Affinity Targeting Schemes for Biomarker Research - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 193

hundred proteins. Mixtures of this complexity
are amenable to high levels of identi

This role will be achieved in at least three ways
( Figure 2 ), all based on stable isotope coding of
samples and internal standards. The stable
isotope labeling with amino acids in cell culture
(SILAC) approach ( Figure 2 A) is a relative quan-
ti

cation
when protein components are above the MS
detection limit. The exception might be phos-
phorylation, in which more complex mixtures
are likely.
One mode of identifying PTM-selected
proteins is by a process similar to shotgun pro-
teomics. Following trypsin digestion, peptides
are identi

cation method in which in vivo coding is
achieved with heavy versions of proteins
produced in cell cultures through growth in
media containing high levels of arginine and
lysine labeled with three 13 C atoms. Light
proteins are produced in cell cultures grown in
normal, unlabeled medium. Thus all tryptic pep-
tides in proteins derived from these 13 C-enriched
cultures should be labeled at the C-terminus and
should be at least 3 amu higher in mass than
peptide isotopomers derived from the normal
culture. When the control and experimental
cultures are grown under identical conditions,
the isotope ratio of heavy and light forms of
peptides is approximately one. Relative differ-
ences in protein concentration between proteins
in the control and experimental cultures are
directly proportional to the isotope ratio of
heavy to light forms of peptides.
A disadvantage of the SILAC method is that
many types of studies cannot be carried out in
cell cultures, particularly those involving
humans. This issue has led to in vitro coding
methods for relative quanti

ed by RPC-MS/MS. Generally, identi-

fication is based on non-PTM-bearing peptides,
the logic being that they outnumber those with
a PTM and there is a greater chance a few of
them will ionize well. These non-PTM-bearing
peptides are in turn traced to a parent protein
or family of protein isoforms. Knowing the
parent protein aids in the identi

cation of
PTM-containing peptides. Peptides with a PTM
can be identi ed in several ways. One is by look-

ing for them in the original tryptic digest of
af

nity-selected proteins. The second is by sub-
jecting peptide digests to a second round of
PTM targeted af nity selection in which PTM-

bearing species will be greatly enriched. This is
illustrated in Figure 1 by the symbol 3. The
advantage of this latter approach is that nonspe-
ci

c peptide binding is diminished by double
selection.
Identi cation of proteins in complexes is also

amenable to af

cation ( Figure 2 C).
The fact that most peptides contain primary
amines, either on lysine or at the N-terminus
that are easily derivatized, has been widely
exploited in isotope coded samples according
to origin. This can be thought of as a chemical
analog of bar coding. Reductive derivatization
with

nity selection as will be dis-
cussed more extensively later in this chapter.
Having selected one protein in a complex, other
members of the complex will be co-puri ed as

well. Finally, there is the case in which
a substrate, inhibitor, binding partner, or dye
can be identi

13 C 2 H

¼

ed that binds to a protein. O is one approach to coding.

Another is to derivatize the primary amine
with an H-hydroxysuccinamide-activated acid
that is coded with some combination of 13 C,
15 N, and 18 O atoms. Deuterium is less attractive
in coding because 2 H and 1 H isotopomers of
a peptide can be partially resolved during
reversed phase chromatography, 6 which compli-
cates quanti

Quanti cation

As we know from enzyme-linked immuno-
sorbent assay (ELISA), quanti cation is a major

objective in most af nity-targeting analyses.

Along with the use of MS to identify af

nity-
selected species, isotope ratio analysis by MS
will play a major role in the future of MALISA.

cation. Control samples can be

coded with a nonisotopically labeled