Affinity Targeting Schemes for Biomarker Research - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 198

approach is used in SISCAPA multiplexing. All
the antibodies can be immobilized together or
on individual sets of particles that are then
mixed.

selected in a second step by calmodulin, after
which elution is accomplished by addition of
Ca(II). The TAP method allows rapid puri

ca-
tion of complexes without prior knowledge of
composition, activity, or function of the indi-
vidual components. Combined with MS, the
TAP strategy allows for the identi

Binding Proteins
Perhaps 40% or more of the proteins in cells
are involved in some type of intermolecular
complex that plays a role in either creating a func-
tional supramolecular complex that regulates
a pathway, controls transcription, directs transla-
tion, enhances transport, acts as a chaperone, or
selects species for degradation. These binding
proteins can often be used in the selection of
a partner with which they co-reside. Af

cation of
proteins interacting with any target protein.
Although conceptually elegant, the TAP
method appears to have some limitations, the
most serious being that different laboratories
get quite different results using what seems
to be the same or very similar methods. A
further limitation common to all the tagging
methods is that the generation of fusion
proteins often does not occur under natural
conditions. This means that the requisite
enzymes necessary for PTMs that lead to
complex formation may not be present in the
biological system being used.

nity
selection can be achieved in several ways. One
is by using a polyclonal antibody (pAb) to target
one of the proteins in the complex, generally in
a pull-down scheme as discussed previously.
Using a pAb has the advantage that there is no
need to know the portion(s) of the protein surface
that are interfaced with partners and unavailable
for binding. A second approach is to add one or
more af

Avidin/Streptavidin
Avidin and streptavidin are both homotetra-
mers of about 68 and about 60 kD, respectively,
with biotin dissociation constants in the range of
10 -15 . Avidin is glycosylated while streptavidin
is not. Deglycosylated avidin is available commer-
cially, sometimes known as NeutrAvidin

nity selectable tags to a protein in the
complex, generally through a plasmid or gene
manipulation ( Figure 4 ).
One of the more widely used approaches is
the tandem af cation (TAP) method 14

illustrated in Figure 4 . This method generally
involves the fusion of calmodulin binding
peptide to the C-terminus of a protein along
with the IgG binding domain from protein A.
It is important that (1) the protein A tag be
attached farthest away, at the end of the fusion
protein, so that it is easily available for binding
by the IgG-Fc column and (2) elution can be
achieved under very gentle conditions that will
not dissociate the complex. This

nity puri .The

pI for natural streptavidin is about 5.0, whereas
that of some recombinant forms is nearly neutral.
The pI of avidin, in contrast, is 10.5. This point is
important because the more nearly neutral pI of
streptavidin tends to give lower nonspeci c

binding in chromatographic applications. The
fact that avidin and streptavidin have such enor-
mous af nity for biotin has been widely exploited

in protein analysis, primarily in af

nity chroma-
tography but also as a means to immobilize bio-
tinylated species. Based on the fact that biotin is
a small molecule to which a variety of functional
groups can be attached means it can be used in
a broad spectrum of tagging reactions. Reagents
for the covalent coupling of biotin to a primary
amine, sulfhydryl, carboxyl, or carbonyl group

result is

achieved in the nity capture

of the complex on an IgG-Fc column. Complex
release is achieved by proteolysis of the linkage
between the protein A binding domain and the
calmodulin binding peptide with tobacco etch
virus protease. The complex is again af

first step after af

nity