Affinity Targeting Schemes for Biomarker Research - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 201

during detection d all of which are critical in quan-
ti

biotinylated pAb* from Ag a / n before proteol-
ysis. Wash volumes of 20- to 100-column
volumes are desirable to remove nonspeci

cation of expression. The ideal would be to
quantify a feature or features in the primary struc-
ture of this protein family common to all forms of
the protein. Mixed-mode MALISA is capable of
doing such quanti

cally
bound proteins. The immune complex is then
dissociated using an acidic mobile phase (0.1 M
glycine/2% acetic acid-HCl solution; pH 2.5)
in step three, leaving pAb* still bound to the
tetrameric avidin column. With a monomeric
avidin column, both pAb* and Ag a / n are eluted
from the af

cation using the protocol out-
lined in the scheme below, the caveat being that
an antibody can be found that binds all members
of the protein family. Steps in the process are seen
in the following scheme in which pAb is a poly-
clonal antibody, Ag a / n represents multiple iso-
forms of the antigen ranging from a to n,
pAb:Ag a / n designates immune complexes of all
these isoforms, e A/G is immobilized protein A
or G, and IMER is an immobilized trypsin reactor.
With samples other than blood, the antigen family
would

nity sorbent by the acidic mobile
phase. The rest of the process is the same as
described earlier. The advantage of the
trypsin-IMER is that it provides quicker and
more reproducible proteolysis in addition to
allowing automation. 25

cation of
peptide(s) can be achieved on either a relative
or absolute basis as described earlier. Relative
differences in expression would easily be
achieved using the SILAC method or derivatiza-
tion with stable isotope coding agents such as
iTRAQ. Absolute quanti

Quanti

first be sequestered in immune complexes
using a pAb.WhenthepAb is added to a sample
solution at a m g/mL or higher this step of immune
complex formation occurs within 15 e 30 min, the
time required for immune complex formation
being related to pAb concentration. The immune
complex is then captured by a protein A/G
column in the second step of analysis, followed
by recovery of the captured antigens with an
acidic mobile phase wash in the third step. Anti-
gens Ag a / n and the capture pAb are then trypsin
digested and the peptide fragments identi

cation would be best
achieved by addition of a 13 C-labeled internal
standard, generally an isotopomer of the signa-
ture peptide(s).
Before a biomarker reaches the point of clin-
ical utility, it must undergo multiple rounds of
screening ranging from discovery to validation
and ed by finally clinical acceptance. Slightly different

analytical methods may be used in these various RPC-MS/MS.

pAb þ Ag a / n / pAb : Ag a / n ð complex formation in-solution Þ

A = G þ pAb : Ag a / n / A = G : pAb : Ag a / n ð affinity chromatography Þ

Ag a / n /

þ þ ð Þ

A = G : pAb : A = G pAb Ag a / n antigen recovery

þ ð Þ

pAb Ag a / n / IMER / RPC MS = MS proteolysis and identifcation

Antigens from blood samples in contrast are
generally selected with a biotinylated antibody
(pAb*) and an avidin/streptavidin af

stages of development. Whereas recognition of
quantitative differences between patients and
controls in discovery may be achieved with
small numbers of subjects and relative quantifi-

nity
column. This approach allows immune
complexes (pAb*:Ag a / n ) to be selected indepen-
dent of the large amount of IgG in blood
samples. Tetrameric avidin is used in cases in
which

-
cation methods such as iTRAQ labeling, valida-
tion must be carried out with large numbers of
subjects, greater statistical rigor, and absolute
quanti the objective is to separate the cation.