Biology Reference
In-Depth Information
Immobilized heparin is also extensively
used
20
as a ligand for the enrichment of
a number of proteins including coagulation
factors and proteins interacting with nucleic
acids (e.g., growth factors).
b)
Phosphoproteins: Phosphorylation is another
very important post-translational mod-
i
generate fractions that must be analyzed sepa-
rately and, more importantly, the recovery for
each step is below 100%, with consequent losses
of proteins, especially those that are the least
abundant.
Depletion: A Biospeci
c Method
with Limited Enrichment
Advances in protein discovery were per-
formed with the use of immunosorbents to
remove the gene products representing the
largest majority in human blood.
27
This so-
called depletion approach under various
versions targeting from 6 to 70 major proteins
in human plasma
28,29
cation associated with pathological
situations to the point that the misregulated
phosphoproteome could be the basis of the
discovery of protein markers for drug design.
Drug-speci
c modulation of signaling
pathways in speci
c metabolic alterations
could in fact predict the response to speci
c
therapy.
21
Phosphopeptides are separated by
af
nity chromatography involving antibodies
against phosphorylated domains of proteins
or by an af
is not immune from sev-
eral dif
culties, as repeatedly reported. Such
processes are all operative at the upper part of
the protein scale and consequently minor protein
components of the initial mixture persist being as
dilute as they were before the removal of high-
abundance species (frequently even more dilute,
though) and thus remain largely undetectable,
unless they are submitted to a concentration
process. Unfortunately, concentration is hard to
implement because (1) the very small sample
volume (typically 20
e
100
nity-based selective separation
method such as metal chelate chroma-
tography involving iron and gallium ions.
22
Titanium or zirconium oxide
23
is used for
phosphopeptide fractionation after having
adjusted physicochemical separation
conditions.
c)
Carbonylated proteins: These proteins
represent another interesting group of post-
translational modi
cations. Carbonylation is
L of serum or
plasma) due to the very low binding capacity
of the grafted antibody columns and (2) massive
protein losses. With immunodepletion, other
proteins are concomitantly removed,
30
probably
due to nonspeci
m
the result of modi
cation of side-chains of
some amino acids such as lysine and arginine
subsequent to either bad protein refolding,
oxidative stress, or aging.
24
Detecting the
identity of low-abundance carbonylated
proteins is an approach to study certain
diseases; this is performed by indirect af
c binding on the one hand, and
to interaction with captured proteins on the
other hand, precluding possibilities to
nity
separations. Due to their low concentration,
their identi
find low-
abundance protein markers. This phenomenon
reaches devastating proportions, as described
by Shen et al.
31
and recently con
cation is simpli
ed after
enrichment treatments.
rmed by other
authors.
32
Co-removed proteins were very
numerous to the point that for biomarker
discovery purposes, the eluted fraction from
immunosorbents was suggested to be analyzed
at the same time. In such an approach, the work-
load is doubled and the eluted fraction is so rich
in high-abundance protein that other species
may escape the detection by the so-called signal
nity-based solid phase
techniques have been used for the removal of
nucleotide-binding proteins by using PolyA
25
and PolyU
26
as immobilized ligands. Despite
numerous approaches describing the separation
of complex proteomes by groups of proteins,
these processes are mostly labor-intensive, they
Finally, special af
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