Mitochondrial changes play important roles in human ovarian cancers. This protocol establish an effective platform to isolate and purify mitochondria from human ovarian cancer and the control tissues for large-scale proteomics analysis. Mitochondria, as a centers of energy metabolism, cell signaling, and oxidative stress, insight into mitochondrial proteome changes in ovarian cancers benefit our understanding molecular mechanism, and is a discovery of effective biomarkers and the therapeutic targets.
This technique use differential-speed centrifugation in combination with density gradient centrifugation to separate mitochondria from human ovarian cancer in the control tissues, which results in high quality mitochondria samples for quantitative proteomics analysis. The mitochondria preparation, coupled with iTRAQ quantitative proteomics, have been successfully used in analysis of human ovarian cancer tissue, which easily translates to analyze other tissue, mitochondria proteomics. Those who have never performed this method may find that it can be difficult to isolate mitochondria from ovarian control than cancer tissues.
It's necessary to add an trypsin to improve control tissue homogenization and add an extra layer of density gradient medium to improve mitochondria separation. To begin this procedure, prepare 250 milliliters of the mitochondrial isolation buffer as outlined in the text protocol. Place approximately 1.5 grams of ovarian cancer tissues in a clean glass dish.
Add two milliliters of pre-chilled mitochondrial isolation buffer to lightly wash the blood from the tissue surface. Repeat this wash three times. Then, use clean ophthalmic scissors to fully mince the tissue into pieces that are approximately one cubic millimeter, and transfer the minced tissues into a 50 milliliter centrifuge tube.
Add 13.5 milliliters of mitochondrial isolation buffer containing nagarse at a concentration of 0.2 milligrams per milliliter. Use an electronic homogenizer to homogenize the minced tissues for two minutes at four degrees Celsius. After this, add another three milliliters of mitochondrial isolation buffer to the tissue homogenates and mix them well by pipetting.
Centrifuge the prepared tissue homogenate at 1, 300 xg, and at four degrees Celsius for 10 minutes. Remove the pellet and keep the supernatant. Next, centrifuge the supernatant at 10, 000 xg and at four degrees Celsius for 10 minutes.
Remove the microsome-containing supernatant, and keep the pellet. Add 2 milliliters of mitochondrial isolation buffer and re-suspend the pellet thoroughly by light pipetting. Centrifuge this pellet suspension at 7, 000 xg and at four degrees Celsius for 10 minutes.
Discard the supernatant, and keep the pellet, which contains the crude mitochondria. Add 12 milliliters of 25%density gradient medium to re-suspend the extracted crude mitochondria. Make a discontinuous density gradient containing the re-suspended crude mitochondria as outlined in the text protocol, and centrifuge it at 52, 000 xg, and at four degrees Celsius for 90 minutes.
Use a long and blunt syringe to collect the purified mitochondria at the interface between the 25%and 30%gradient mediums, and transfer this to a clean tube. Add mitochondrial isolation buffer to the collected mitochondria to dilute it to a three-fold volume. Centrifuge at 15, 000 xg, and at four degrees Celsius for 20 minutes.
Discard the supernatant, and keep the pellet. Collect the final pellet which contains the purified mitochondria, and store it at 20 degrees Celsius. First, prepare 250 milliliters of the mitochondrial isolation buffer as outlined in the text protocol.
Add approximately 1.5 grams of the normal control ovarian tissues to a clean glass dish. Add two milliliters of pre-chilled mitochondrial isolation buffer to lightly wash the blood from the tissue surface. Repeat this wash three times.
Using clean ophthalmic scissors, fully mince the tissue into pieces that are approximately 1 cubic millimeter, and transfer the minced tissues into into a clean 50 milliliter tube. Add eight milliliters of a solution containing 0.05%trypsin and 20 millimolar EDTA and PBS to the minced control tissues, and digest at room temperature for 30 minutes to help lyse the cells and release the mitochondria. Centrifuge at 200 xg for five minutes.
Discard the supernatant, and keep the tissues and cells. Add 13.5 milliliters of mitochondrial isolation buffer containing nagarse at a concentration of 0.2 milligrams per milliliter, and use an electric homogenizer to homogenize the minced tissues at four degrees Celsius for two minutes. Add another three milliliters of mitochondrial isolation buffer to the tissue homogenates and mix thoroughly by pipetting.
Centrifuge the prepared tissue homogenate at 1, 300 xg and at four degrees Celsius for 10 minutes. Remove the pellet and keep the supernatant. Centrifuge the supernatant at 10, 000 xg and at four degrees Celsius for 10 minutes.
Remove the supernatant, which contains the microsomes, and keep the pellet. Then, add 2 milliliters of mitochondrial isolation buffer and re-suspend the pellet thoroughly by light pipetting. Centrifuge this pellet suspension at 7, 000 xg and at four degrees Celsius for 10 minutes.
Discard the supernatant and keep the pellet which contains the crude mitochondria. Add 12 milliliters of 25%density gradient medium to re-suspend the extracted crude mitochondria. Make a discontinuous density gradient containing the crude mitochondria as outlined in the text protocol, and centrifuge it at 52, 000 xg and at four degrees Celsius for 90 minutes.
Use a long and blunt syringe to collect the purified mitochondria in the range from the interface between the 25 and 30%gradient mediums to the interface between the 34 and 38%gradient mediums. Transfer the purified mitochondria to a clean tube. Add mitochondrial isolation buffer to the collected mitochondria to dilute it to a three-fold volume.
Centrifuge at 15, 000 xg and at four degrees Celsius for 20 minutes, and discard the supernatant. Add 2 milliliters of mitochondrial isolation buffer to re-suspend the pellet and centrifuge at 15, 000 xg and at four degrees Celsius for 20 minutes. Discard the supernatant and keep the pellet.
Collect the final pellet and store it at 20 degrees Celsius. In this study, high quality mitochondrial samples are prepared from human ovarian cancer and control tissues for large-scale quantitative proteomics. There are a few differences in the preparation of the mitochondria from ovarian cancer tissues and control ovarian tissues, including using a different discontinuous density gradient.
After centrifugation, the purified mitochondria from ovarian cancer tissues are found at the interface between 25%and 30%while those from control ovarian tissues are found in the range from the interface between 25%and 30%to the interface between 34%and 38%The quality of the prepared mitochondria is then evaluated with differential speed centrifugation and density gradient centrifugation via electron microscopy. The electron microscope images demonstrate that in both ovarian cancers and control ovarian tissues, the main organelles isolated are mitochondria, except for a small quantity of peroxisomes. However, the morphology of the mitochondria is seen to change more in ovarian cancers than control ovarian tissue.
The quality of the prepared mitochondria is also evaluated via western blot. The western blot images also demonstrate that the major component in prepared mitochondrial samples from ovarian cancers and control ovaries is mitochondria, except for a small quantity of peroxisomes, which is consistent with the electron microscopy analysis. It is reasonable for peroxisomes to be contained in the prepared mitochondria, because mitochondria interact extensively with peroxisomes, which in turn reflects the functional completeness of the mitochondria.
These results demonstrate the high quality of the prepared mitochondrial samples. When I'm performing this procedure, it's very important that you fully mince tissues before homogenization, and add the trypsin to the minced control tissues. It's also important that you make and or prepared discontinuous density gradient for the cancer samples and the controls.
Following this procedure, iTRAQ or TMT quantitative proteomics or phosphoproteomics can be performed to clarify ovarian cancer mitochondria proteome profile, and phosphoproteome profile can be established to clarify the roles of mitochondria in ovarian cancer in great depths. This technique pave the way for researchers to systematically study mitochondria proteome and the phosphoproteome in ovarian cancer, construct the signaling pathway network system, and discover the reliable biomarkers and the therapeutic targets.
