Biology Reference
In-Depth Information
directly and are trapped at the beginning RP
section of the biphasic column for separation.
This iterative process is repeated multiple times
using increasing salt gradients. An advantage of
this separation technology is that the entire
system is coupled directly online with the
mass spectrometer enabling a large number of
peptides to be directly identi
the column and the protein(s) of
interest
is
captured by the af
nant. Proteins that do not
possess a complementary binding site for the
bound ligand will either pass directly through
the column or be eluted by a low-stringency
washing step. The bound proteins are recovered
by washing the column with a competitive
substrate or a solution that disrupts the interac-
tion between the protein and the af
ed in a high-
throughput manner. Other chromatographic
modes have been used for peptides, proteins,
and metabolites
nant (e.g.,
denaturants). Although an antibody is directed
to a speci
fractionation, as given in
nity
methods have been developed to capture a class
of proteins. These methods include immobilized
metal af
c protein, many other af
Table 2
.
Although most fractionation techniques have
focused on the development of methods to frac-
tionate the entire proteome samples, methods
have also been developed to interrogate a speci
nity columns containing nickel to
capture histidine-containing peptides,
5,6
gallium
to isolate phosphopeptides,
7
c
subset of compounds. For example, Regnier and
coworkers
3
used lectin af
or titanium di-
for phosphopeptides.
8
oxides
In addition,
nity chromatography
to select glycosylated peptides, signi
af
nity methods have been developed to select
peptides containing speci
cantly
reducing the complexity of the peptide mixture
prior to MS analysis. Dr. Xia Xu et al.
4
have
developed liquid
c types of residues
such as cysteine, tryptophan, or methionine.
9,10
There are a variety of different lectins that have
been used to selectively separate glycoproteins
based on the composition of the carbohydrate
side-chain.
11
A method was also developed
that utilizes phosphoprotein isotope af
liquid extraction and MS
methods for analyzing 15 different estrogen
metabolites in bio
e
uid samples such as serum
and urine.
nity
tags, combining stable isotope and biotin
labeling for proteome-wide af
nity separation
and quantitation of phosphoproteins.
12
AF
FINITY CHROMATOGRAP
HY
nity chromatography is primarily used to
capture a speci
Af
ISOELECTRIC FOCUSING
c protein or a class of proteins
within complex mixtures. The principle of
af
nity chromatography is based on the ability
of a biologically active molecule to bind specifi-
The principle of isoelectric focusing (IEF) is
simple to understand and perform using either
gel or liquid phase medium. The protein sample
is mixed with the desired pH range carrier
ampholyte mixture, or other carrier buffer, in
a focusing cell or spotted on an SDS-PAGE plate.
If an electric potential is applied, the proteins
migrate to a position in the established pH
gradient equivalent to their respective isoelectric
point (pI). If a protein diffuses away from this
pH region, its net charge will change and the
resulting electrophoretic forces will in
-
cally and reversibly to a complementary mole-
cule, which is often bound to a solid support.
These ligand molecules may include antibodies,
metals, lectins, biotin, aptamers, and other mole-
cules. The binding sites of the immobilized
substances must be sterically accessible after
their coupling to the solid support and should
not be deformed by immobilization. In the case
of speci
nant is attached to
the active surface of the column packing material
or column surface. The sample is injected onto
c proteins, an af
uence its
migration back to its pI point. The net result is
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