Oxidative stress is recognized as an important underlying factor in the
pathogenesis of many degenerative diseases as well as normal senescence. The
free radicals, reactive oxygen species (ROS) and electrophiles produced during
oxidative stress are capable of modifying nucleic acids, lipids and proteins. There
are a variety of oxidative modifications that occur to proteins including: cleavage
of the protein backbone, direct oxidation of amino acid side chains by ROS, and
adduction by electrophilic species such as lipid peroxidation products. Many of
these oxidative modifications result in the introduction of carbonyl groups into the
proteins. Protein carbonylation levels are commonly used as a biomarker to
assess the degree of oxidative damage to a system. However the most
commonly employed methods for measuring oxidative modifications to proteins,
typically fail to provide any information about the identity of the modified protein,
site of modification, or the chemical nature of the modification.
In the present study we develop an analytical technique based on affinity labeling
with N'-aminooxymethylcarbonylhydrazino-D-biotin (aldehyde reactive probe,
ARP), along with mass spectrometric analysis which allows for the full
characterization of protein carbonylation modifications. The ability of the ARP
method was first demonstrated for the case of oxylipid peptide and protein
conjugates formed by Michael addition-type conjugation reactions with α,β-
unsaturated aldehydic lipid peroxidation products with nucleophilic amino acid
residue side chains. ARP was used to label a 4-hydroxy-2-nonenal (HNE)
modified cysteine containing model peptide, and HNE modified E. coli
thioredoxin, which were characterized using ESI-MS/MS and MALDI-MS/MS.
ARP was also used to label the oxidative modifications alpha-aminoadipic
semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS), formed during
the metal catalyzed oxidation of GAPDH.
After demonstrating the utility of the technique on model systems, it was then
applied to complex biological systems. In one case, subsarcolemmal
mitochondria (SSM) isolated from rat cardiac tissue. Mitochondria are well
known to be a major source of ROS within the cell. They are therefore important
mediators of oxidative stress, as well as regulators of cell death. We were able
to identify 39 unique sites on 27 mitochondrial proteins which were modified by
six different α,β-unsaturated aldehydes, including acrolein, β-hydroxyacrolein,
crotonaldehyde, 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal and 4-oxo-2-
nonenal. Additionally we identified nine Lys residues on four mitochondrial
proteins that were oxidized to AAS and subsequently labeled with ARP. The
proteins identified with oxidative modifications include members of the
mitochondrial electron transport chain, TCA cycle, membrane transport, lipid
metabolism, and other important mitochondrial enzymes.
The ARP technique was also applied to identify protein targets of 4-hyroxy-2-
nonenal in human monocytic THP-1 cells that were exogenously exposed to
HNE. It was shown previously that exposure of THP-1 cells to HNE resulted in
apoptosis, necrosis and protein carbonylation. We applied a multi-pronged
proteomic approach involving electrophoretic, immunoblotting and mass
spectrometric analysis to unequivocally identify eighteen sites of HNE
modification on sixteen proteins. It was also demonstrated in this study that
pretreatment of THP-1 cells with ascorbic acid resulted in decreased levels of
HNE-protein conjugate formation