CARDIOVASCULAR JOURNAL OF AFRICA • Vol 22, No 4, July/August 2011
176
AFRICA
ised to prevent blood clotting. Mitochondria were isolated from
mouse heart tissues (for both obese and age-matched lean
controls) using a mitochondrial isolation kit (Sigma-Aldrich, St.
Louis MO) that allows for rapid isolation of enriched mitochon-
drial fractions for proteomic studies.
6,7
Approximately 120 mg of
left ventricular tissue was dissected out from control and obese
mice and weighed, cut into smaller pieces and resuspended in
10 volumes of extraction buffer A (10 mM HEPES: pH 7.5, 200
mM mannitol, 70 mM sucrose, 1 mM EGTA).
We added 0.25 mg/ml trypsin followed by incubation on ice
for three minutes. After the samples were centrifuged for 15
seconds in a microfuge, the supernatant was removed by aspira-
tion, eight volumes of extraction buffer A (containing 0.25 mg/
ml trypsin) was added to the pellet and it was incubated on ice
for 20 minutes. To quench proteolytic reactions, an albumin solu-
tion was added to a final concentration of 10 mg/ml. Samples
were thereafter centrifuged for 15 seconds in a microfuge,
the supernatant was aspirated, and the pellet was washed with
eight volumes of extraction buffer A and re-centrifuged for 15
seconds. The latter step was repeated once more, where after the
pellet was homogenised 20 to 30 times using a glass homogen-
iser.
The homogenised sample was subsequently centrifuged at
1 000
×
g
for five minutes, and the supernatant was collected
and re-centrifuged at 3 500
×
g
for 10 minutes. The mitochon-
drial pellet was resuspended (10 mM HEPES: pH 7.4, 250 mM
sucrose, 1 mM ATP, 0.08 mM ADP, 5 mM sodium succinate,
2 mM K
2
HPO
4
, 1 mM DTT) and protein concentrations were
determined using the Bradford assay. We typically obtained
1 000
µ
g of mitochondrial proteins for our proteomic analyses.
The ReadyPrep 2D CleanUp kit (Bio-Rad, Hercules CA) was
used to concentrate mitochondrial proteins by precipitation and
also to wash away compounds that may have interfered with
the isoelectric focusing (IEF) step. After precipitation, proteins
were washed and then resuspended in an IEF/2D-compatible
sample buffer [8 M urea, 2% CHAPS, 50 mM DTT, 0.2% (w/v)
Bio-Lyte 3/10 ampholyte, 0.002% (w/v) bromophenol blue] and
protein concentrations were determined using the RC/DC assay
(Bio-Rad, Hercules CA). The complete mitochondrial isolation
procedure was performed at 4ºC with ice-cold solutions.
Two-dimensional polyacrylamide gel electrophoresis
(2D-PAGE)
The first dimension was performed using the PROTEAN-IEF
cell (Bio-Rad, Hercules CA). Samples containing 100
µ
g mito-
chondrial protein were loaded onto 11-cm immobilised pH
gradient strips (pH 5–8) (Bio-Rad, Hercules CA). Three experi-
ments were performed for each condition (i.e. control vs obese).
Strips were rehydrated under passive conditions for 12 hours at
20ºC and focused at 200 V for 20 minutes. Thereafter they were
linearly increased over two hours to a maximum of 8 000 V and
then run (rapid ramping) to accumulate a total of 40 000 V/h.
Prior to the second dimension, the immobilised pH gradient
strips were first equilibrated for 15 minutes in a buffer contain-
ing 0.375 M Tris-HCL (pH 8.8), 6 M urea, 2% SDS, 20% glyc-
erol and 2% (w/v) DTT, followed by equilibration for another
15 minutes in a buffer containing 0.375 M Tris-HCL (pH 8.8), 6
M urea, 2% SDS, 20% glycerol and 2.5% (w/v) iodoacetamide.
Subsequently, the strips were embedded in 0.5% low-melting
point agarose containing 0.003% bromophenol blue (Bio-Rad,
Hercules CA) on the top of Criterion XT 4–12% precast Bis
Tris gels (Bio-Rad, Hercules CA), containing a 4% stacking
gel. Electrophoresis was performed at 200 V; constant for 55
minutes. Gels were stained overnight with Brilliant Blue G
colloidal concentrate (Sigma-Aldrich, St. Louis MO) in order to
visualise the protein spots.
Identification of mitochondrial proteins
To obtain gel images, the gels were scanned with a GS-800 cali-
brated densitometer (Bio-Rad, Hercules CA) using the Quantity
One-4:5.2 (basic) software program (Bio-Rad, Hercules CA).
The gel images were subsequently analysed using the PDQuest
version 8 software program (Bio-Rad, Hercules CA) to identify
differentially expressed protein spots on the gels from control
and obese mice samples.
Protein spots of interest (i.e. only differentially expressed
ones) were manually excised, de-stained and subjected to
in-gel digestion by trypsin, followed by ElectroSpray-Injection-
Quadrupole Time of Flight (ESI-Q-TOF) mass spectrometry.
Fig. 1. Two-dimensional PAGE patterns of mitochondrial proteins. Heart mitochondria were isolated from normal
and obese female mice and purified from interfering substances with the first-dimension separation. Proteins were
separated by two-dimensional PAGE and detected by Coomassie staining. The spots containing proteins that were
subsequently identified are numbered. A: control and B: obese. Molecular weight is given in kDa.
