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Published in final edited form as:
J Proteomics. 2011 May 1; 74(5): 683–697. doi:10.1016/j.jprot.2011.02.013.
Analysis of proteome changes in doxorubicin-treated adult rat
cardiomyocyte
Suresh N. Kumar1, Eugene A. Konorev2, Deepika Aggarwal3, and Balaraman
Kalyanaraman4,#
1Department of Pathology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA
4Department
of Biophysics and Free Radical Research Center, Medical College of Wisconsin,
Milwaukee, Wisconsin, USA
Abstract
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Doxorubicin-induced cardiomyopathy in cancer patients is well established. The proposed
mechanism of cardiac damage includes generation of reactive oxygen species, mitochondrial
dysfunction and cardiomyocyte apoptosis. Exposure of adult rat cardiomyocytes to low levels of
DOX for 48 h induced apoptosis. Analysis of protein expression showed a differential regulation
of several key proteins including the voltage dependent anion selective channel protein 2 and
methylmalonate semialdehyde dehydrogenase. In comparison, proteomic evaluation of DOXtreated rat heart showed a slightly different set of protein changes that suggests nuclear
accumulation of DOX. Using a new solubilization technique, changes in low abundant protein
profiles were monitored. Altered protein expression, modification and function related to oxidative
stress response may play an important role in DOX cardiotoxicity.
Keywords
Doxorubicin; cardiomyopathy; proteomics
1. Introduction
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The anthracycline antibiotic, doxorubicin (DOX), is an anticancer drug that is used widely in
treating leukemias, lymphomas, lung, breast, ovary and uterine cancers. However, DOX
chemotherapy causes a cumulative and delayed cardiomyopathy in cancer patients (2% in 2year versus 5% in 15-year survivors) as a major side effect [1,2]. Human case study reports
and preclinical data in animal models indicate that DOX treatment caused mitochondrial
damage and decrease in ATP / ADP ratio [3]. The proposed mechanism of cardiotoxicity
involves free radical generation [4], lipid peroxidation [5], cardiomyocyte apoptosis [6,7]
and somatic mitochondrial alteration and dysfunction [8-10]. DOX undergoes a one-electron
redox cycling at the complex I site of the mitochondrial electron transport chain (ETC),
© 2010 Elsevier B.V. All rights reserved.
#
Corresponding author: B. Kalyanaraman, PhD, Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank
Road, Milwaukee, WI, 53226, USA; balarama@mcw.edu; Telephone: 414-456-4000; Fax: 414-456-6512.
2(present address) Department of Pharmaceutical Sciences, University of Hawaii at Hilo, Hilo, Hawaii, USA
3(present address) Dr. Reddy's Laboratories Ltd., Hyderabad, India
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forming a semiquinone that reduces molecular oxygen to superoxide that dismutates to form
hydrogen peroxide [11-13]. DOX can be reduced by a variety of flavoproteins including
cytochrome P450 reductase, mitochondrial NADH (nicotinamide adenine dinucleotide –
reduced) dehydrogenase and nitric oxide synthase (reductase domain) [12,14]. In the
presence of redox-active metal ions (iron and copper), highly reactive hydroxyl radicals or
perferryl iron are formed from DOX redox activation [15]. These oxidants target lipid,
protein, and DNA causing extensive modifications [16-18]. Reactive oxygen species formed
from DOX redox cycling also affect iron uptake and iron homeostasis [19,20], inhibit
aconitase and iron regulatory protein (IRP) function and alter the transferrin / ferritin mRNA
(messenger ribonucleic acid) levels. Another mechanism by which aconitase was inactivated
is via the interaction with the secondary alcohol derivative of DOX [21,22].
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Doxorubicin caused a dose-dependent cell death in isolated adult rat cardiomyocyte [23,24].
Studies with antioxidants [25] and iron chelators [26] suggest that mechanisms other than
peroxidative stress contribute to cardiac impairment in DOX chemotherapy [27,28]. In
addition, DOX accumulates in the nucleus causing DNA damage. DOX also selectively
inhibits gene expression and interferes with the transcription factor's ability to bind to DNA
due to formation of GC adducts at the 5’-UTRs (untranslated regions). We have previously
demonstrated the critical role of activated transcription factor kappa-B in DOX-mediated
apoptosis in endothelial cells and in isolated adult rat cardiomyocyte [29-33]. The DNA
repair process in DOX-treated cells is different from that of hydrogen peroxide-treated cells
[34-36]. Overall, published data suggest that oxidative stress appears to play a key role in
DOX-mediated cardiotoxicity and apoptotic myocyte death. In this study, we report the
protein expression changes observed in isolated cardiomyocyte and adult rat heart during
DOX treatment and also provide a methodological improvement that identifies changes in
non-abundant protein levels.
2. Materials and methods
2.1. Reagents
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All reagents of the highest quality commercially available were purchased. TRIS base,
glycine, sodium dodecyl sulfate (SDS), doxorubicin-HCl, dithiotheritol (DTT), Urea, 3-[(3Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), ammonium
bicarbonate, α-cyano-4-hyroxycinnamic acid (CHCA), deoxyribonuclease (DNAse), bovine
serum albumin (fraction V), 80 mesh screen filter, medium 199, iodoacetamide, creatine,
taurine, insulin, cytosine β-D-arabinofuranoside, vitamin solution (100x), sodium
deoxycholate, Triton x-100, glycerol, urea, thiourea, ethylenediaminetetraacetic acid
(EDTA), ethyleneglycoltetraacetic acid (EGTA), diethylene triamine pentaacetic acid
(DTPA), phenylmethylsulphonyl fluoride (PMSF), aprotinin, leupeptin, β-glycerophosphate,
sodium fluoride, sodium orthovanadate, sodium pyrophosphate, 4-(2-Hydroxyethyl)-1piperazineethane sulfonic acid (HEPES), sucrose, mannitol, calcium chloride (CaCl2),
tween-20, acetonitrile (ACN), dimethyl sulfoxide (DMSO), acetone and HPLC pure water
were purchased from Sigma-Aldrich Co. USA. DMEM, fetal bovine serum (FBS),
collagenase (type II, the preparation with low collagenase activity of 200-250U/mg) and
Dulbecco phosphate buffered saline (DPBS), complete amino acid mixture (50x) and MEM
non-essential amino acid solution (100x) were from Gibco, USA. Immobilized IPG (3-10)
strips, Bio-lyte 3-10 ampholytes and prestained protein molecular weight marker were
purchased from Biorad, USA. 2D-quant kit and advanced ECL kit were purchased from GE
Life Sciences. Syringe filters (5 μ) and Zip-Tip C18 was purchased from Millipore, USA. All
other general chemicals such as potassium dihydrogen phosphate (KH2PO4), sodium
bicarbonate (NaHCO3), sodium chloride (NaCl), potassium chloride (KCl), magnesium
sulfate (MgSO4) were purchased from Fisher Scientific, USA.
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2.2. Buffers
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All buffers involved in cardiomyocyte isolation were prewarmed to 37°C before use.
Perfusion buffer contains 25 mM HEPES buffer, 110 mM NaCl, 11 mM glucose, 1 mg/mL
BSA, 5 mM creatine, 20 mM taurine, 2.6 mM KCl, 1.2 mM MgSO4, 20 μl/ml of 50x
complete amino acid mixture, 10 μl of 100x stock non-essential amino acid mixture, 10 μl/
mL of 100x stock of vitamin solution and 1.2 mM KH2PO4 and 5 mM pyruvate sodium (pH
7.4). The pH of the perfusion buffer was adjusted to 7.4 and sterile-filtered using 0.2 μm
cellulose acetate filter. Digestion buffer contain perfusion buffer, 25 μM CaCl2 and 200 U/
mL of collagenase [37]. Isolation buffer contains digestion buffer, 20 μg/mL of DNAse and
1% BSA. Wash buffers I, II and III are made up of perfusion buffer containing 200 μM, 500
μM and 1 mM CaCl2 , respectively. Isolated cardiomyocytes were cultured in M-199
medium containing 25 mM NaHCO3, 25 mM HEPES, 2 mg/mL BSA, 0.5 mM creatine, 5
mM taurine, 0.1 μM insulin, 10 μM cytosine β-D-arabinofuranoside, 10% FBS, 100 U/mL
penicillin and 100 μg/mL streptomycin. Protease inhibitor cocktail contains 5 mM EDTA, 2
mM EGTA, 5 mM DTPA, 2 mM PMSF, 5 μg/mL aprotinin, 5 μg/mL leupeptin, 1 mM βglycerophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 2.5 mM sodium
pyrophosphate and 5 mM DTT. Radio-immunoprecipitation assay (RIPA) buffer was made
with 50 mM Tris pH 7.6, 150 mM NaCl, 1% triton x-100, 1% sodium deoxycholate, 0.1%
SDS and protease inhibitor cocktail. Rehydration buffer is made up of 7 M urea, 2 M
thiourea, 4% CHAPS, 0.2% ampholytes (3-10) and 100 mM DTT. Equilibration buffer I is
made up of 6 M urea, 375 mM Tris-HCl, pH 8.8, 2% SDS, 20% glycerol and 2% DTT.
Equilibration buffer II is made up of 6 M urea, 375 mM Tris-HCl, pH 8.8, 20% glycerol and
2.5% idoacetamide. Cytoplasmic extraction buffer is made up of 1x PBS pH 7.0 containing
0.1% digitonin and protease inhibitor cocktail. Membrane fraction I extraction buffer is
made up of 1x PBS pH 7.0 containing 0.3% triton x-100 and protease inhibitor cocktail.
Membrane fraction II extraction buffer is made up of 1x PBS pH 7.0 containing, 1% triton
x-100 and protease inhibitor cocktail. Filamentous protein extraction buffer is made up of 1x
PBS pH 7.0 containing 0.5% triton x-100 and 0.6 M KCl and protease inhibitor cocktail.
2.3. Adult rat cardiomyocyte isolation and culture
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The ventricular cardiomyocytes were isolated according to a previously described method
[38,39] that was modified in our laboratory. Adult male Sprague-Dawley rats weighing
approximately 180-220 g were anesthetized with intraperitoneal injections of pentobarbital
(60 mg/kg body weight) followed by intraperitonial injection of 250 U/kg of heparin. The
heart was excised, mounted on aortic cannula and perfused (in a Langendorff nonrecirculating mode) with the oxygenated perfusion buffer supplemented with 1 mM CaCl2.
After 15 min of perfusion, it was switched to calcium-free oxygenated buffer in a nonrecirculating mode for 5 min followed by recirculation at a constant flow rate for 30 min. At
the end of perfusion the ventricular tissue was dissected, minced and incubated with gentle
shaking in the isolation buffer at 37°C for 10 min. The single cells released were dispersed
from the remaining undigested tissue by mixing the suspension with a 5 ml serological
pipette, and the cells were collected using 80 mesh screen filter. The cells were washed by
centrifugation at 150 rpm in succession with wash buffers I, II, and III and resuspended in a
small volume of wash buffer III. This cell suspension was layered on a 4% BSA - 1 mM
CaCl2 cushion and centrifuged at 200 rpm for 2-3 min. Ventricular myocytes settled down
and the resulting cell pellet was resuspended in growth medium and plated. Cardiomyocytes
were separated from other cells by differential plating at 1.5 × 106 cells / mL on to laminin
coated 150 mm dishes. This isolation procedure generally yields 75-80% rod-shaped
cardiomyocyte [40] that exclude trypan blue.
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2.4. Doxorubicin treatment of cardiomyocyte and isolation of cell lysate
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The isolated cardiomyocytes were placed in the incubator at 37°C with 5% CO2 for
overnight incubation. DOX was added to the medium at 0.5 μM concentration and the cells
were lysed in RIPA buffer at 0, 24, or 48 h after treatment. At these concentrations the cells
did not release significant lactate dehydrogenase (marker for cellular damage) [40] but
exhibit apoptosis as seen by a large amount of floating cells and positive TUNEL assay [41].
The cell lysate was left to stand on ice for 30 min and centrifuged at 4°C at 13,000 rpm for
30 min. The clear supernatant was transferred to a fresh tube and aliquots in triplicate were
analyzed for protein concentration using 2D-quant kit (bicinchoninic acid method) using
BSA. Protein concentration equivalent to 600 μg was precipitated by incubating with 8
volumes of ice cold acetone and left to stand at -20°C overnight. The precipitated protein
was collected by centrifuging at 4°C at 13,000 rpm for 30 min, and the pellet washed with
ice cold acetone twice. The final pellet was dried under vacuum and was stored at -80°C
until use.
2.5. Fractionation of cell lysate
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For fractionation experiments, DOX-treated (0.5 μM for 48 h and 20 μM for 24 h) and
untreated control cells (vehicle for 48 h) were collected in 1x DPBS and washed twice in the
same buffer. The cell pellet was resuspended in 2 mL of cytoplasmic extraction buffer,
incubated on ice for 1 hr and spun at 16,000 rpm for 15 min at 4°C. The resultant
supernatant was spun at 100,000x g for 20 min at 4°C (supernatant collected). The pellets
from 16,000 rpm and 100,000x g were pooled and reextracted with membrane fraction I
extraction buffer and spun at 16,000 rpm for 20 min at 4°C (supernatant collected). The
resultant pellet was extracted with membrane fraction II extraction buffer and spun at 16,000
rpm for 20 min at 4°C (supernatant collected). The final pellet was again extracted with
filamentous protein extraction buffer and spun at 16,000 rpm for 20 min at 4°C (supernatant
collected). Protein estimation of the extracts was performed as above.
2.6. DOX-treated adult rat
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Animal experiments were performed in accordance with the animal protocol approved by
the Medical College of Wisconsin Animal Care and Use Committee. Adult male SpragueDawley rats were segregated into groups as control (n=16) and DOX-treated (n=11). The
control group was injected through the tail vein with saline and the DOX-treated group
received 2.5 mg/kg of DOX in saline. Echocardiographic monitoring of cardiac function was
performed at various time points and the animals were sacrificed at 8 wks with
intraperitoneal injection of sodium pentabarbitol (100 mg/kg body weight). The heart tissue
was collected, flash frozen and stored at -80°C for further analysis. Approximately 500 mg
of tissue was homogenized in RIPA buffer, protein estimated, and sample processing for 2D
electrophoresis was performed as described above.
2.7. 2D gel electrophoresis and silver staining
To each 600 μg of the dried precipitated protein extract, 600 μl of rehydration buffer was
added to solubilize the protein, and 300 μl (equivalent to 300 μg protein sample) of this
sample was loaded on 17 cm IPG strips (pH 3-10, 0.5 mm thick and 4% T / 3% C
composition). Isoelectric focusing was performed using Protean IEF system (Biorad). After
initial rehydration at 20°C for 16 h and at 50 V constant, isoelectric focusing was done in
three stages. The settings were 20°C, 250 V for 0.5 h (rapid ramping – conditioning step),
10,000 V for 3 h (linear ramping – voltage ramping step) and 10,000 V for 60,000 V-hr
(rapid ramping – final focusing step) with maximum current set at 50 μA/gel strip. After the
first dimensional run the gels were washed to remove the excess mineral oil, and reduced
using equilibration buffer I for 15 min at room temperature, followed by alkylation using
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equilibration buffer II for 15 min at room temperature in the dark. Finally the gels were
washed and equilibrated in the Laemmli SDS-running buffer (Tris / glycine / SDS) and
layered on a 12% SDS-PAGE gel. The gel was sealed with 1% agarose containing 0.001%
Bromophenol blue dye and ran at 35 mA/gel constant and at 4°C using precooled (4°C)
running buffer. Gels were stained with modified silver staining method [42] that is
compatible with mass spectroscopic analysis. Briefly, after running the gels, they were
marked and fixed overnight at 4°C with shaking in fixative solution. The gels were
sensitized with sensitizer containing 30% methanol, 4% sodium thiosulphate solution (stock
– 50 mg/mL) and 68 g/L sodium acetate for 30 min at room temperature. The gels were
washed with water thrice for 10 min each, followed by silver solution (2.5 g/L silver nitrate)
treatment for 25 min at room temperature. This was followed by water wash thrice and
developer (25 g/L sodium carbonate and 0.04% formaldehyde solution) until all spots were
developed, and the reaction was terminated using 14.6 g/L EDTA for 10 min followed by
water wash. The stained gels were documented using a Kodak gel documentation system.
2.8. Mass spectrometry - MALDI-TOF detection
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2.8.1. In-gel digestion—Spots of interest were punched out, transferred to fresh tubes and
trypsin digested according to [43] with modifications. The gel pieces were destained using
15 mg/mL of sodium thiosulphate and 10 mg/mL of potassium ferricyanide (1:1 ratio) for 30
min at room temperature. The destained slices were washed twice with 50 mM ammonium
bicarbonate, reduced, and S-alkylated with 20 mM DTT (20 min at 60°C) and 100 mM
iodoacetic acid (20 min at room temperature in the dark) in 50 mM ammonium bicarbonate.
The gel slices were washed and dehydrated with ACN and dried under vacuum. The dried
gels were rehydrated with 12.5 ηg/μl trypsin and allowed to swell for 1 h on ice. Finally the
excess buffer was removed and trypsin-free buffer was added and allowed to digest
overnight at 37°C. The digested peptides were eluted with 0.1% TFA followed by 50%
ACN in 0.1% TFA and dried down using vacuum. The dried material was resuspended in
0.1% TFA and used for the desalting procedure.
2.8.2. Sample preparation—Matrix solution was prepared using recrystallized α-CHCA
at 10 mM to prepare saturated solution in 50% ACN / 0.1% TFA (1:1) by vortexing and
centrifuging at 13,000 rpm for 5 min at room temperature. The clear supernatant was
transferred to a new amber tube and used for co-crystallizing the peptides. The in-gel
digested peptides were desalted using C18 reverse phase Zip-Tips according to
manufacturer's protocol. After the final wash, the bound peptides were eluted using the
matrix solution and directly deposited onto the MALDI target plate. The plate was dried and
immediately used for data collection.
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Recrystallization of CHCA was done by dissolving approximately 5 mg/mL in ultrapure
ethanol and warmed to 40°C for 2 h with stirring. The bright yellow liquid was filtered using
Whatman # 1 paper and diluted to 50% with ultrapure water and left overnight at 4°C.
CHCA precipitates out of the solution. The precipitated material was collected by filtering
and vacuum dried.
2.8.3. Data collection—Mass spectroscopic data collection was done using Applied
Biosystem's PerSeptive voyager DE-PRO mass spectrometer (MALDI-TOF). Data was
collected at 3 GHz laser repetition rate, in reflector mode, positive polarity, delayed
extraction (resolution = 8000) mode at 25 kV accelerating voltage. The ion signals were
recorded under delay time, and grid voltages set depending on the standards for accurate m/z
values (routinely the extraction delay time is at 150-180 ηsec, grid voltage at 70-77%, and
grid wire voltage set at 0.01-0.02%). Nitrogen laser pulse at 337 nm was used for sample
ionization and 200 laser shots / spectrum were averaged. After correcting for background
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and noise levels the m/z values of the collected spectrum were externally calibrated with
bradykinin (monoisotopic [M+H]+ - 1060.57 Da), angiotensin I (monoisotopic [M+H]+
-1296.69 Da), ACTH (1-17) - (monoisotopic [M+H]+ - 2093.08 Da), ACTH (18-39) –
(monoisotopic [M+H]+ - 2465.20 Da) and internally calibrated using known trypsin auto
digest peptides (3 point calibration, m/z 842.51, m/z 1045.564 and m/z 2211.105). The
trypsin that is used in our in-gel digestion protocol is TPCK (N-p-tosyl-L-phenylalanine
chloromethyl ketone) treated and reductively methylated to limit auto digestion. An empty
gel spot was always digested with excess of trypsin in order to identify the trypsin auto
digest peptides under our experimental conditions.
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2.8.4. Database search—Database search was done with MASCOT (Matrix Science)
peptide mass fingerprint software with peptides in the monoisotopic [M+H]+ mass range of
800-3500 Da. The target protein was identified using NCBI database, taxonomy restricted to
Rattus, missed cleavage set at maximum of four (in-gel digestions are often incomplete),
enzyme chosen, cysteine modification with iodoacetamide and monoisotopic tolerance at 50
ppm. This protocol had consistently given us correct identification of known standards, and
therefore was applied for identifying unknown peptides. Any identification with more than 6
peptides matching, and at least 3 overlapping peptides, were considered positive
identification in order to eliminate bias and maintain stringency in data analysis. The masses
of those fragments were not included in the search if the isotopic pattern did not follow the
typical mass range pattern. Each fragment mass pattern was individually analyzed manually
prior to accepting for inclusion in database search, and the known trypsin peptides were
manually eliminated from the search.
3. Results
3.1. Proteome changes in DOX-treated adult rat cardiomyocytes
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The changes in protein levels in doxorubicin-treated cardiomyocytes were documented by
using the 2D gel analysis (Figure 1). We have previously shown that treatment with DOX at
clinically relevant concentrations (0.5 μM) induced ROS production and subsequently
apoptosis in a caspase-3-dependent manner. We analyzed protein expression changes using
2D gel resolution in isolated adult rat cardiomyocytes under the same conditions. Significant
changes occurred after a 48 h DOX treatment, as compared with untreated control and 24 h
DOX treatment. Proteins identified include those that are involved in apoptosis, energy
metabolism, stress response and signaling. We identified four types of protein changes that
were either upregulated, differentially upregulated, differentially downregulated, or
downregulated after DOX treatment. The upregulated proteins were detected only in DOXtreated cells but not in control cells, representing treatment response proteins and modified
proteins. Differentially upregulated proteins were those that were present at higher quantities
in DOX-treated cells; the differentially downregulated were quantitatively less in DOXtreated cells. The differentially regulated proteins could also be representing a group of
stress response proteins that are different from the upregulated or downregulated proteins.
Finally, the downregulated proteins (minor) were lost due to DOX treatment. These changes
were pronounced at 48 h treatment as shown in Figure 1 and to the best of our knowledge,
this represents the first documented DOX induced changes in proteome in adult
cardiomyocytes.
Figure 2 demonstrates differentially downregulated and downregulated proteins. There were
several spots that yielded very good mass spectral data (not shown) but did not yield
accurate identification. It is likely that the spot represents a complex mixture of modified
proteins. Table 1 indicates that the proteins identified are stress responsive (ATP synthase,
enolase alpha, Alpha B-crystallin, translocation protein 1, Stress induced phosphoprotein 1)
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or apoptotic / cell damage markers (p38 alpha, lipocortin, voltage dependent anion selective
channel protein 2, creatine kinase, MTUS1). Also, F1-ATP synthase upon DOX treatment
was observed in differentially downregulated (P10719), differentially upregulated
(AAA40778) and upregulated (AAA40778) list. This is due to the fact that protein P10719
is the precursor of protein AAA40778 that probably undergoes modification (differentially
upregulated) and cleavage (differentially downregulated). The amount of the observed
protein that in the modified or cleaved form were quantitatively very small as compared to
the P10719, indicating the occurrence of a dynamic process. Another differentially
downregulated protein, ETF-β (Spot 22, Table 1), might also be downregulated through a
putative nitrative modification on the single tyrosine residue (data not shown).
Figure 3 shows differentially downregulated protein displayed on 2D gel, along with the
densitometric scan to focus on the level of expression changes (due to the limitation of silver
staining technique, the data is to be considered representation of the gels shown here). It is
evident from this data that the effect of DOX induced proteome changes were profound at
48 h treatment under the conditions described. The largest drop in the expression is
MMSDH involved in valine and pyrimidine catabolic pathway.
3.2. Proteome changes in DOX-treated adult rat model
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In order to better understand the proteome changes observed in the DOX-treated
cardiomyocyte and DOX-treated adult rats, adult male rats were injected with DOX over a
period of 8 weeks. The analysis of the heart proteome revealed that although the protein
profile are strikingly similar on a 2D gel, the protein changes occurring are somewhat
different than those recorded in the cellular model (Figure 4). MALDI-TOF analyses of the
proteins indicated that major protein changes involve stress response proteins (Table 2). The
upregulated proteins in the animal model included molecular chaperone, hydroxylase,
oxidoreductases, cytoskeletal proteins, kinases, phospholipid transporter, endopeptidase and
transcription coactivator. The common protein changes between the in vitro cellular model
and the in vivo animal model are overexpression of beta enolase, troponin and alpha Bcrystallin. Other major protein changes were quite different from those identified in the cell
culture model.
3.3. Proteome analysis of DOX-treated cardiomyocytes with prefractionation of the cell
lysate
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The above models indicate that there are significant differences in proteome changes in
cellular and in vivo models of DOX cardiomyopathy. We investigated fractionation of
protein by solubilization technique in order to enrich less abundant proteins which might be
difficult to identify by traditional methods. In our solubilization methods and 2D gel
analysis of our cellular model, we were able to resolve many more proteins that were not
easily detected before (Figure 5). This is the result of enrichment of the protein; we are able
to load sufficient quantities of those proteins that were previously below the detection limit.
In addition, the changes on protein profile are quite dramatic for 0.5 μM DOX treatment for
48 h as compared to untreated control; however, DOX treatment at 20 μM concentration for
24 h had quantitatively higher level of a small number of proteins and this difference is more
pronounced in the cytoplasmic fraction than in other fractions.
4. Discussion
The cytotoxic drugs such as DOX are capable of causing cardiotoxicity due to generation of
free radicals, lipid peroxidation, immune modulation and apoptosis. DOX also binds to
several cellular and plasma proteins, modulating their functions [44,45]. Several models
including isolated cardiomyocyte culture [40,46], mitochondrial preparation [47] and animal
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model [9] have been used to study the mechanism of DOX-mediated cardiotoxicity. Protein
changes in any model may depend on the kind of secondary metabolites it generates [48].
Also, DOX accumulates in the nucleus besides mitochondria, thus interfering with nuclear
functions [49]. The excessive amount of oxidant generated caused oxidative stress that is
beyond the cellular antioxidant capacity, and therefore damages cell membranes due to lipid
peroxidation and alters the signaling pathways and protein expression. As a consequence of
this process, several proteins undergo changes that are critical for understanding DOXinduced damage. This study, using proteomic analysis, attempts to give insight into this
process.
Among the proteins identified, lipocortin V (annexin V) is an annexin family protein that is
found on the cytosolic side of the plasma membrane, and it is known to exhibit antiinflammatory effects by inhibiting the cytosolic phospholipase A2 activity [50]. Annexin V
(along with other isoform II and VI) is overexpressed in end stage heart failure [51], and it
binds to phosphatidylserine (PS) exposed on the apoptotic cell surface [52]. Alpha Bcrystallin, a 22 KDa protein found in rat heart, is a stress inducible molecular chaperone and
has been shown to salvage cardiomyocyte apoptosis [53]. It is actually a functional small
heat-shock protein induced by heat and other physiological stress and hyper-induced in
neurodegenerative disease such as Alzheimer's, Creutzfeldt-Jacob, and Parkinson's diseases
[54].
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The ETF-QO / ETFDH couple accepts electrons (2 × 1e-) from five acyl Co-A
dehydrogenases and four amino acid catabolism products through ETF-β. DOX treatment is
known to inhibit long chain fatty acid oxidation and transport across mitochondrial
membrane [55,56]. Loss of ETF-β in the DOX-treated samples and low levels of ETFDH
overexpression / modification detected in the 48 h sample could be a defense mechanism, as
ETFDH is a nuclear encoded mitochondrial protein and is regulated by signaling from
AMP-activated protein kinase that detect the low level ATP / AMP involved in fatty acid
oxidation and amino acid oxidation. Both of these are catabolic processes that result in
generation of energy to compensate for the loss of mitochondrial function [57]. MMSDH is
an aldehyde dehydrogenase in the valine and pyrimidine catabolic pathway. Fatty acid
acylation via myristate (C14:0) is a covalent modification of the active site cysteine that
inhibits the enzyme activity, and the level of inactivity varies with the metabolic state of the
mitochondria [58]. MMSDH has been identified as a marker for aging heart where it is
nitrated [59]. Deregulation of this protein was not previously reported, and thus the large
extent of downregulation reported here by DOX is a novel finding. The significance of its
downregulation might be consistent with the problems with fatty acid metabolism involving
ETF-β / ETFDH, deregulation of ETC, and loss of mitochondria that is damaged with DOX
treatment. The alternative explanation is that the modified (acylated / nitrated) protein
migrated differerently than the original protein. The role of nitrative stress in DOX-induced
cardiotoxicity has been reported (60,61). Taken together, protein modification by nitration
appears to play a crucial role in DOX mediated cardiomyocyte cell death.
The cardiomyopathy results also from a loss of F-actin network (due to depolymerization)
which coordinates troponin (F-actin binding protein) involved in controlling contraction.
Thus the modifications of troponin T such as phosphorylation could be a manifestation of
DOX-induced injury. DOX treatment suppresses alpha actin, troponin I (resulting in
myofibrillar loss) [29], sarcoplasmic reticulum Ca2+ ATPase, calcium gated Ca2+ release
channel (resulting in impaired Ca2+ handling and perturbation of contraction and relaxation
cycle) [62], Rieske Fe-S protein, ADP / ATP translocase, phosphofructokinase, creatine
kinase M isoform (resulting in energy impairment) [31]. It is worth noting that the animals
treated for 10 wks with DOX did not survive well with the treatment plan. In this animal
model, the anti-apoptotic protein HSP27 that confer resistance to DOX and other anticancer
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drugs was downregulated [63,64]. Suppression of this protein in the animal model in the
presence of DOX is of interest with respect to the apoptotic mechanism. In our
cardiomyocyte model, p38 is highlighted in the overexpressed protein list, and the, blot
analysis shows that this protein gets phosphorylated with time upon DOX treatment (data
not shown). Phosphorylation activates p38 kinase, and it in turn phosphorylates many
substrates including HSP27 thus regulating apoptosis [65,66].
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Doxorubicin-treated cardiomyocyte mitochondrial preparation showed a small but
significant decrease in the complex III activity, while the complex I activity remained
unaffected (data not shown). It is possible that under our experimental conditions,
superoxide generated could be at both complexes I and III, but complex III is also affected
by DOX effects on ETFDH and ETF-β. We attempted to confirm if the change in ETFDH
levels are true overexpression of ETFDH or some modification by testing the untreated
control and DOX-treated sample on a Western blot (data not shown) using an ETFDH
antibody (kindly donated by Rikke K.J. Olsen, Aarhus University Hospital and Faculty of
Health Sciences, Denmark). We could not detect any marked modification. There is,
however, a quantitative increase in ETFDH levels in DOX-treated samples. Therefore it is
possible that the ETF-QO protein levels and loss of ETF-β protein levels drastically affect
complex III ability to function properly, thus increasing the chances of superoxide generated
at this site. In addition, generation of superoxide at complex I site could be due to redox
cycling of DOX. It would be interesting to obtain a direct evidence to prove this point, as
superoxide generated at complex I and complex III (Qi center) is released towards the matrix
where glutathione and other oxidant protection machinery are present, while the superoxide
generated from complex III (Qo center- quinol oxidase) are released to the inner membrane
side [67,68]. DOX also affects highly unsaturated fatty acids, desaturating and elongating
enzymes that are involved in the biosynthesis of essential fatty acids [69]. Increased ROS
formation in the mitochondria triggers the intrinsic pathway that leads to the opening of
transition pores, and the process is favored by oxidation of glutathione and other sulfhydryls
[70]. Events such as activation of BAK (BCL-2 antagonist / Killer 2) by BCL2-Homology
(BH3)-only proteins translocate to mitochondrial outer membrane. This results in the
translocation of cytochrome C to cytoplasm where it triggers the formation of apoptosome in
association with Apaf1 (apoptotic protease activating factor 1) protein. Apoptosome
activates caspase 9 which then activates the effector caspase 3 resulting in apoptosis.
NIH-PA Author Manuscript
Another important downregulated protein in response to DOX treatment was VDAC2. It is
well known that VDAC2 plays a critical role in mitochondrial apoptosis. VDAC2 is a
member of anion channel proteins (porins) residing in the outer mitochondrial membrane.
These proteins are involved in regulation of metabolic interactions and solutes exchange
between mitochondria and cytosol, and in regulating the permeabilization of outer
mitochondrial membrane during apoptosis. Permeabilization of the membrane is known to
initiate the release of cytochrome c into the cytosol and initiate mitochondrial apoptotic
pathway. VDAC2 is an isoform that is present in low abundance in the outer mitochondrial
membrane and specifically interacts with a multidomain proapoptotic member of Bcl-2
family of proteins BAK (71). This interaction prevents oligomerization of BAK, the event
that leads to the formation of outer mitochondrial membrane pores and leaking of
cytochrome c into the cytosol (71). Genetic depletion of VDAC2 resulted in excessive BAK
oligomerization and apoptotic cell death, while overexpression of this protein prevented
BAK activation and inhibited mitochondrial pathway (71,72). On the other hand, other
studies performed on mouse embryonic fibroblasts and other cell types, have shown that
VDAC2 recruits BAK to the mitochondrial membrane and is required for BAX- and BIDinduced apoptosis (73,74). The role of VDAC2 in the regulation of mitochondrial apoptotic
pathway is cardiomyocytes is not known. Interestingly, another proteomic study reported
upregulation of VDAC2 abundance in hepatoma cells as a result of the action of
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hepatocarcinogenic dioxin compound (75). The fate of the regulation of VDAC2 expression
in cardiomyocytes has not been described previously. It has been known from our previous
publication that doxorubicin induces an abundant release of cytochrome c into the cytosol in
both adult and neonatal cardiomyocytes (76). Changed expression of this protein by
doxorubicin could clearly be an important factor regulating the mitochondrial apoptotic
pathway in cardiomyocytes.
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It is interesting to note that the proteomic changes in DOX-treated cardiomyocyte and rat
heart are not similar. The exact reasons for this difference are not clear. However, the
cultured myocytes were treated with DOX for 24 or 48 h, whereas in vivo cardiac tissues
were harvested 7 days after the last injection. Under these conditions, no DOX was present
in the cardiac tissues isolated from rats. In contrast, DOX was continuously present during
in vitro cardiomyocyte cell culture experiments (thus generating oxy-radicals continuously).
Thus, the difference between DOX-induced effects in cardiomyocyte culture and in vivo
animals could be due to differences in the amount and duration of DOX exposure. Another
reason for the observed difference might also be possibly due to the fact that the adult rat
heart tissue has more protein in the whole tissue extract. Therefore, we investigated other
methods of sample preparation to improve protein identification. The subcellular
fractionation is a useful technique, but isolated fractions are often contaminated with other
subcellular organelles. Charge fractionations have protein overlaps, and chromatographic
fractionation requires a large amount of sample. On the contrary, solubilization techniques
are simple, highly reproducible, and enrich low abundant proteins in an inexpensive and
reproducible manner. It is also advantageous to further separate them on narrow-range pH
strips to further resolve the proteins. Our experimental results in Figure 5 clearly document
the advantage of such a technique which will help in identifying the changes occurring in the
low abundant proteins.
5. Conclusions
The global proteomic analyses of DOX-treated cardiomyocytes and heart tissues isolated
from DOX-treated adult rat indicate different changes in proteome. The common protein
changes between the in vitro cellular model and the in vivo animal model are overexpression
of beta enolase, troponin and alpha B-crystallin. Other major protein changes detected in
tissues were much different from those identified in the cell culture model.
Acknowledgments
This work was made possible with the help of a NIH grants (R01CA152810) and (1UL1RR031973).
NIH-PA Author Manuscript
Abbreviations
ACTH
adrenocorticotropic hormone
ADP
adenosine diphosphate
AMP
adenosine monophosphate
ATP
adenosine triphosphate
DOX
doxorubicin
ETC
electron transport chain
ETF
electron transfer flavoprotein
ETF-QO / ETFDH
electron transfer protein dehydrogenase
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HSP27
heat shock protein 27 KDa
MALDI-TOF
matrix assisted laser desorption and ionization – time of flight
MMSDH
methylmalonate semialdehyde dehydrogenase
PBS
phosphate buffered saline
p38 alpha
Mitogen activated protein kinase p38 alpha
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Figure 1. Upregulated and differentially upregulated proteins in DOX-treated adult rat
cardiomyocytes
Silver stained 12% SDS-PAGE (first dimension pI 3-10) analysis of adult rat cardiomyocyte
cultured for 48 h without treatment (control), 0.5 μM DOX treatment for 24 h and 0.5 μM
DOX treatment for 48 h. Spots are marked as upregulated and differentially upregulated
(DUR) protein. Upregulated proteins are those protein spots that are present in the 48 h
DOX treatment and not present in the untreated control gel and DUR are the proteins present
in both untreated and treated cells but present quantitatively more in the DOX treatment.
Some of the spots were represented in bold to indicate the pI / molecular weight matches
across the gel.
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Figure 2. Downregulated / differentially downregulated proteins in DOX-treated adult rat
cardiomyocytes
Silver stained 12% SDS-PAGE demonstrates the differentially downregulated (DDR) and
the downregulated (DR) protein changes in DOX-treated cardiomyocytes. DR proteins are
proteins present only in the untreated control but not in 48 h DOX treatment and DDR are
the proteins present in both untreated and treated cells but present quantitatively more in the
control gels.
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Figure 3. Quantitative evaluation of DOX-treated adult rat cardiomyocytes
Representative changes in protein levels with DOX treatment for 24 h and 48 h as compared
to control. The changes are also expressed as densitometric quantification values (average of
two independent assays). The spots were quantified from 300 dpi images from the Kodak
gel documentation system. Prior to staining, the gels were run in duplicate and stained in an
identical manner.
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Figure 4. Proteome changes in DOX-treated adult rat
Adult rats were treated with weekly injections of saline or DOX for 8 weeks as described in
materials and methods. The heart tissue was collected at the end of experiment and whole
tissue extract was prepared and resolved on a 2D gel and silver stained. Protein
concentration equivalent to 300 μg was used for each gel.
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Figure 5. Prefractionated samples of DOX-treated adult rat cardiomyocytes – 2D gel analysis
Two dimensional gel analysis of different solubilization extracts. Protein extract of 300 μg
was resolved and silver stained. The controls were 48 h treated with DMSO and DOX
treatment were for the indicated period.
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Table 1
Protein changes in DOX-treated adult rat cardiomyocytes
Accession #
Molecular function
Matched / Measured peptides
score
Kumar et al.
Proteins identified to undergo changes due to 48 h DOX treatment. Some of the unknown proteins are included since their mass spectrum is very good yet
the data base search does not yield a significant ID.
Upregulated proteins (DOX treated cardiomyocytes)
J Proteomics. Author manuscript; available in PMC 2012 May 1.
Spot # 1
Alpha B-crystallin (19.9KDa, pl 6.8)
CAA42911
Structural protein / Molecular chaperone
11 / 46 - 22% coverage
68
Spot # 2
Alpha B-crystallin (19.9KDa, pl 6.8)
AAP31996
Structural protein / Molecular chaperone
11 / 46 - 22% coverage
68
Spot # 3
Potassium dependent sodium-calcium exchanger (NCKX2)
(19.7KDa, pl 6.5)
NP_113931
Transporter
26/44 - 54% coverage
212
Spot # 4
DNA damage inducible transcript 4-like / SMHS1 (Ddit4) (21.9KDa,
pl 6.8)
NP_536324
Energy metabolism / apoptosis regulator
9 / 33 - 12% coverage
64
Spot # 9
Troponin-T (TnnT2) (35.7KDa, pl 5)
NP_036808
Actin binding motor protein
16 / 46 -36% coverage
116
Spot # 11
Protease (prosome, macropain) 28 subunit, alpha (PSME1)
(28.8KDa, pl 5.6)
AAH60574
Proteosome activator
6 / 42 - 17% coverage
74
Spot # 19
Translocation protein 1 / AB2-292 (Tloc1, 45.9KDa, pl 6.9)
NP_001029301
Protein transporter activity
8/28 - 16% coverage
71
Spot # 22
MTSG1 346 amino acid isoform (Mtusl, 40.2KDa, pl 5.8)
AAO60218
Mitochondrial tumor suppressor gene may be
involved in regulating cell proliferation
17 / 42 - 34% coverage
124
Spot # 23
MTSG1 346 amino acid isoform (Mtusl, 40.2KDa, pl 5.8)
AAO60218
Mitochondrial tumor suppressor gene may be
involved in regulating cell proliferation
17 / 43 - 34% coverage
124
Spot # 24
Lamin C2 (Lmna, 52.6KDa, pl 6.2)
CAA67641
Intermediate filament (structural protein)
9 / 29 - 20% coverage
194
Spot # 25
Electron transfer flavoprotein ubiquinone oxidoreductase precursor
(ETFDH, 68.1KDa, pl 7.4)
AAQ67364
Oxidoreductase
8 / 42 - 16% coverage
71
Spot # 26
Stress induced phosphoprotein I (Stipl, 63.2 Kda, pl 6.4)
AAH61529
Chaperone
14 / 63 - 22% coverage
92
Spot # 29
MAP kinase 14 / p38 alpha (MAPK14, 40KDa, pl 5.9)
NP_112282
Non receptor serine threonine protein kinase
12 / 36 - 28% coverage
107
Spot # 31
F1-ATPase beta subunit (ATP5b, 38.8KDa, pl 5.1)
AAA40778
Ion channel hydrogen transporter
15 / 29 - 45% coverage
170
Spot # 34
Lactate dehydrogenase B (Ldhb, 36.9KDa, pl 5.7)
AAH59149
Dehydrogenase (oxidoreductase)
10 / 44 - 26% coverage
64
Differentially upregulated proteins (DOX treated cardiomyocytes)
DUR # 11
Beta enolase (ENO3, 47KDa, pl 7.53)
CAA68788
Lyase
19 / 48 - 40% coverage
173
DUR # 14
F1- ATPase beta subunit (ATP5b, 56.3KDa, pl 4.9)
AAA40778
ATP synthase
9 / 48 - 24% coverage
104
DUR # 15
Actin gamma -2 (Actg2, pl 5.2)
NP_037025
Actin and actin related protein
7 / 38 - 17% coverage
97
Enolase alpha (Eno1, pl 6.5)
AAH78896
Lyase
18 / 38 - 43% coverage
176
Lipocortin V (Anxa5, pl 4.65)
AAC06290
Transfer/carrier protein
10 / 32 - 26% coverage
158
DUR # 18
Creatine kinase (Ckm, 43KDa, pl 7.1)
AAA40936
Transferase
7 / 46 - 14% coverage
148
DUR # 20
Lactate dehydrogenase B (Ldhb, pl 5.92)
AAH59149
Dehydrogenase
8 / 44 - 22% coverage
159
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DUR # 16
DUR # 17
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Accession #
Molecular function
Matched / Measured peptides
score
NIH-PA Author Manuscript
DDR # 21
ATP synthase, H+ transporting, mitochondrial F1 complex, beta
precursor (Atp5b, 56.3KDa, pl 4.92)
P10719
ATP synthase
21 / 45 - 60% coverage
209
DDR # 22
Electron transfer flavoprotein beta subunit (ETF-β, pl 7.9)
NP_001004220
Hydroxylase
11 / 29 - 44% coverage
110
DDR # 25
Methylmalonate semialdehyde dehydrogenase (MMSDH,
ALDH6A1, 57.8KDa, pl 7.9)
Q02253
Aldehyde dehydrogenase member
16 / 30 - 34% coverage
163
DDR # 26
Voltage dependent anion selective channel protein 1 (Vdac1, pl 8.6)
AAH72484
Anion channel / Voltage gated ion channel
10 / 29 - 54% coverage
201
Kumar et al.
Differentially downregulated proteins (DOX treated cardiomyocytes)
Downregulated proteins (DOX treated cardiomyocytes)
J Proteomics. Author manuscript; available in PMC 2012 May 1.
DR # 27
Voltage dependent anion selective channel protein 2 (Vdac2,
31.8KDapl 7.5)
P81155
Anion channel / Voltage gated ion channel
8 / 77 - 34% coverage
74
DR # 28
Pleckstrin homology domain containing family A (PI binding
specific) member 8 (Plekha8, pl 4.7)
NP_001102705
Nucleic acid binding / Transfer protein
6 / 41 - 28% coverage
81
Protein score is -10*log(P) where P is the probability that the observed match is a random event.
Protein scores greater than 61 are significant (P<0.05).
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Table 2
Protein changes in DOX-treated adult rat
Accession #
Molecular function
Matched / Measured peptides
Score
Alpha B-crystallin
CAA42911
Structural protein / Molecular chaperone
5 / 32 - 28% coverage
70
Kumar et al.
Protein changes in 8 weeks DOX-treated and control animal model. Downregulated proteins were fewer than the upregulated or modified proteins.
Upregulaled proteins (DOX treated adult rat)
Spot # 1
J Proteomics. Author manuscript; available in PMC 2012 May 1.
Spot # 3
Similar to β-enolase
CAA68788
Lyase
13 / 36 - 28% coverage
132
Spot # 4
14-3-3 protein ε-chain
A60031
Cytoplasmic chaperones / Activator of tyrosine and
tryptophan hydroxylases
8 / 53 - 36% coverage
116
Spot # 5
Tyrosine 3-monooxgenase / tryptophan 5-monooxgenase activation
protein, β-polypeptide
AAH76502
Cytoplasmic chaperones / Activator of tyrosine and
tryptophan hydroxylases
6 / 59 - 24% coverage
68
Spot # 6
Catalase
P04762
Aminoacylase activity / Oxidoreductase activity
5 / 28 - 22% coverage
67
Spot # 7
Actin
Spot # 8
H+-transporting
two-sector ATPase β-chain, mitochondrial - rat
NP_062056
Structural protein
22 / 47 - 45% coverage
170
A28701
ATP-Synthase
12 / 36 - 35% coverage
122
P07943
Aldehyde reductase / Oxidoreductase
5 / 55 - 12% coverage
71
(fragment)
Spot # 9
Aldose reductase (AR) (Aldehyde reductase)
Spot # 10
Proteasome subunit-α type 3 (Proteasome component C8)
P18422
Endopeptidase (threonine type)
5 / 12 - 20% coverage
84
Spot # 11
Macropain / Transactivating protein BRIDGE
AAD32925
Transcription co-activator
7 / 43 - 26% coverage
74
Spot # 12
Macropain / Transactivating protein BRIDGE
AAD32925
Transcription co-activator
7 / 47 - 26% coverage
74
Spot # 13
Apolipoprotein A-I precursor (Apo-AI)
CAA25224
Phospholipid transporter activity
7 / 31 - 25% coverage
71
Spot # 14
Creatine kinase, sarcomeric mitochondrial precursor (S-MtCK)
P09605
Kinase / Nucleotide binding
9 / 65 - 19% coverage
63
Spot # 15
Similar to CCAAT/enhancer binding protein-α
CAA31242
Transcription factor
6 / 42 - 22% coverage
68
Spot # 16
Lactate dehydrogenase
P42123
Oxidoreductase
14 / 44-28% coverage
98
Spot # 18
ATP synthase beta subunit
AAB02288
ATP-Synthase
12 / 42-35% coverage
128
Spot # 20
Neurofilament triplet L protein
A21762
Cytoskeleton
10 / 74 - 31% coverage
104
Spot # 31
Troponin T2
NP_036808
Cytoskeleton / Protein binding
15 / 32 - 36% coverage
92
Downregulated proteins (DOX treated adult rat)
Spot # 24
HSP27
NP_114176
Heat shock protein / Anti-apoptosis
7 / 44-11% coverage
64
Spot # 35
Phosphocholine cytidylyltransferase A
P19836
Regulatory rate limiting enzyme
11 / 38-22% coverage
86
Protein score is -10*log(P) where P is the probability that the observed match is a random event.
Protein scores greater than 61 are significant (P<0 05).
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Table 3
Accession #
Molecular function
Matched / Measured peptides
Score
J Proteomics. Author manuscript; available in PMC 2012 May 1.
Spot # 1
Alpha B-crystallin
CAA42911
Structural protein / Molecular chaperone
5 / 32 - 28% coverage
70
Spot # 3
Similar to β-enolase
CAA68788
Lyase
13 / 36 - 28% coverage
132
Spot # 4
14-3-3 protein ε-chain
A60031
Cytoplasmic chaperones / Activator of tyrosine and
tryptophan hydroxylases
8 / 53 - 36% coverage
116
Spot # 5
Tyrosine 3-monooxgenase / tryptophan 5-monooxgenase activation
protein, β-polypeptide
AAH76502
Cytoplasmic chaperones / Activator of tyrosine and
tryptophan hydroxylases
6 / 59 - 24% coverage
68
Spot # 6
Catalase
P04762
Aminoacylase activity / Oxidoreductase activity
5 / 28 - 22% coverage
67
Spot # 7
Actin
NP_062056
Structural protein
22 / 47 - 45% coverage
170
Spot # 8
H+-transporting two-sector ATPase β-chain, mitochondrial - rat
(fragment)
A28701
ATP-Synthase
12 / 36 - 35% coverage
122
Spot # 9
Aldose reductase (AR) (Aldehyde reductase)
P07943
Aldehyde reductase / Oxidoreductase
5 / 55 - 12% coverage
71
Spot # 10
Proteasome subunit-α type 3 (Proteasome component C8)
P18422
Endopeptidase (threonine type)
5 / 12 - 20% coverage
84
Spot # 11
Macropain / Transactivating protein BRIDGE
AAD32925
Transcription co-activator
7 / 49 - 26% coverage
74
Spot # 12
Macropain / Transactivating protein BRIDGE
AAD32925
Transcription co-activator
7 / 47 - 26% coverage
74
Spot # 13
Apolipoprotein A-I precursor (Apo-AI)
CAA25224
Phospholipid transporter activity
7 / 31 - 25% coverage
71
Spot # 14
Creatine kinase, sarcomeric mitochondrial precursor (S-MtCK)
P09605
Kinase / Nucleotide binding
9 / 65 - 19% coverage
63
Spot # 15
Similar to CCAAT/enhancer binding protein-α
CAA31242
Transcription factor
6 / 42 - 22% coverage
68
Spot # 16
Lactate dehydrogenase
P42123
Oxidoreductase
14 / 44-28% coverage
98
Spot # 18
ATP synthase beta subunit
AAB02288
ATP-Synthase
12 / 42-35% coverage
128
Spot # 20
Neurofilament triplet L protein
A21762
Cytoskeleton
10 / 74 - 31% coverage
104
Spot # 31
Troponin T2
NP_036808
Cytoskeleton / Protein binding
15 / 32 - 36% coverage
92
Kumar et al.
Upregulated proteins (Doxorubicin-treated adult rat)
Downregulated proteins (Doxorubicin-treated adult rat)
Spot # 24
HSP27
NP_114176
Heat shock protein / Anti-apoptosis
7 / 44-11% coverage
64
Spot # 35
Phosphocholine cytidylyltransferase A
P19836
Regulatory rate limiting enzyme
11 / 38-22% coverage
86
Protein score is -10*log(P) where P is the probability that the observed match is a random event.
Protein scores greater than 61 are significant (P<0.05)
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