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Michael adducts in oxidized LDL and samples
were then delipidated and digested with trypsin
to generate proteolytic fragments and the tryptic
peptides analyzed by LC-MS/MS.
107
A diag-
nostic product ion of m/z 268 corresponding to
the histidine immonium ion modi
CONCLUSIONS
It can be concluded from the discussion in this
chapter that targeted proteomics has many
advantages relative to more global proteomic
strategies used in the identi
ed by HNE
is generated upon fragmentation of peptides
modi
cation of thousands
of proteins. One is that structure-based targeting
greatly reduces the number of proteins in
complex samples
d
generally to less than
a hundred proteins unless multiple targeting
agents are being used in tandem. Identi
ed
peptides are located on the surface of LDL mole-
cules. In addition to the HNE Michael addition
studies mentioned earlier, HNE and malondial-
dehyde (MDA) can form Schiff base adducts
with lysine. In fact, mass spectrometry easily
discriminates between these two mechanisms
of protein modi
ed with HNE. Generally, these modi
cation
and quanti
cation become much simpler as
a consequence. A second conclusion is that
most of the targeting schemes involve some
type of adsorption, which in turn allows enrich-
ment of the targeted species. Still another is that
selection and enrichment can be achieved
quickly
d
often within a few minutes, which
will be an enormous asset in clinical diagnostics.
Finally, targeting allows proteins to be selected
for identi
cation. Schiff base formation
results in the increase of the mass in peptides
by 138 amu with MDA and that from Michael
addition of HNE is 180 amu. A study with model
proteins in plasma (hemoglobin and
b
-lactoglob-
ulin) showed that the ratio of Michael adducts to
those of Schiff base formation is 99:1.
108
cation based on
their biological function. Again, this ability is of
great value in studying biological phenomena.
Banfor PN, Preusser LC, Campbell TJ, et al.
Comparative effects of levosimendan, OR-1896,
OR-1855, dobutamine, and milrinone on
vascular resistance, indexes of cardiac function,
and O2 consumption in dogs. Am J Physiol Heart
Circ Physiol 2008; 294: H238-48.
cation and quanti
ABU
NDANT PROTEIN REMOV
AL
Matrix suppression of ionization is a major
problem in proteomics, the culprit frequently
being high-abundance proteins. With blood
samples being examined by shotgun proteomics,
attempts to circumvent this problem have
focused on the use of commercial immunosor-
bents to remove the 10 to 20 most abundant
proteins. Although effective, many low-
abundance proteins are partially or totally
selected as well. At least 129 proteins have
been reported to have been captured during
abundant protein removal.
109
This phenomenon
is the result of what is sometimes referred to as
the abundant protein
References
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d
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intermolecular complexes are an issue as well.
Af
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'
nity selection of any member of a complex
captures all the members of the complex. A
concern in both of these cases is that proteins
of interest will be removed.
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