Biology Reference
In-Depth Information
high-mannose type glycans (those with terminal
α
-
D
-mannopyranoside
or
-
D
-glucopyranoside residues). Many additional lectins are
available, with glycan-specifi cities that encompass most of the typi-
cal complex-type glycans. The soybean
α
-conglycinin SSP are
known to be N-glycosylated [
66
], and at least one glycan is of the
high-mannose type which should allow use of con A in a lectin-
based affi nity-depletion step [
55
,
58
]. Using con A-affi nity chro-
matography removes all high-mannose type glycoproteins from a
complex mixture (Fig.
4c
), including low-abundance non-SSP
proteins. These can be eluted from the lectin with the hapten
α
β
-conglycinin by electro-
phoresis, digested, and analyzed by LC-MS. Immobilized lectins
can be reused multiple times, reducing the disadvantages of initial
expense and capacity (Table
1
).
Removal of supra-abundant proteins with antibodies is the
most specifi c of all of the depletion methods. The effi cacy of immu-
noremoval depends upon the avidity of the antibodies used. In
general, antibodies prepared against a native antigen are more use-
ful in immunoremoval than antibodies prepared against a dena-
tured antigen. This can be problematic with SSP, some of which
are only slightly soluble in aqueous solutions. However, at least
some of the diffi culties in antigen solubility can be overcome
through the use of synthetic peptide-antibodies [
21
] (Fig.
4d
).
There are several potential diffi culties that must be considered
before committing to an immunoremoval strategy (Table
1
) [
55
].
Antibody preparation can be expensive, and is not always effi cient.
Nonetheless, the selectivity and effi ciency of immunoremoval make
it a good fi rst choice when designing an experimental strategy.
Combinatorial-ligand random-peptide beads allow a protein
to be removed from a complex mixture, up to the point of satura-
tion of the ligand [
67
]. With suffi cient diversity, it is theoretically
possible to have an immobilized ligand complementary to each
protein in a complex mixture, ensuring that they would all be
adsorbed. When a biological sample is incubated with such a
ligand-library under capacity-restrained conditions, abundant pro-
teins will saturate all available high-affi nity ligands and the remain-
ing non-binding majority of the protein will remain in solution
[
68
]. In contrast, a low abundance protein will not saturate the
corresponding high-affi nity ligand, and most of this protein will be
removed from solution [
55
]. Based on this saturation-overload
principle, use of a combinatorial library should enrich for low-
abundance proteins relative to those of high abundance [
69
].
Elution of the entire population of proteins adsorbed to the beads
should result in a solution with a smaller dynamic range than the
starting material but still including representatives of all of the
original proteins.
The combinatorial-peptide beads, in the format of the
ProteoMiner kit [
70
], have been productively used in analyses of
-methyl-mannoside, separated from the
β
