Affinity Targeting Schemes for Biomarker Research - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 195

derivatizing reagent while experimental samples
are coded with a heavy isotope labeled version
of the derivatizing reagent. The iTRAQ (isobaric
tags for relative and absolute quanti

limit peptide sequences being used in quanti ca-

tion. Nonsignature peptides can be used in quan-
ti

cation, but only after stringent af nity

cation)
reagents, for example, exist in eight different iso-
topomers, allowing eight or more samples to be
isotopically coded and analyzed in a single
RPC-MS/MS analysis. Isotope ratios are again
used to determine relative concentration differ-
ences in proteins according to sample origin.
Coef

selection.
There is also a mass spectrometry component
to quanti cation. Isotope ratios can be deter-

mined with any mass spectrometer, either in the

first MS dimension or in the second using frag-
ment ions. It is important to note, however, that
duty cycle has a large impact on sensitivity.
Instruments that spend a longer period of time
measuring speci

cients of variation range from 5% to 8%
with in vitro isotope coding reagents.
Absolute quanti

c fragments will accumulate
more ions and be more sensitive. It is for this
reason that the triple quadrupole instruments, or
similar instruments with an extended duty cycle,
have lower limits of detection. It is also desirable
to base quanti

cation is most widely
achieved by comparing the isotope ratio of
signature peptides from a protein parent to
isotopically labeled internal standards added at
a known concentration to samples during anal-
ysis. The strategy and point of addition are
seen in Figure 2 B. Although this multiple reac-
tion monitoring (MRM) method has been used
for half a century in other

cation on area of an eluted people
as opposed to a few measurements of isotope
ratios taken during the course of analyte elution.

fields of chemistry, it
has recently come to be popular in proteomics. 7
The concept is that upon trypsin digestion,
a protein will be quantitatively converted into
a series of

AFFI NITY TARGETING METH ODS

Structural features have been targeted in
a number of different, unique ways. It is for
this reason that a list of targeting methods and
the manner in which they are used is included
in this chapter. Knowledge of targeting tools
and how to employ them is of critical importance
in designing af

limit peptides; the term “ limit

peptides means that the peptide is fully

digested to the limits possible with the digestive
enzyme. When this occurs, the concentration of
the limit peptide is stoichiometrically related to
that of its parent. But not all limit peptides are
of this type. Some limit peptides are derived
from other tryptic peptides that are only
partially digested. The quantitative relationship
between these limit peptides and their protein
parent is unknown, making them of little value
in protein quanti

”

nity methods and solving pro-
teomics problems.

Immune-Selection and Mass-Linked
Immuno-Selective Analysis (MALISA)
Immune-selection of proteins and peptides
can be achieved in at least four ways ( Figure 1 ):
two by immune complex formation in solution
and two by capture with immobilized anti-
bodies. The oldest is by immune complex forma-
tion in solutions and precipitation. Following
addition of excess antigen targeting antibody to
a sample, an immune complex is formed in solu-
tion that is subsequently precipitated by addi-
tion of a second,

cation. When the sequence of
a limit peptide appears in another peptide from
the protein parent, it cannot be used in quantifi-

-
cation. Another caveat is that the limit peptide
being used in quanti cation should be a signa-

ture of the parent protein and not part of any
other protein in the proteome of the organism
being examined. When this is not true, it is neces-
sary that protein level af

nity selection ( Figure 1 )
has eliminated all other proteins that might bear immunoglobulin G (IgG)