Affinity Targeting Schemes for Biomarker Research - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 211

targetable with antibodies. N-methyllysine 95,96 -
targeting antibodies are diverse in being able to
target the various unique types of lysine methyla-
tion, a PTM and the surrounding amino acid
sequence, or a group of lysine modi

Proteins are oxidized in multiple ways.
Although reversible in many cases, carbonyla-
tion is not. Protein carbonylation is a nonenzy-
matic PTM that often leads to loss of protein
function. It occurs most frequently in one of
three ways: (1) by direct oxidation of amino
acid side chains or the polypeptide backbone of
a protein, (2) through formation of adducts
with lipid peroxidation end products, and (3)
by oxidative cleavage of advanced glycation
end products. It is possible that all of these forms
of carbonylation can occur within the same
protein and even at the same site. A general
property of in vivo carbonylation is that it occurs
on hundreds of protein species within a cell and
generally at a small number of speci

cations. The

type of modi cation can even affect protein func-

tion in the case ofmono-, di-, and trimethylation of
the same lysine residue. 97,98 For example, di- and
tri- but notmonomethylation on histoneH3 lysine
36 marks active transcription of

the genes

involved in flowering time regulation and other

processes in Arabidopsis thaliana. Being able to
select proteins bearing a particular PTM or a
PTM on a particular sequence is an enormous
asset indetermining the biological role of the PTM.
Methylation on arginine is equally diverse and
antibody selectable. Using antibodies that target
lysine methylated residues, 59 methylation sites
have been identi

c sites. At

least 35 different types of modi cations have

been observed in protein carbonylation.
Carbonylated proteins have been selected in
multiple ways, the most common involving deri-
vation of the carbonyl groups with either biotin
or dinitrophenyl hydrazine. One of the higher
resolution strategies is fractionation of the
selected protein before proteolysis, 106 as illus-
trated in Figure 8 . Subsequent to biotinylation
with biotin hydrazide and reduction with
sodium cyanoborohydride biotinylated, proteins
were selected by mono-avidin af

ed in proteins by RPC-MS/
MS. Arginine methylation in contrast to lysine
methylation results in the formation of either
asymmetric or symmetrical dimethylation of
arginine residues located in RG-rich clusters.
Dimethyl-arginine speci c antibodies have been

used in the identi cation of approximately 200

proteins that are putatively arginine-methyl-
ated. 99 Major protein complexes identi ed in this

way include components required for pre-
mRNA splicing, polyadenylation, transcription,
signal transduction, and cytoskeleton and DNA
repair.

nity chroma-

tography. Protein identi cation was achieved

through unmodi ed peptides. Oxidation sites

were identi ed by the presence of biotinylation.

Oxidation of lipids can result in the genera-
tion of reactive degradation products such as
4-hydroxynonenal (HNE) and malondialde-
hyde. These compounds frequently react with
proteins through either Michael addition or
Schiff base formation. For example, apolipopro-
tein B-100 (Apo B-100) is a single low-density
lipoprotein (LDL) that solubilizes fatty acids by
adsorption. Oxidation of Apo B-100 makes it
susceptible to uptake and accumulation in
receptor cells, leading to formation of atheroscle-
rotic plaques inside blood vessels. To study
advanced lipid peroxidation adducts of this
molecule, NaBH 4 was used to stabilize the

Oxidation
As part of normal metabolism, reactive oxygen
species (ROS) are generated widely in cells. ROS
are also derived from the environment. At high
concentrations of ROS cells, oxidative stress
(OS) becomes a serious problem. Excessive OS
leads to many forms of damage in cells, one being
protein oxidation. Pathological levels of OS have
been implicated in a plethora of diseases ranging
from diabetes mellitus 100 and neurodegenerative
diseases 101

ammatory diseases, 102 athero-
sclerosis, 103 cancer, 104 and even aging. 105

to in