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In-Depth Information
in glycosylation.
37,38
One of the problems in
studying these diseases is in recognizing which
glycoform on a speci
There is also the issue in validation of how to
screen anywhere from ten to several hundred
candidate biomarkers simultaneously. Analysis
of multiple biomarker candidates is done the
same way as individual proteins except with
multiple antibodies. Many MS instruments
now allow peptides from 10 to 100 proteins to
be identi
c protein is associated
with the disease. With cancer, the associated
structural changes can alter the antigenic and
adhesive properties of tumor cells,
39,40
their
capacity to metastasize,
41,42
and their potential
for secondary tumor colonization.
43,44
This associ-
ation couples structural changes to the disease.
Important aspects of glycoprotein biomarkers
are that (1) glycosylation is a PTM, (2) genes not
associated with expression of the protein are
responsible for the aberrations, (3) a speci
ed and quanti
ed in a single analysis
using MRM methods.
POST-TRANSLATIONAL
MODIFICATIONS
c
site (perhaps among several) in the glycoprotein
is involved, (4) aberrations in glycosylation
occur on speci
Among the many types of PTMs, only a few
have currently been targeted by af
nity selec-
tors. Glycosylation is one of the most easily
selected modi
c proteins, and (5) a small
number of pathways are affected. It is critical
to reiterate that very speci
cations based on the availability
of a broad range of lectins that target a wide
variety of glycan features. A much smaller
subset of glycans can be selected by anti-
bodies, the most common being Lewis
antigens. The same is true with phosphoryla-
tion; although tyrosine phosphorylation can
be targeted with a monoclonal antibody,
immune-speci
c modi
cations occur
at speci
c proteins. The great
strength of glycoproteomics over glycomics is
that all this speci
c sites on speci
city is lost when glycans are
removed from their protein parent in glycomic
types of analysis. Many of these aberrations
and ways to target them are known, although
the proteins involved have not been identi
ed.
c targeting of other forms of
phosphorylation is poor. Methylation and
acetylation in contrast seem to be well targeted
by antibodies.
It is for these reasons that af
nity selection of
glycoproteins based on the presence of a speci
c
PTM has been widely exploited in identifying
glycoprotein biomarkers. The value of anti-
bodies
45
and lectins
46,47
nity selecting
glycoproteins has been well established. Both
have been shown to reduce the complexity of
blood samples suf
in af
Glycosylation
Among the several hundred types of PTMs,
glycosylation is the most common. It is for this
reason that the development of proteomics
methods for the study of PTMs is becoming
increasingly important.
26
Glycosylation of the
N-type on asparagines and O-type on serine
or threonine are thought to occur in over 50% of
all proteins.
27
Alzheimer
ciently that glycoproteins
can be identi
ed by shotgun proteomics without
abundant protein removal.
4,48,49
Binding sites
are identi
nity
selected glycopeptides with PNGase F in the
case of N-glycosylation and RPC-MS/MS.
50,51
Asparagine in the N-X-S/T sequon is converted
to aspartic acid subsequent to treatment with
PNGase F. PNGase F hydrolysis can also be
carried out in H
18
O, causing the mass of aspartic
acid to increase by 4 amu. There is no universal
enzymatic method for deglycosylating O-linked
proteins.
ed by deglycosylation of the af
s disease,
28
certain types
'
of heart disease,
29
respiratory illnesses,
30
dia-
betes,
31
stress,
32
some autoimmune diseases,
33
fibrosis,34
34
some renal function diseases,
35
arthritis,
36
cancer,
6
and cellular adhesion
e
related
diseases have all been associatedwith aberrations
cystic
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