grafted onto an individualized bead in
many copies similarly to an af
the
entire process. The
first essentially impacts the
enrichment of low-abundance species: the larger
the sample loading, the more dilute species
become concentrated and thus detectable. The
second point is also important, but for other
reasons: (1) it must be strong enough to fully
desorb all captured species, (2) elution agents
can be selected to be compatible with subsequent
analytical determinations, and (3) desorption of
proteins can be performed sequentially, thus gen-
erating fractions. The entire process and related
variants are described in a recent review.
36
Reproducibility of the process when per-
formed under comparable conditions was
demonstrated by a number of authors.
37
The
question of absolute quantitative protein is
biased by the modi
nity chromatog-
raphy solid phase. Depending on the number
of amino acids used, the resulting library
contains a population of millions of different
ligands (e.g., 11, 24, or 64 million starting from
15, 17, or 20 different amino acids, respectively).
When a protein mixture, such as blood serum, is
exposed to such a peptide library under large
overloading, several interesting phenomena
take place. First, each bead acts as an af
nity
sorbent for partner proteins and, due to the
very large ligand diversity, all proteins are
captured by the beads. Second, high-
abundance proteins saturate rapidly the corre-
sponding af
nity beads and the protein excess
remains in the supernatant. The third phenom-
enon, related to the af
cation of
the dynamic
protein concentration range.
In spite of this change, the proportionality
between species of two or more samples is main-
tained, allowing a relative quantitation.
38
This
behavior is an important feature for the detection
of upregulated and downregulated conditions
when searching for protein markers.
Another important point to consider is the
volume of sample involved in the process.
Although it is generally ideal to start from large
nity of several proteins
for a same peptide ligand, but with different
af
nity constants, induces a displacement effect
that depends on the respective concentration of
competing proteins and their dissociation
constants. An additional phenomenon is the
behavior of low-abundance proteins. Because
the amount of these proteins is by de
nition
low, the partner beads are not saturated, but
Search WWH ::
Custom Search