Proteins modified by the lipid peroxidation aldehyde 9,12-dioxo-10(E)- dodecenoic acid in MCF7 breast cancer cells

Article abstract of DOI:10.1021/tx9002808

The hydroperoxide of linoleic acid (13-HPODE) degrades to 9,12-dioxo-10(E)-dodecenoic acid (DODE), which readily modifies proteins. This study identified the major proteins in MCF7 cells modified by DODE. To reduce false positives, three methods were used to identify DODE-modified proteins. First, cells were treated with a synthetically biotinylated 13-HPODE (13-HPODE-biotin). Modified proteins were enriched by neutravidin affinity and identified by two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS). Second, cells were treated with native 13-HPODE. Protein carbonyls were biotinylated with an aldehyde reactive probe, and modified proteins were enriched by neutravidin affinity and identified by 2D LC-MS/MS. Third, using a newly developed DODE antibody, DODEmodified proteins were located by 2D sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot and identified by in-gel digestion and LC-MS/MS. Analysis of the proteins characterized by all three methods revealed a significant overlap and identified 32 primary proteins modified by DODE in MCF7 cells. These results demonstrated the feasibility for the cellular formation of DODE protein-carbonyl adducts that may be future indicators of oxidative stress.

Full text of DOI:10.1021/tx9002808

Chem. Res. Toxicol. 2010, 23, 557–567
557
Proteins Modified by the Lipid Peroxidation Aldehyde
,12-Dioxo-10(E)-dodecenoic Acid in MCF7 Breast Cancer Cells
9
†
†
†
†
†
Peter G. Slade, Michelle V. Williams, Viral Brahmbhatt, Ajit Dash, John S. Wishnok,
,
†,‡
and Steven R. Tannenbaum*
Departments of Biological Engineering and Chemistry, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139
ReceiVed August 14, 2009
The hydroperoxide of linoleic acid (13-HPODE) degrades to 9,12-dioxo-10(E)-dodecenoic acid (DODE),
which readily modifies proteins. This study identified the major proteins in MCF7 cells modified by
DODE. To reduce false positives, three methods were used to identify DODE-modified proteins. First,
cells were treated with a synthetically biotinylated 13-HPODE (13-HPODE-biotin). Modified proteins
were enriched by neutravidin affinity and identified by two-dimensional liquid chromatography-tandem
mass spectrometry (2D LC-MS/MS). Second, cells were treated with native 13-HPODE. Protein carbonyls
were biotinylated with an aldehyde reactive probe, and modified proteins were enriched by neutravidin
affinity and identified by 2D LC-MS/MS. Third, using a newly developed DODE antibody, DODE-
modified proteins were located by 2D sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
Western blot and identified by in-gel digestion and LC-MS/MS. Analysis of the proteins characterized
by all three methods revealed a significant overlap and identified 32 primary proteins modified by DODE
in MCF7 cells. These results demonstrated the feasibility for the cellular formation of DODE
protein-carbonyl adducts that may be future indicators of oxidative stress.
Introduction
been shown to be a breakdown product of 9-hydroperoxy-10,12-
octadecadienoic acid in lentil seeds (4). The oxidation of
R-linolenic acid by lipoxygenase has been shown to produce
the 13-OOH of this PUFA (13), and the possibility exists for
this hydroperoxide to also degrade to DODE, but that has yet
to be studied. These R,ꢀ-unsaturated aldehydes have been shown
to covalently modify DNA (14, 15), cellular glutathione (16, 17),
and proteins (18–22). Protein modification occurs through
conjugate addition (often Michael addition) to nucleophilic
amino acid side chains (Lys, Cys, and His), and HNE, ONE,
and DODE also form carbonyl-containing adducts and EDE a
pyrrole adduct (Scheme 1B). Of the R,ꢀ-unsaturated aldehydes
shown in Scheme 1A, only DODE is exclusive to linoleic acid
decomposition, retaining the carboxylic acid functional group.
We have recently shown that DODE is the most reactive of the
protein-modifying carbonyls occurring from 13-HPODE de-
composition, and only DODE modifications were observed when
cytochrome c (cyt c) was incubated with its precursor 13-
HPODE (22). Therefore, because of DODE’s reactivity and
uniqueness to linoleic acid, the focus of this study was to identify
the major cellular proteins modified by DODE in MCF7 breast
cancer cells and demonstrate feasibility for the cellular formation
of these potential indicators of oxidative stress.
Cellular oxidative stress can result in the damage of biom-
acromolecules such as proteins, DNA and polyunsaturated fatty
acids (PUFAs). The oxidation of PUFAs results in the formation
of a variety of lipid hydroperoxides occurring from reactive
oxygen species (1) or the enzymatic action of cyclooxygenases
(
2) and lipoxygenases (3). These hydroperoxides are prone to
decomposition into R,ꢀ-unsaturated aldehydes through multiple
pathways (4–10). Our previous studies have demonstrated that
lipid peroxidation chemistry dominates DNA adduct formation
in a mouse inflammation model (11). It therefore seemed likely
that lipid-derived carbonyl protein adducts are also a dominant
chemistry of inflammation and would make potential biomarkers
of inflammation and cellular oxidative stress.
Linoleic acid is the most abundant PUFA found in mammals
and its lipid peroxidation products likely account for the majority
of lipid-derived protein carbonyls (12). The lipid hydroperoxide
formed from the oxidation of linoleic acid, 13-hydroperoxy-
,11(Z,E)-octadecadienoic acid (13-HPODE), reductively de-
grades to 4-hydroperoxy-2(E)-nonenal (HPNE) and 4,5-epoxy-
(E)-decenal (EDE). HPNE undergoes dehydration or reduction
to form the unsaturated aldehydes 4-oxo-2(E)-nonenal (ONE)
and 4-hydroxy-2(E)-nonenal (HNE), respectively (Scheme 1A).
The formation of the unsaturated aldehyde, 9,12-dioxo-10(E)-
dodecenoic acid (DODE), is derived directly from the linoleic
acid hydroperoxide (13-HPODE) degradation. DODE has also
1
9
2
To reduce the number of false positives and to identify only
DODE-modified proteins, three independent methods were used
for modified protein identification. First, MCF7 cells were
treated with a biotin-linked 13-HPODE (Scheme 3). The biotin
group was coordinated to the carboxylic acid of the hydroper-
oxide. Because DODE is the only reactive carbonyl to retain
the acid functional group, only proteins modified by DODE
would be biotinylated using this tag. Modified proteins were
avidin affinity purified and identified by two-dimensional liquid
chromatography-tandem mass spectrometry (2D LC-MS/MS).
Second, MCF7 cells were treated with standard 13-HPODE.
*
To whom correspondence should be addressed. Tel: 617- 253-3729.
E-mail: srt@mit.edu.
†
Department of Biological Engineering.
Department of Chemistry.
Abbreviations: 13-HPODE, 13-hydroperoxy-9,11(Z,E)-octadecadienoic
‡
1
acid; ARP, aldehyde reactive probe (aminooxymethylcarbonylhydrazino
D-biotin); cyt c, cytochrome c; DODE, 9,12-dioxo-10(E)-dodecenoic acid;
EDE, 4,5-epoxy-2(E)-decenal; HNE, 4-hydroxy-2(E)-nonenal; ONE, 4-oxo-
2
(E)-nonenal.
1
0.1021/tx9002808  2010 American Chemical Society
Published on Web 02/04/2010

5
58 Chem. Res. Toxicol., Vol. 23, No. 3, 2010
Scheme 1. Decomposition of 13-HPODE
Slade et al.
Scheme 2. Reaction of ARP with Carbonyls
Scheme 3. Biotin-Linked 13-HPODE
Proteins were separated by 2D chromatography, and DODE-
modified proteins were located by Western blot using a newly
developed polyclonal DODE antibody. These proteins were
identified by in-gel digestion and LC-MS/MS. Third, proteins
from 13-HPODE-treated cells were reacted with an aldehyde
reactive probe (ARP). ARP is a biotinylated hydroxylamine that
reacts preferentially with aldehyde/keto groups (23), and Maier
et al. have demonstrated the usefulness of this reagent to label
and affinity purify carbonyl-modified proteins from rat heart
mitochondria (24) and HNE-modified thioredoxin (25). The
hydroxylamine of ARP forms either an aldoxime or a ketoxime
with the aldehyde or keto functionality of the lipid-derived
carbonyl adduct as shown in Scheme 2. Carbonyl-containing
proteins from the MCF7 cells were derivatized with ARP and
selectively enriched via the biotin side chain. Modified proteins
were identified by 2D LC-MS/MS.
Comparison of the proteins identified from the three tech-
niques resulted in the identification of a total of 32 major DODE-
modified proteins. These proteins were classified into five major
functional groups: structural, regulatory, molecular chaperones,
glycolysis, and nucleic acid binding. The protein location was
mainly cytoplasmic, although a number of membrane-associated
and nuclear proteins were also identified. These results char-
acterized the major proteins modified by DODE in MCF7 breast
cancer cells and introduced DODE-modified proteins as possible
target biomarkers for oxidative stress. This study verified biotin
labeling, affinity chromatography, and 2D LC-MS/MS as a
successful method to identify DODE-modified proteins from a
complex biological system.
Materials and Methods
Materials. ARP was purchased from Invitrogen (Eugene, OR),
and biotin-PEO-LC-amine was purchased from Pierce (Rockford,
IL). Diisopropylethylamine (DIPEA), dimethylformamide (DMF),
2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexaflu-
orophosphate methanaminium (HATU), lipoxidase, cyt c (equine
heart), ammonium bicarbonate, acetic acid, trifluoroacetic acid, and
ascorbic acid were purchased from Sigma Chemical Co. (St. Louis,
MO). Trypsin was purchased from Promega (Madison, WI).
Linoleic acid was purchased from Cayman Chemical. Gases were
supplied by AirGas (Salem, NH). DODE was a generous gift from
Prof. Ian A. Blair (University of Pennsylvania).
Synthesis of 13-HPODE. 13-HPODE was synthesized by the
enzymatic oxidation of linoleic acid with soybean lipoxidase (26).
Briefly, 50 mg of linoleic acid was oxidized by the addition of
6
00000 units of soybean lipoxidase in 50 mL of 0.2 M borate buffer,
pH 9. The reaction was stirred on ice for 1.5 h under O . The
solution was acidified with concentrated HCl to pH 3, and
3-HPODE was extracted with 2 × 25 mL of ether. Ether
extractions were washed with 50 mL of deionized H O, dried with
2
1
2
sodium sulfate, and filtered. Ether was evaporated, and the
13-HPODE oil residue was dissolved in a minimal amount of
hexane. 13-HPODE was further purified by flash chromatography

Proteins Modified by DODE in MCF7 Breast Cancer Cells
Chem. Res. Toxicol., Vol. 23, No. 3, 2010 559
using a 15 cm × 2 cm packed silica gel column with a mobile
plate, followed by incubation for 0.5 h at 37 °C. The medium was
then removed, and the cells were washed with PBS. MCF7 growth
medium (10 mL, containing 1.0 mM ascorbic acid) was added, and
the plates were incubated at 37 °C for 4 h. After incubation, the
medium was removed, and the cells were washed with PBS. Cells
were lysed with 0.5 mL of nondenaturing lysis buffer (Sigma,
CelLytic M Cell Lysis Reagent) containing a cocktail of protease
inhibitors: AEBSF (1 mM), E-64 (10 µM), pepstatin A (1.4 µM),
EDTA (1 mM), and bestatin (40 µM). Lysed cells were scraped
from the plates and transferred to 1.5 mL Eppendorf tubes and
aspirated through a 20-gauge needle eight times to ensure complete
cell lysis. Cell debris was pelleted, and the supernatants were then
removed, combined, and dialyzed (Pierce Slide-A-Lysed 3000
MWCO) into PBS for 48 h at 4 °C. Control MCF7 cells were not
treated with 13-HPODE-containing media. To 40 plates of cells
(90% confluence), 10 mL of growth media was added (final
concentration, 1.0 mM). Plates were incubated at 37 °C for 4 h.
Control cells were lysed and treated as described above.
Immunostaining and Imaging of MCF7 Cells Treated with
13-HPODE-Biotin. Cells treated with 13-HPODE-biotin and
untreated controls were cultured as described above, after which
medium was aspirated and the cells were washed with sterile PBS
(three times). The cultures were then incubated with Cy3-conjugated
streptavidin (Invitrogen, catalog no. 43-4315; 1:100 dilution) for
an hour, and Hoechst 33342 (Invitrogen, catalog no. H-3570; 1:5000
dilution) was added into the medium for 30 s before it was washed
with PBS (three times). Imaging was done with an epifluorescence
microscope (Zeiss Axiovert-100) using Chroma Set CZ 915 and
CZ 902 filters for Cy-3 and Hoechst, respectively.
Neutravidin Affinity Chromatography of Cell Lysates. Im-
mobilized Neutravidin (Pierce) was packed into a column with the
following dimensions: ID, 9 mm; OD, 7 mm; and height, 60 mm.
The final column volume was 2 mL. All buffers and samples were
allowed to warm to room temperature. The column was washed
with 4 column volumes of PBS. A protease inhibitor cocktail was
added to protein samples: AEBSF (1 mM), E-64 (10 µM), pepstatin
A (1.4 µM), EDTA (1 mM), and bestatin (40 µM). Samples (in
PBS) were added directly to the neutravidin column. To remove
nonbiotinylated proteins, the column was washed with 10 column
volumes of PBS-2% 3-[(3-cholamidopropyl)dimethylammo-
nio]propanesulfonic acid (CHAPS), 5 column volumes of PBS, and
5 column volumes of deionized water. Wash fractions were
analyzed by UV-vis spectrometry for baseline absorbance (280
nm). Bound proteins were eluted by the addition of 4 column
volumes of 0.4% TFA/80% acetonitrile. Elution fractions were
combined, frozen, lyophilized, and stored at -20 °C.
Gel Electrophoresis for Biotinylated and Nonbiotinylated
Proteins. One hundred micrograms (Coomassie) or 30 µg (Western)
of sample was processed by 2D gel electrophoresis for purposes
of protein identification/Western blotting. For antibiotin Western
blotting and Coomassie, proteins were focused on 11 cm 4-7
immobilized pH gradient (IPG) strips (Immobiline DryStrip gels,
Amersham Biosciences) using an IPGphor focusing apparatus
phase of 70% hexane and 30% ethyl acetate at a flow rate of about
2
mL/min. The product was eluted in 5 mL fractions around
fractions 6-10. The purity of 13-HPODE was checked by normal
phase HPLC-DAD using a Regis Rexchrome silica column (25 cm
×
100 mm, 5 µm) and an isocratic mobile phase of 98.5% hexane,
1
.5% isopropanol, and 0.1% acetic acid at a flow rate 3 mL/min.
The final purified 13-HPODE was dried, redissolved in ethanol to
give a 20 mg/mL or 65 mM concentration (ε234nm ) 25600 M-
1
-
1
cm ), and stored at -80 °C.
Synthesis of 13-HPODE-Biotin. 13-HPODE was synthesized
and purified as described above. To allow for complete coupling
to the biotin, a small excess of 13-HPODE was used to give a molar
ratio of 1.1:1 for 13-HPODE:biotin-PEO-LC-amine. 13-HPODE
(
1
6.5 µmol; 20 mg/mL) and 6.5 µmol of HATU were dissolved in
00 µL of DMF. Six micromoles of biotin-PEO-LC-amine, dis-
solved in DMF, was added along with 32.5 µmol (5 × molar excess)
of DIPEA to give a final reaction volume of ∼200 µL. The reaction
was stirred for 1 h at room temperature, dried, and redissolved in
1
00% ethanol. The purity of 13-HPODE-biotin was checked by
reverse phase HPLC-ESI-TOF-MS using a Microsorb-MV 2.5 mm
250 mm, 5 µm C18 column and a mobile phase gradient of
-80% B over 60 min at 0.3 mL/min: A ) water/0.5% acetic acid
×
2
and B ) acetonitrile/0.5% acetic acid. The mass spectrum of the
major product showed m/z 713, corresponding to [M + H]+ for
the biotinylated 13-HPODE. A second minor signal at m/z 313 was
+
also detected, corresponding to [M + H] for unreacted 13-HPODE.
From the peak heights, it was determined that the sample contained
about 98% 13-HPODE-biotin and 2% 13-HPODE. No unreacted
biotin-PEO-LC-amine was seen, and no further purification was
necessary. The sample was dissolved in 100% ethanol to a
concentration of about 20 mg/mL.
Reaction of Cyt c with 13-HPODE and 13-HPODE-Biotin.
Cyt c (1 mg/mL, 100 µL in pH 7.0 chelex-treated 100 mM HEPES
buffer) was reacted with 13-HPODE or 13-HPODE-biotin (224
nmols, 10 µL of ethanol) in the presence of vitamin C (10 mM) at
3
7 °C for 24 h. The sample was filtered (regenerated cellulose 3000
Da MWCO; Amicon) to remove unreacted 13-HPODE or 13-
HPODE-biotin and vitamin C and brought up in PBS to a
concentration of 1 mg/mL.
ARP Derivatization of DODE-Modified Cyt c. Cyt c samples
1 mg/mL) treated with 13-HPODE were incubated with ARP (10
(
mM) at a final volume of 1.0 mL in phosphate buffer, pH 5-6.
The reaction was stirred vigorously for 12 h at room temperature.
The samples were filtered (regenerated cellulose 3000 Da MWCO;
Amicon) to remove unreacted ARP.
ARP Derivatization MCF7 Cellular Protein Carbonyls. Ex-
tracted cellular proteins were dialyzed (4 °C) for a minimum of
4
8 h into PBS (2000 Da MWC, 3 mL cassette; Pierce). To the
dialyzed sample, a protease inhibitor cocktail was added as follows:
-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF)
1 mM), E-64 (10 µM), pepstatin A (1.4 µM), EDTA (1 mM), and
4
(
bestatin (40 µM). None of these protease inhibitors contain ketones
or aldehydes to interfere with the ARP oxime formation reaction.
ARP was added to a final concentration of 3.0 mM. The pH was
adjusted to 6.0 with 5% HCl, and the reaction was stirred at room
temperature for 12 h. Derivatized protein was dialyzed against PBS
for at least 48 h to remove any unreacted ARP. The total protein
concentration was measured by BCA assay (Pierce) before avidin
affinity chromatography.
(
Amersham Biosciences). For anti-DODE Western blotting and
Coomassie staining, proteins were focused on a 11 cm 3-10 pH
gradient strip. Samples were applied by cup loading. IPG strips
were then equilibrated in equilibration buffer (50 mM Tris-HCl, 6
M urea, 30% glycerol, and 2% SDS) supplemented with 1%
dithiothreitol (DTT) to maintain the fully reduced state of proteins,
followed by 2.5% iodoacetamide to prevent reoxidation of thiol
groups during electrophoresis. Proteins were separated on 12.5%
Tris-glycine gels (BioRad) using a Criterion System (BioRad).
Proteins were visualized by Coomassie, SimplyBlue SafeStain
(Invitrogen).
Development of Anti-DODE Antibody. DODE was conjugated
to Mariculture keyhole limpet hemocyanin (mcKLH) by incubating
a 1:1 (w/w) ratio of DODE to mcKLH in 100 mM HEPES buffer
pH 7.1; the reaction was stirred overnight at 37 °C. Conjugated
mcKLH was spin filtered (regenerated cellulose 10000 Da MWCO;
Amicon) and sent to Covance Research Products Inc. for the
development of an antibody to DODE in rabbits. After antibody
Treatment of MCF7 Cells with 13-HPODE and
3-HPODE-Biotin. MCF7 cells were purchased from Invitrogen.
1
Cells were grown in a media containing Dulbecco’s modified
Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/
streptomycin, MEM nonessential amino acids, sodium pyruvate,
and L-glutamine. Cells were grown to 90% confluence on 10 cm
plates before treatment and protein extraction.
The media from 40 plates (90% confluence) were removed, the
cells were washed with PBS, and 10 mL of DMEM (no FBS) was
added to each plate. Either 13-HPODE or 13-HPODE-biotin (10
µL of 20 mg/mL or 64 µM final concentration) was added to each

5
60 Chem. Res. Toxicol., Vol. 23, No. 3, 2010
Slade et al.
development, the IgG DODE antibody was purified from the rabbit
antisera using immobilized A/G protein beads (Pierce). Eluted/
purified DODE antibody was buffer exchanged and concentrated
to 10 mg/mL in PBS and stored at -80 °C for Western blot analysis.
Anti-Biotin and Anti-DODE Western Blots. Proteins were
transferred to a polyvinylidine fluoride (PVDF) membrane (Bio-
Rad). Precision Plus protein standard (BioRad) was used to estimate
molecular weights. Antibiotin (Cell Signaling) 1:20000 was used
to detect biotinylated proteins. Polyclonal anti-DODE (Covance)
charged states (most of the nonpeptide background constituents are
singly charged). The accumulation time and pulsar frequency were
maintained at 3 s and 6.99, respectively; the mass tolerance was
50 mmu. MS/MS was performed using nitrogen as the collision
gas. Rolling collision energies were calculated (in this study) as
follows: z ) 1, (collision energy) ) 0.06 × (m/z) + 8; z ) 2,
(collision energy) ) 0.05 × (m/z) + 5.6; z ) 3, (collision energy)
) 0.04 × (m/z) + 6.7; and z > 3, (collision energy) ) 0.015 ×
(m/z) + 25. Chromatography was performed using an Agilent 1100
capillary LC system with fused-silica capillary columns (75 µm
i.d. × 360 µm o.d.; 14 cm length, tip 8 µm, New Objective,
Woburn, MA) that were packed in-house, with 5 µm C18 reverse-
phase material (Vydac, Hesperia, CA). The flow rate from the
pumps was 3-5 µL/min, and the flow was split before introduction
of the sample on the column to 100-200 nL/min. Solvent A was
0.5% acetic acid in water, and solvent B was 0.5% acetic acid in
acetonitrile. A linear gradient was performed as follows: 2% B at
0 min, 2% B at 3 min, 65% B at 70 min, 80% B at 80 min, 80%
B at 90 min, 2% B at 130 min, and 2% B at 170 min.
1
:1500 was used to detect DODE-modified proteins.
In-Gel Digestion. Protein spots were picked and washed in
Milli-Q water for 15 min and then washed three times in 25 mM
NH HCO /50% CH CN for 30 min. Gel plugs were dehydrated in
00% CH CN for 10 min while vortex mixing. The supernatant
was removed, and gel plugs were dried in the SpeedVac. Trypsin
1 µg/50 µL) (Sigma) suspended in 25 mM NH HCO was added,
4
3
3
1
3
(
4
3
and gel plugs were allowed to rehydrate for 30 min on ice and
allowed to digest overnight at 37 °C. The samples were then
centrifuged, and the supernatant was removed. The pellet was
resuspended in CH
3
CN with 1% TFA, vortexed, and sonicated for
Agilent QTOF MS/MS. Capillary ESI was conducted using a
capillary voltage of 3000 V and fragmentor voltage of 205 V.
Nitrogen was the drying (5 L/min) gas, and the heated capillary
was at 325 °C. Multiple MS/MS experiments were carried out with
increasing collision energies over 0-50 V to evaluate fragmentation
of modified and unmodified peptides. Liquid chromatography was
performed using an Agilent 1200 capillary LC system with a Zorbax
3
0 min to release hydrophobic peptides. The supernatant was
removed and combined with the previous supernatant and stored
at -20 °C until ready for MS/MS analysis.
Strong Cation Exchange (SCX) Separation for MCF7
Proteins. Neutravidin elutions of proteins from cell lysates from
1
3-HPODE-treated cells and 13-HPODE-biotin-treated cells were
digested in solution with trypsin as follows: To 200 µg of protein,
µg of sequencing grade trypsin (Promega) was added in ∼200
µL of 25 mM ammonium bicarbonate; proteins were digested for
h at 37 °C. Digested proteins were desalted by SPE (Strata 50um,
3
00SB-C18 5 µm 0.3 × 150 mm (Agilent Technologies) at a flow
rate of 10 µL/min. Solvent A was 0.5% acetic acid in water, and
solvent B was 0.5% acetic acid in acetonitrile. The gradient profile
was as follows: 2% B at 0 min, 2% B at 3 min, 70% B at 20 min,
4
4
7
0% B at 25 min, 95% B at 30 min, 2% B at 35 min, and 2% B
tri-Func, C18-E), dried, and redissolved in 100 µL of mobile phase
A (described below) for SCX separation. Purified peptides were
separated by SCX liquid chromatography using an Agilent 1100
LC system with a Polysulfethyl A 100 mm × 4.6 mm, 5 µm,
column and a flow rate of 0.25 mL/min. Mobile phase A was 10
at 40 min.
Database Searches and Protein Identification. Data were
analyzed using Spectrum Mill proteomics software (Rev A.03.02.060;
Agilent) and verified using Mascot version 2.2 (Matrix Science)
(
27).
Spectrum Mill Identification. Raw data were extracted under
mM sodium phosphate (Na
and mobile phase B was 10 mM sodium phosphate (Na
5% acetonitrile, and 0.4 M KCl, pH 2.8. A linear gradient was
2
HPO
4
) and 25% acetonitrile, pH 2.8,
2
HPO ),
4
default conditions and searched against human sequences in the
NCBInr database using trypsin as the protease, allowing two missed
cleavages and including variable modifications of oxidized me-
thionine. Peptides were considered valid with a forward-reverse
score >2 and a rank 1-rank 2 score >2, a score threshold >7.67,
and percentage-scored peak intensity >70%. Only proteins with two
or more validated peptides and a total score >25 were considered
valid for reporting. To compare identified proteins between treat-
ment groups, the number of spectra and the summed ion intensity
of peptides for each protein (total ion intensity) were used as
indicators of protein amounts. The spectral intensity of each ion
was calculated from the extracted ion chromatogram of each
precursor ion. The total intensity was calculated as the sum of
spectral intensity (Spectrum Mill default settings). Total spectral
intensity values between proteins were compared only within each
experiment to give a qualitative value for total protein abundance
in the samples.
Mascot Identification. Raw data were extracted to mzData or
mgf files for Agilent or Applied Biosystems instruments, respec-
tively, using either the MassHunter or the Analyst software. For
protein identification, either the NCBInr and/or the SwissProt human
databases were searched. Parameters included the following:
enzyme as trypsin; max missed cleavages ) 2; fixed modifications
carbamidomethyl (C) for 2D gel analysis only; variable modifica-
tions of methionine oxidation (M); precursor tolerance set at 0.1
Da; ansd MS/MS fragment tolerance set at 0.2 Da. Proteins were
identified with two or more peptides. Significance of a protein match
for Mascot was based on an expectation value of <0.05 and a
combined peptide score >50.
2
used as follows: 0% B from 0-10 min, 100% B at 70 min, 100%
B at 75 min, and 0% B at 80 min. Peptides were collected in 1
min fractions to give about 40-60 fractions per sample. Fractions
were desalted by ZipTips and redissolved in 5 µL of 0.4% acetic
acid before LC-ESI-QTOF MS/MS analysis.
LC-ESI-TOF/MS. Whole protein cyt c analyses were performed
on an Agilent ESI-TOF mass spectrometer (Palo Alto, CA). ESI
was conducted using a needle voltage of 3.5 kV. Nitrogen was used
as the drying (10 L/min) and nebulizer (15 psig) gas with the heated
capillary at 325 °C. Chromatography was performed using an
Agilent 1200 LC system. LC separations were performed using a
Zorbax 300SB-C18 (2.1 mm × 100 mm; 3.5 µm; Agilent
Technologies Palo Alto, CA) column with a flow rate of 0.2 mL/
min. Solvent A was 0.25% acetic acid in water, and solvent B was
0
.25% acetic acid in 90% acetonitrile. A linear gradient was
performed as follows: 5% B at 0 min, 50% B at 45 min, 80% B at
0 min, 5% B at 55 min, and 5% B at 65 min. Data analysis: To
5
evaluate modification of cyt c with either DODE or DODE-biotin,
the intact protein mass spectrum was deconvoluted using the
BioConfirm function in the Agilent MassHunter Workstation
software.
LC-ESI-QTOF MS/MS. Peptide analyses for protein ID were
performed with either the Applied Biosystems (Framingham, MA)
QSTAR Elite quadrupole-TOF mass spectrometer equipped with
a nanospray source (Proxeon Biosystems, Odense, Denmark) or
the Agilent 6520 Accurate-Mass Q-TOF.
ABS QSTAR Elite MS/MS. The electrospray interface for this
instrument used a microtee (Upchurch Scientific, Oak Harbor, WA)
with a 1 in. piece of platinum rod, inserted into one arm of the
microtee, to supply the electrical connection. The electrospray
voltage was typically 1600-1700 V applied just upstream of the
column. Data-dependent MS/MS analysis was performed on the
three most intense peaks in each full-scan spectrum, using multiply
Results
ARP Modification of 13-HPODE-Treated Cyt c. The
ability of ARP to derivatize the DODE protein-carbonyl was

Proteins Modified by DODE in MCF7 Breast Cancer Cells
Chem. Res. Toxicol., Vol. 23, No. 3, 2010 561
Figure 1. ARP derivatization of DODE-modified cyt c. (A) Deconvoluted mass spectrum of unmodified cyt c. (B) Deconvoluted mass spectrum
of DODE-modified cyt c. (C) Deconvoluted mass spectrum of DODE-modified cyt c after derivatization with ARP.
evaluated. DODE-modified protein was synthesized by treating
purified cyt c with 13-HPODE and ascorbic acid as described
above (Materials and Methods). We have previously identified
DODE as the major modification of cyt c treated with 13-
HPODE (22). Treated protein was then incubated with ARP
and analyzed by LC-MS. The deconvoluted mass spectrum of
unmodified cyt c (m/z ) 12359) is represented in Figure 1A.
After treatment with 13-HPODE, the appearance of m/z ) 12585
(
Figure 1B) indicated the DODE modification of cyt c with the
addition of 226 Da, equivalent to the mass of DODE. The
derivatization of the DODE-carbonyl modification by ARP
(
Figure 1C) is indicated by m/z ) 12899, corresponding to the
addition of 314 Da, equivalent to the mass of ARP with the
loss of water. Comparison of peak heights in Figure 1C suggests
>
75% derivatization efficiency of ARP to the protein-carbonyl
DODE.
Treatment of Cyt c with 13-HPODE-Biotin. To determine
Figure 2. Deconvoluted mass spectrum of cyt c modified by 13-
HPODE-biotin.
if the 13-HPODE-biotin would successfully create a DODE-
biotin adduct, cyt c was treated with 13-HPODE-biotin and
ascorbic acid as described above (Materials and Methods) and
the modified cyt c analyzed by LC-MS. The deconvoluted mass
spectrum of the biotin modified cyt c can be seen in Figure 2,
with unmodified cyt c at m/z ) 12359 and the major modifica-
tion at m/z ) 12985, a difference of 626 Da corresponding to
the mass of the DODE-biotin adduct. The peak at m/z )
staining (Figure 3A). 13-HPODE-biotin was located using Cy3
conjugated streptavidin (Figure 3B). Overlaying the Cy3 and
Hoechst stains (Figure 3C) demonstrated the 13-HPODE-biotin
to have been taken up by the MCF7 cells. Cy3 fluorescence
was only seen in the 13-HPODE-biotin-treated cells, and no
fluorescence was observed in the controls.
Validation of DODE Antibody. To test antibody specificity
for DODE modifications, cyt c was modified with each of the
four different lipid peroxidation adducts HNE, ONE, EDE, and
DODE as previously described (22). Modified and unmodified
cyt c were separated by one-dimensional SDS-PAGE, and the
modifications were analyzed by Western blot, using the poly-
clonal anti-DODE antibody. As seen in Figure 4, the DODE
antibody recognized only DODE-modified cyt c. The DODE
antibody showed no cross-recognition to other lipid peroxidation
modifications or to unmodified cyt c. This result established
the ability of the DODE antibody to recognize only DODE-
modified protein.
1
3611san addition of 1252 Daswas equivalent to the addition
of two DODE-biotin modifications on cyt c. This result
confirmed the major protein modification product from the13-
HPODE-biotin tag to be the DODE carbonyl modification.
Fluorescent Staining of MCF7 Cells Treated with
1
3-HPODE-Biotin. To determine if the 13-HPODE-biotin
would successfully transverse the cellular membrane, MCF7
cells were treated with 13-HPODE-biotin and ascorbic acid as
described in the Materials and Methods; control cells were left
untreated. Plated cells were located using nuclear Hoechst

5
62 Chem. Res. Toxicol., Vol. 23, No. 3, 2010
Slade et al.
Figure 3. Immunostaining and imaging of MCF7 cells treated with 13-HPODE-biotin. (A) Hoechst nuclear staining. (B) Cy3 conjugated streptavidin
staining. (C) Merged picture of panels A and B.
Figure 4. Western blot using a DODE antibody. The antibody was tested against unmodified cyt c and DODE-, ONE HNE-, and EDE-modified
cyt c. Only DODE-modified cyt c was recognized.
Figure 5. 2D SDS-PAGE MCF7 proteins treated with ARP and affinity purified by neutravidin. (A) Coomassie blue-stained gel. (B) Antibiotin
Western blot.
Neutravidin Affinity Chromatography Validation. To
determine the ability of the neutravidin chromatography to
enrich for only biotinylated proteins from a cellular lysate,
MCF7 cells were treated with 13-HPODE and ascorbic acid as
described earlier. Cells were lysed, and the proteins were purified
Proteins Identified by 2D SDS-PAGE and Anti-DODE
Western Blots. MCF7 breast cancer cells were treated with 13-
HPODE and ascorbic acid; control cells were treated only with
ascorbic acid. Both control and treated cells were lysed and
pelleted, and the protein fractions were then buffer-exchanged
into deionized water by spin filtration. Cellular proteins were
separated by 2D SDS-PAGE. Two gels were run in tandem; an
anti-DODE Western to locate DODE-modified proteins and a
Coomassie blue-stained gel for in-gel digestion and LC-MS/
MS identification of modified proteins. Western blots of control
cells showed no significant DODE-modified proteins. Anti-
DODE Western blots of 13-HPODE-treated cells revealed a
number of DODE-modified proteins (Figure 6). The corre-
sponding spots were located on the Coomassie blue gel, in-gel
digested, and analyzed by nano-LC-MS/MS. Proteins were
(
Materials and Methods). Proteins were reacted with ARP and
biotinylated proteins then enriched using a neutravidin affinity
column (Materials and Methods). Enriched proteins were
analyzed by 2D SDS-PAGE using both anti-Biotin Western blot
and Coomassie staining. Figure 5A shows the Coomassie-stained
gel, and Figure 5B shows the Western blot. The similarity
between the two gels demonstrated that the majority of the
enriched proteins were biotinylated. These results verified both
the neutravidin affinity technique and further validated the ARP
reaction with cellular proteins.

Proteins Modified by DODE in MCF7 Breast Cancer Cells
Chem. Res. Toxicol., Vol. 23, No. 3, 2010 563
tissue, resulting in protein modification and the generation of
protein-carbonyls. Proteins are considered most likely to be
ubiquitously affected by disease (28), and carbonyl-modified
proteins shed from affected tissue are therefore potential early
disease markers. Numerous inflammatory disease studies have
measured the formation of protein-carbonyls as a consequence
of heart disease, lung disease, aging, neurodegenerative disor-
ders, and inflammatory bowel disease (29–34). A major pathway
for the formation of protein carbonyls comes from the oxidation
of PUFAs, leading to a variety of unsaturated lipid hydroper-
oxides. These hydroperoxides break down in the presence of
reductants to generate R,ꢀ-unsaturated aldehydes, which readily
modify proteins, to form lipid peroxidation protein-carbonyls.
We have previously demonstrated the lipid peroxidation alde-
hyde DODE to be the most reactive protein-modifying aldehyde
arising from linoleic acid oxidation and degradation (22).
DODE-modified proteins are also of interest since this carbonyl
modification is uniquely formed from linoleic acid oxidation
(8); evidence of its endogenous formation can therefore be linked
back to a single pathway of lipid peroxidation. This research
evaluated the ability of DODE protein modifications to occur
in MCF7 breast cancer cells and identified the major proteins
modified by DODE.
Figure 6. 2D SDS-PAGE of MCF7 cells treated with 13-HPODE. Anti-
DODE Western blot showing the presence of DODE-modified proteins;
circled proteins (red) were picked for mass spectrometry identification.
identified by Mascot database searching. A complete list of
DODE-modified proteins identified is shown in the Supporting
Information (Table 1). A list of each identified protein’s PI,
molecular weight, and 2D gel spot location can be seen in the
Supporting Information (Table 2).
Proteins Identified by ARP Derivatization and 2D
LC-MS/MS. MCF7 breast cancer cells were treated with 13-
HPODE and ascorbic acid as described (Materials and Methods);
control cells were treated only with ascorbic acid. Both control
and treated cells were lysed and pelleted, and the protein fraction
was buffer exchanged into PBS (pH 6) by spin filtration.
Carbonyl-modified proteins in both control and treated samples
were derivatized with biotin using ARP as described earlier.
ARP-derivatized proteins were retained on a neutravidin column,
unmodified proteins were washed from the column, and ARP-
derivatized/carbonyl-modified proteins were eluted with 80%
acetonitrile and 0.4% TFA. Eluted proteins from both control
and treated samples were separated into 40-60 fractions by
strong cation exchange. Fractions were desalted and analyzed
by capillary-LC-MS/MS. Proteins were identified by database
searching with both Mascot and Spectrum Mill. A total list of
modified proteins identified in both control and treated cells
can be seen in the Supporting Information (Table 1).
Proteins Identified by 13-HPODE-Biotin Treatment
and 2D LC-MS/MS. MCF7 breast cancer cells were treated
with 13-HPODE-biotin and ascorbic acid as described (Materials
and Methods). Cells were lysed and pelleted, and the protein
fraction was buffer exchanged into PBS (pH 7) by spin filtration.
Modified proteins were retained on a neutravidin column,
unmodified proteins were washed from the column, and ARP-
derivatized/carbonyl-modified proteins were eluted with 80%
acetonitrile and 0.4% TFA. Eluted proteins were separated into
Identification of protein modification generally requires the
characterization of the modified peptide and the site and nature
of the modification, involving affinity purification of the
modified peptide followed by mass spectrometry. Unfortunately,
the ARP-biotin and the 13-HPODE-biotin tags created numerous
fragment ions during MS/MS that hampered peptide identifica-
tion by database searching, and we were unsuccessful at
identifying specific DODE-modified peptides. The polyclonal
antibody used in this study was effective in Western blots but
bound the DODE antigen too tightly to be used for purification
by immunoaffinity chromatography. Therefore, to avoid the
necessity of identifying specific DODE-modified peptides, this
study used three distinct methods to identify DODE-modified
proteins, which effectively reduced the number of false positives
and decreased the number of false negatives, eliminating the
need to identify a DODE-modified peptide for each protein
identified. First, cells were treated with a synthetically bioti-
nylated hydroperoxide of linoleic acid (13-HPODE-biotin),
which readily forms a biotin-DODE protein modification in vitro
(
Figure 2). This method allowed for proteins modified only by
DODE to be affinity purified and identified by mass spectrom-
etry. Second, cells were treated with the native hydroperoxide
of linoleic acid (13-HPODE) to determine if the protein
modification pattern differed from 13-HPODE-biotin treatment.
This method used an ARP to biotinylate all carbonyl-modified
proteins since proteins could potentially be modified by all three
HNE, ONE, and DODE aldehydes occurring from 13-HPODE
degradation (Scheme 1). This method also allowed for the
identification of false positives by reacting proteins with ARP
from control cells that were not treated with 13-HPODE.
Controls established a set of background proteins caused by
nonspecific binding to affinity columns or from carbonyl
modification not specific to the 13-HPODE treatment. Third, a
polyclonal DODE antibody was used to locate, by Western blot,
DODE-modified proteins in cells treated with 13-HPODE. Spots
from these proteins were cut from Coomassie blue-stained gels
and identified by mass spectrometry.
4
0-60 fractions by strong cation exchange. Fractions were
desalted and analyzed by capillary-LC-MS/MS. Proteins were
identified by database searching with both Mascot and Spectrum
Mill. The 13-HPODE-biotin produces a DODE-biotin modifica-
tion; the proteins identified using this tag were therefore
considered to be DODE-modified. A total list of modified
proteins identified using the 13-HPODE-biotin tag can be seen
in the Supporting Information (Table 1).
Discussion
A consequence of inflammation is the formation of reactive
oxygen and nitrogen species from activated phagocytes, which
are used to combat disease or infection. If inflammation is
chronic, these reactive chemicals can damage surrounding host
The ability of the R,ꢀ-unsaturated aldehydes HNE, ONE,
EDE, and DODE (methyl ester) to induce apoptosis in endot-
helial cells has been shown by Jian et al. to be in the low
micromolar concentration (16). The concentration of 13-HPODE

5
64 Chem. Res. Toxicol., Vol. 23, No. 3, 2010
Slade et al.
from 13-HPODE decomposition in vitro, their existence in a
cells remains feasible, and this type of modification may further
explain the greater number of proteins identified with the 13-
HPODE-biotin method.
To highlight DODE modification, a master list of likely
DODE-modified proteins was created (Figure 8). To create this
list, proteins were selected only if they were identified by all
three techniques (HPODE-biotin, HPODE ARP, and anti-DODE
Western). Proteins were also selected if they were identified by
both the HPODE-biotin and the HPODE ARP techniques but
were not seen in the ARP controls. A final set of 31 were
identified (Figure 8) and were considered the major proteins
modified by DODE in MCF7 cells treated with 13-HPODE. A
greater overlap of protein identification was seen between the
two biotin methods (HPODE-biotin and HPODE ARP) than
with the antibody method. This is not surprising since the biotin
methods used 2D LC to identify proteins, which is generally
considered a more sensitive technique than the 2D SDS-PAGE
method used with the DODE antibody. To emphasize this point,
Figure 8 shows these proteins ordered by their Spectrum Mill
total spectrum intensity (Materials and Methods); generally, the
higher abundant proteins were more likely to be identified by
all three methods andsas abundance decreasedsthe SDS-PAGE
method appeared to be less likely to identify the protein. As
described in the Materials and Methods, this type of spectral
counting was used only in a qualitative manner to give a general
estimate of relative protein abundance.
Figure 7. Venn diagram representing the number of overlapping
proteins identified by all three methods.
and 13-HPODE-biotin used in this study was about a 10-fold
increase over these previously established levels. As described
(
Materials and Methods), cells were treated for 30 min with 10
mL of FBS-free media containing 64 µM 13-HPODE or 13-
HPODE-biotin. This concentration is high and not necessarily
physiologically relevant, but treatment showed no observable
effect on the cell growth and viability, suggesting that cellular
DODE concentrations were below the apoptosis-inducing levels
of 2-5 µM, as established by Jian et al. (16). The kinetics for
the cellular uptake and degradation of 13-HPODE is not well
understood, and we were therefore unable calculate the true
cellular concentration of DODE. Because we were able to
identify a number of DODE-modified proteins, the cellular
concentration of DODE must have been either high enough to
overcome any detoxification mechanisms (such as glutathione
scavenging) or protein modification may have occurred during
a specific aldehyde/protein interaction (as described below). Any
future studies should analyze cellular lysates for glutathione-
DODE adducts; this would help establish the ability of these
cells to take up 13-HPODE as well as give a relative cellular
DODE concentration from 13-HPODE degradation.
Using all three methods (13-HPODE ARP, 13-HPODE-biotin,
and anti-DODE Western blot), 98 proteins were characterized
as potentially modified by DODE (Supporting Information,
Table 1). As represented in Figure 7, there was substantial
overlap, with 18 proteins identified in all three methods and 31
proteins identified by the two biotin methods (ARP and 13-
HPODE-biotin). Only six proteins were exclusively identified
by the DODE antibody (Figure 7), and the majority of antibody-
identified proteins overlapped with the two biotin systems,
demonstrating biotin tagging as a successful technique for the
enrichment of carbonyl-modified proteins. As previously stated,
the 13-HPODE-biotin method was designed to identify only the
DODE modification. For a number of reasons, this technique
identified the highest number of modified proteins (Figure 7
and Supporting Information, Table 1). The biotinylation of the
carboxylic acid group of 13-HPODE likely facilitated its ability
to penetrate cell walls by elimination of the charge on the
carboxyl group, and a greater uptake of this species would allow
for more protein modification. A recent study by Szapacs et al.
identified new carboxylated electrophile adducts occurring from
oxidation of a biotin-modified phospholipid containing linoleate
The DODE-modified protein cellular locations are shown in
Figure 9A. Proteins were mainly cytoplasmic (Figure 9A), which
was expected. The number of membrane-bound proteins identi-
fied was limited since the extraction methods used in this study
were not optimal for the analysis of this class of proteins. Future
studies to characterize proteins endogenously modified by
inflammation should probably take greater account of membrane
proteins, since the nitric oxide produced during the inflammatory
process has a greater solubility in the lipid membrane than in
the aqueous cytoplasm (37). Surprisingly, a significant number
(17%) of proteins had locations in the nucleus. Jian et al. have
shown that DODE was unable to translocate across plasma
membranes (16). Therefore, in this study, for proteins to be
DODE-modified in the nucleus, 13-HPODE would have to
remain intact long enough to cross the nuclear membrane. Two
of these nuclear proteins (valosin-containing protein and elonga-
tion factor 2) are involved in nucleotide binding and transport
into the cytoplasm (38, 39), and it is therefore likely that they
were modified outside the nucleus in the cytoplasm. DODE
modification of the nuclear proteins nucleolin, ribosomal protein
L5, and proliferating cell nuclear antigen is also somewhat
surprising. All three proteins are involved in cell growth and
are likely abundant in MCF7 cells, increasing the probability
of modification even in the nucleus.
(
35). Furthermore, the carboxylic acid containing reactive
aldehyde 9-hydroxy-traumatin has been identified as an enzy-
matic degradation product of 13-HPODE in soybean and alfalfa
seeds (36). The degradation of 13-HPODE can also lead to the
formation of the hydroxy version of DODE or 9-hydroxy-12-
oxo-10(E)-dodecenoic acid (HODE or HODA) (35). Through
keto-amide formation, HODE creates a noncarbonyl-containing
protein modification, which would therefore be undetectable
using the ARP method, but protein modification by Michael
addition of HODE retains the carbonyl functional group,
allowing for derivatization/detection with ARP. Therefore, in
our experiments using 13-HPODE-biotin, some of the proteins
that we identified may have arisen from decomposition products
other than DODE. Although we have not detected these adducts
DODE-modified protein functions are shown in Figure 9B.
Overall, proteins modified by DODE in this study had a limited
number of functions, suggesting some protein specificity for
DODE modification in cells. We expected a general nonspecific
protein modification, resulting largely in the identification of
highly abundant structural proteins. This, however, was not the
case, and DODE modification was distributed among five major
protein groups: molecular chaperones, glycolysis, regulatory,
nucleotide binding, and structural. The lower-than-expected
number of structural proteins may indicate some resistance to
lipid peroxidation adduction by these proteins, but more likely,
DODE modification of other protein types was enhanced due
to specific protein-adduct interactions, such as binding and

Proteins Modified by DODE in MCF7 Breast Cancer Cells
Chem. Res. Toxicol., Vol. 23, No. 3, 2010 565
Figure 8. Major proteins modified by DODE in MCF7 cells treated with 13-HPODE.
Figure 9. Location and function of major proteins modified by DODE in MCF7 cells treated with 13-HPODE. (A) Protein location. (B) Protein
function.
transport. The specificity for DODE modification may be
influenced by the detoxification of cellular reactive aldehydes
by glutathione (GSH) or other abundant reductants. GSH has
been shown to react in vivo with the lipid peroxidation aldehyde
ONE (17), and it is likely that GSH or other cellular reductants
would also react with DODE to decrease the amount of cellular
DODE available, thus limiting modification primarily to proteins
directly interacting with 13-HPODE. This study was designed
to monitor only high molecular weight proteins, and any lower
molecular weight DODE adducts (such as DODE-GSH) were
removed by ultrafiltration. For a more complete picture of
cellular DODE interactions, future studies should monitor the
formation of a DODE-GSH species.
modification was a probable result of their high cellular
abundance as previously mentioned. The nucleotide binding
proteins involved in transport may additionally bind to or interact
with lipids, which would explain the preferential DODE
modification of this class of proteins. In fact, valosin-containing
protein binds both nucleotides and lipids during cellular mitosis
(39). The identification of a number of molecular chaperone
proteins, such as heat shock proteins (HSP) 27, 70, and 90, was
not surprising since these multifunctional proteins are involved
in the transport of a large array of molecules. HSPs contain
binding domains that bind exposed hydrophobic residues of
substrate proteins or peptides; they also bind nonprotein
molecules, including lipid-based TLR ligands, LPS, and lipo-
proteins, with exposed hydrophobic residues (40–44). It is
therefore likely that a specific binding between 13-HPODE and
these molecular chaperones occurs, enhancing their modification
The structural proteins identified as DODE-modified were
actin, ankyrin, spectrin R, tropomyosin, vitamin D-binding
protein, and myristoylated alanine-rich C-kinase substrate. Their

5
66 Chem. Res. Toxicol., Vol. 23, No. 3, 2010
Slade et al.
by DODE. Furthermore, a recent study by Vila et al. identified
HSP 70 and 90 as two of the major protein targets of the
linoleate decomposition product HNE (45), and our studies
further emphasize the potential importance of these proteins with
regards to lipid peroxidation modification.
Acknowledgment. This work was supported by grants from
the Harvard-MIT HST Center for Experimental Pharmacology
and Therapeutics, the National Institutes of Health (NCI Program
Project Grant CA26731), and the MIT Center for Environmental
Health Sciences (NIEHS Grant P30 ES002109).
Another group of proteins modified by DODE were enzymes
involved in glycolysis (pyruvate dehydrogenase kinase, glyc-
eraldehyde-3-phosphate dehydrogenase, glucosidase II, fructose-
bisphosphate aldolase, glucose-6-phosphate dehydrogenase, and
lactate dehydrogenase). Their enhanced modification may be
specific to the MCF7 cells, sinceslike most cancer cellssthey
exhibit an increased rate of glycolysis (46, 47), and DODE-
specific modification may be explained by higher cellular
concentrations of these enzymes. Increased expression of lactate
dehydrogenase (LDH) is common in tumor cells under hypoxic
conditions. The MCF7 cells used in this study likely ac-
cumulated LDH since they were grown to 90% confluence, thus
creating a lowered oxygen environment. It is thought that the
increased expression of LDH in tumor cells may be a critical
part of the metabolic reprogramming that occurs during tum-
origenesis (48), and the specific modification of LDH by reactive
aldehydes may therefore be of some interest with respect to
this hypothesis. Preferential DODE modification of these
proteins may involve some overlap in substrate recognition
between glycolysis enzymes and fatty acid metabolism enzymes.
Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme
in the pentose pathway, a major producer of NADPH (49), and
is also involved in lipid and cholesterol synthesis (50, 51). G6PD
is up-regulated during oxidative stress providing cellular protec-
tion by generating NADPH to maintain reduced glutathione
levels (23). G6PD is also inhibited by and presumably binds
PUFAs, such as linoleate, which may potentially explain a direct
interaction between G6PD and 13-HPODE (52, 53).
Supporting Information Available: Tables of total number
of carbonyl-modified proteins identified in MCF7 cells and
DODE-modified proteins in MCF7 cells identified by 2D SDS-
PAGE and tandem mass spectrometry. This material is available
free of charge via the Internet at http://pubs.acs.org.
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