Biology Reference
In-Depth Information
Glycan-Targeting Antibodies
Antibodies have been widely exploited by
pathologists
52
to differentiate between normal
and malignant cells in targeting aberrant glyco-
sylation.
53
Combinations of Lewis x (Le
x
),
sialyl-Lewis x (s-Le
x
), sulfosialyl-Lewis x (Ss-
Le
x
), and sialyl-Lewis a (s-Le
a
) antigens (
Figure 5
)
along with
b
-(1,6)-branching, sialylation, and
fucosylation of glycans
54
are elevated in N- and
O-linked glycoproteins on the surface of malig-
nant cells and in the blood of cancer
patients.
41,55,56
Cancer-associated glycoproteins
carrying these glycans have been reported to be
shed into blood and lymph.
57,58
These tumor-
associated glycans have also been connected to
tumor grade, metastasis, and poor prognosis.
59
Sialyl-Lewis x (s-Le
x
)-bearing species have
been selected from plasma with the monoclonal
antibody CHO-131. This antibody selects s-Le
x
antigen at points of branching in which the
GlcNAc residue of s-Le
x
is coupled in a
b
-(1,6)
linkage.
60
At N-linked glycosylation sites, s-Le
x
is conjugated to the glycan matrix through a
-GlcNAc-
b
-(1,6)-Man- linkage and GlcNAc of
s-Le
x
is conjugated in a -GlcNAc-
b
-(1,6)-GalNAc-
linkage to core 2
b
-(1,6)-O-glycan structures.
CHO-131 has been used in af
to sample origin and their relative concentra-
tions compared by isotope ratio measurements
during mass spectral analysis. The Le
x
glycotype
of s-Le
x
-bearing glycoproteins has been found to
be elevated in breast cancer patients.
62
Although the proteins bearing Lewis antigens
have been most widely examined by immunoaf-
finity chromatography, others will follow. It has
been determined that glycans with branched oli-
gomannose side chains are immunogenic. As
antibodies targeting these mannose rich glycans
become available, they are likely to be used in
glycoproteomics. Proteins that bear an advanced
glycation end-product (AGE) are immunogenic
as well. Increasing evidence points to the
adverse effects of AGE proteins in cataract
formation, Alzheimer
s disease, osteoarthritis,
myocardial dysfunction, diabetes, and other
aging-related diseases. AGE-modi
'
ed proteins
result from an initial non-enzymatic glycation
of free protein amino groups on proteins via
a Maillard reaction. Subsequent to Amadori
rearrangements a stable product is formed that
over the course of time can be oxidized by
radical oxygen species to a series of carbonyl-
containing AGE species. With appropriate anti-
bodies, the proteins conjugated to the AGE can
be isolated and identi
nity chromatog-
raphy systems to probe the possibility that
glycoproteins bearing such s-Le
x
antigens are
released into plasma. Subsequent to capture by
the CHO-131 immunosorbent, proteins are de-
sorbed with an acidic mobile phase, then tryptic
digested, and the peptide cleavage fragments
further fractionated by RPC before identi
ed.
Lectins
Lectins have played a major role in recog-
nizing that aberrations in glycosylation are asso-
ciated with a variety of diseases,
rst as
histochemical staining agents 50 years ago and
more recently as af
cation
by tandem mass spectrometry. This particular
analytical strategy identi
nity chromatography selec-
tors of glycans.
63,64
The fact that large numbers
of glycan types can be captured with lectins
makes them extremely value in the isolation,
identi
es glycoproteins based
on nonglycosylated peptides instead of glyco-
peptides.
61
This protocol can be used to capture
and identify both O- and N-glycosylated
proteins carrying s-Le
x
antigen without deglyco-
sylation of peptides. Through derivatization
with a stable isotope labeled coding agent such
as iTRAQ, peptides in tryptic digests of af
cation of glycoprotein
biomarkers.
65
Lectins have many advantages,
among the most useful being that they are abun-
dant, vary widely in selectivity (
Table 1
), provide
af
cation, and quanti
nity
captured samples were isotope coded according
nity-selected glycoproteins and glycopep-
tides for characterization and quanti
cation,
66,67
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