Biology Reference
In-Depth Information
10-fold puri
cation in a single step. It also
means that million-component mixtures require
many levels of fractionation to purify an analyte.
The unique difference in af
a disease-associated SAAP. Individuals with an
SNP, causing isoleucine to be substituted for
methionine-26, are more likely to get Parkinson
s
disease,
3
the result of DJ-1 failing to act as a chap-
erone and protect
'
nity selection
methods is that the feature being targeted is
structure. Structural features are much more
speci
a
-synuclein from disease-
associated
post-translational modi
cations.
c than general features. Achieving a 10
3
-
fold puri
These modi
cations in
a
-synuclein structure in
turn lead to its participation in the formation of
Lewy bodies.
Splice variant biomarkers are a similar
example. Subsequent
cation in a single step is frequently
possible with af
nity selection methods, which
allows preliminary fractionation to be achieved
in fewer steps and with a better idea of what
has been selected.
nity selection of
a known protein family, it is easy to search for
splice variant sites that change in concentration
with disease progression. CD-44 is a good
example; splicing variants of this protein have
been associated with cancer through modern
proteomics methods.
4
The fact that the splice
variant tryptic peptide is glycosylated is fortu-
itous in its selection. Biomarkers can also be sug-
gested by pathways known to be associated with
a biological phenomenon. Proteins associated
with the BRCA-1 mutation and linked pathways
in breast cancer are examples.
5
Again antibodies
against proteins in a disease pathway can be
used
to af
Prerequisites
Structure-based selection means that one
must have two things: (1) some idea of the struc-
ture being targeted and (2) a structure-speci
c
selector, which is why af
nity is not as widely
used in discovery. Af
nity methods are of
much greater utility in veri
cation, validation,
and routine analyses in which the structure of
potential biomarkers has been elucidated in
discovery.
But there are still cases in which af
nity selec-
tion can be used in discovery, such as when
some element or feature of the biomarker struc-
ture is known. A disease-associated single-
nucleotide polymorphism (SNP) is such a case.
The SNP predicts where in the primary structure
of a protein one should
to
recognize
associated
differential
expression.
Another approach is to select classes of
proteins based on a speci
c post-translational
modi
cation (PTM) associated with a disease
or phenomenon. It is frequently the case that
modi
find a single amino acid
polymorphism (SAAP).
2
The question is whether
the SAAP variant is expressed. Antibodies
against the protein are easily produced, all forms
of
cations ranging from glycosylation and
phosphorylation to lysine acetylation, lysine
methylation, arginine methylation, amino acid
oxidation, carbonylation, and ubiquitination
have been reported to change during disease
progression without knowing the protein to
which the PTM is connected. Based on the avail-
ability of a variety of af
the protein immune-selected,
the SAAP-
bearing peptide identi
ed by MS if present,
and the absolute amount of the mutated protein
determined. The only problem would be when
the SAAP-bearing peptide is not produced by
proteolysis or does not ionize in the MS. Protein
level identi
nity selectors ranging
from antibodies, lectins, and binding protein to
aptamers and immobilized metal af
nity chro-
matography, it is possible to isolate proteins
and peptides bearing speci
cation would be the best choice in
this case but would require the MS to have suffi-
-
cient resolution to differentiate between the
native and SAAP-bearing proteins. The M26I
variant of the protein DJ-1 is an example of
c PTMs, as seen in
Figure 1
. Subsequent to selecting PTM-bearing
proteins from a proteome, the selected fraction
generally contains no more than 50 to a few
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