This work was supported by the Hunan Provincial Hundred Talent Plan (to X.Z.), the Xiangya Hospital Funds for Talent Introduction (to XZ), the National Natural Science Foundation of China (Grant No. 81572278 and 81272798 to XZ), the grants from China &#34;863&#34; Plan Project (Grant No. 2014AA020610-1 to XZ), and the Hunan Provincial Natural Science Foundation of China (Grant No. 14JJ7008 to XZ). X.Z. conceived the concept for the present manuscript, obtained the iTRAQ quantitative proteomics data of mitochondria samples, wrote and revised the manuscript, coordinated the pertinent work, and was responsible for the financial support and corresponding work. H.L. prepared mitochondria samples. S.Q. participated in partial work. X.H.Z participated in writing and edited English language. N.L. analyzed the iTRAQ proteomics data. All authors approved the final manuscript.

Ovarian tissue samples including ovarian cancer tissues (n = 7) and normal control ovarian tissues (n = 11) were used for this protocol. The present protocol3,4,5 is approved by the Xiangya Hospital Medical Ethics Committee of Central South University, China.
1. Preparation of mitochondria from human ovarian cancer tissues
<ol>
	<li>Prepare 250 mL of the mitochondrial isolation buffer by mixing 210 ...

<ol>
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F., Islinger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisome-mitochondria+interplay+and+disease.">Peroxisome-mitochondria interplay and disease.</a> Journal of Inherited Metabolic Disease. 38, 681-702 (2015).</li><li>Bartol&#225;k-Suki, E., Imsirovic, J., Nishibori, Y., Krishnan, R., Suki, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mitochondrial+structure+and+dynamics+by+the+cytoskeleton+and+mechanical+factors.">Regulation of mitochondrial structure and dynamics by the cytoskeleton and mechanical factors.</a> International Journal of Molecular Sciences. 18, 1812 (2017).</li><li>Rezaul, K., Wu, L., Mayya, V., Hwang, S., Han, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+characterization+of+mitochondrial+proteome+from+human+T+leukemia+cells.">A systematic characterization of mitochondrial proteome from human T leukemia cells.</a> Molecular &amp; Cellular Proteomics. 4, 169-181 (2005).</li><li>Zhan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+many+proteins+can+be+identified+in+a+2DE+gel+spot+within+an+analysis+of+a+complex+human+cancer+tissue+proteome?.">How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?.</a> Electrophoresis. 39, 965-980 (2018).</li><li>Zhan, X., Li, N., Zhan, X., Qian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revival+of+2DE-LC/MS+in+proteomics+and+its+potential+for+large-scale+study+of+human+proteoforms.">Revival of 2DE-LC/MS in proteomics and its potential for large-scale study of human proteoforms.</a> Med One. 3, e180008 (2018).</li></ol>

The authors have nothing to disclose.

Mitochondrial alterations are a hallmark of ovarian cancer. Preparation of high-quality mitochondrial samples from human ovarian cancer and control tissues for large-scale quantitative proteomics benefit the in-depth understanding of mitochondrial function in ovarian cancer pathogenesis and mitochondrial molecular network changes, and help clarify its molecular mechanism for subsequent discovery of target therapy and effective biomarkers based on mitochondria4,5<...

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism1,2. Most of ovarian cancers are diagnosed in the advanced stage, which seriously hampers therapy. Mitochondrial changes are a hallmark of human ovarian cancers, and mitochondria are the centers of energy metabolism, cell signaling, and oxidative stress3,4,5,6,7. In-depth insights into the changes of the mitochondrial proteome in ovarian cancers compared to control ovarian tissue will benefit in-depth understanding of the molecular mechanisms of ovarian cancer, and the discovery of effective and reliable biomarkers and therapeutic targets. Mitochondrial metabolism has been proposed and recognized as a target for cancer therapy, and antimitochondrial therapy might ultimately be very beneficial for preventing the recurrence and metastasis of cancer8. Individual metabolic profiling is also already practiced as a useful tool for cancer stratification and predictive strategies9,10.
The long-term goal of this research is to develop and use a quantitative mitochondrial proteomics method to study ovarian cancer for clarification of mitochondrial proteome alterations between ovarian cancer and control ovarian tissues, and their molecular network alterations from a systematic multi-omics angle11,12, which will result in the discovery of mitochondria-targeted molecular biomarkers13 for clarification of the molecular mechanisms of ovarian cancer, prediction, and personalized treatment of ovarian cancer patients. Isobaric tags for relative and absolute quantification (iTRAQ) labeling3,4 are an effective method to quantify the mitochondrial protein changes. Preparation of high-quality mitochondrial samples from human ovarian cancer and control ovarian tissues are the prerequisite for iTRAQ quantitative analysis of mitochondrial proteomes3. Mitochondrial preparation coupled with iTRAQ quantitative proteomics has been successfully used in long-term research programs about the human ovarian cancer mitochondrial proteome, including the establishment of mitochondrial proteome reference maps3, the analysis of differentially expressed mitochondrial profiles4,14 and post-translational modifications, including phosphorylation, which has already resulted in the discovery of important signaling pathway network changes in human ovarian cancers5, including alterations in energy metabolism4, lipid metabolism, and mitophagy pathway-systems3.
Previous studies have found that differential-speed centrifugation in combination with density gradient centrifugation is an effective method to isolate and purify mitochondria from human ovarian cancer and control ovarian tissues3,4,5,14. The iTRAQ labeling coupled with strong cation exchange (SCX)-liquid chromatography (LC)-tandem mass spectrometry (MS/MS) is the key technique to detect, identify, and quantify the proteins from the prepared mitochondrial samples.
Here, detailed protocols for mitochondrial preparation coupled with iTRAQ quantitative proteomics are described. These have been successfully used in the analysis of human ovarian cancer tissue mitochondrial proteomes. The protocols include preparation of samples, differential-speed centrifugation, density gradient centrifugation, quality assessment of mitochondrial samples, protein digestion with trypsin, iTRAQ labeling, SCX fractionation, LC, MS/MS, database searching, and quantitative analysis of mitochondrial proteins. Moreover, this protocol easily translates to analyze other human tissue mitochondrial proteomes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Vazyme</td>
			<td>E112</td>
			<td>BCA protein assay kit is a special 3-component version of our popular BCA reagents, optimized to measure (A562nm) total protein concentration of dilute protein solutions (0.5 to 20 micrograms/ml).</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Solarbio</td>
			<td>A8020-5G</td>
			<td>Heat shock fraction, Australia origin, protease free, low fatty acid, low IgG, pH 7, &#8805;98%</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>XiangYi</td>
			<td>TDZ4--WS</td>
			<td></td>
		</tr>
		<tr>
			<td>CHAPS</td>
			<td>Sigma</td>
			<td>C9426-5G</td>
			<td>BioReagent, suitable for electrophoresis, &#8805;98% (HPLC) (Sigma-Aldrich)</td>
		</tr>
		<tr>
			<td>Diamine tetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>798681-100G</td>
			<td>Anhydrous, free-flowing, Redi-Dri, &#8805;98%</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>10197777001</td>
			<td>1,4-Dithiothreitol</td>
		</tr>
		<tr>
			<td>Easy nLC</td>
			<td>Proxeon Biosystems (now Thermo Fisher Scientific)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylen glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA)</td>
			<td>Sigma</td>
			<td>E0396-10G</td>
			<td>BioXtra, &#8805;97 .0%</td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>SilentShake</td>
			<td>HYQ-3110</td>
			<td></td>
		</tr>
		<tr>
			<td>iTRAQ reagent kit</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td>Applied Biosystems iTRAQ Reagents&#8211;Chemistry Reference Guide, P/N 4351918A</td>
		</tr>
		<tr>
			<td>Low-temperature super-speed centrifuger</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Macklin</td>
			<td>M813424-100G</td>
			<td>Mannitol is a polyol (polyhydric alcohol) produced from hydrogenation from fructose that functions as a sweetener, humectant, and bulking agent. It has low hygroscopicity and poor oil solvency.</td>
		</tr>
		<tr>
			<td>MASCOT search engine</td>
			<td>Matrix Science, London, UK; version 2.2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nagarse</td>
			<td>Solarbio</td>
			<td>P9090</td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide (SDT)</td>
			<td>Sigma</td>
			<td>56480-25G</td>
			<td>Purum, &#8805;97.0% (T)</td>
		</tr>
		<tr>
			<td>Nycodenz</td>
			<td>Alere/Axis-Shield</td>
			<td>1002424-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF) protease inhibitor</td>
			<td>Solarbio</td>
			<td>P0100-1ML</td>
			<td>PMSF is a protease inhibitor that reacts with serine residues to inhibit trypsin, chymotrypsin, thrombin, and papain.</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Macklin</td>
			<td>P816354-25G</td>
			<td>Potassium chloride, KCI, also known as potassium muriate and sylvite, is a colorless crystalline solid with a salty taste that melts at 776&#176;C (1420 OF). It is soluble in water, but insoluble in alcohol. Potassium chloride is used in fertilizers, pharmaceuticals, photography, and as a salt substitute.</td>
		</tr>
		<tr>
			<td>Proteome Discover 1.4</td>
			<td>Matrix Science, London, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>05317</td>
			<td>It is 1 roll, 26.5 cm x 1.875 m, 0.45 &#181;m pore size, hydrophobic PVDF transfer membrane with low background fluorescence for western blotting. It is compatible with visible and infrared fluorescent probes.</td>
		</tr>
		<tr>
			<td>Q Exactive mass spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SCX column</td>
			<td>Sigma</td>
			<td>58997</td>
			<td>It is 5-&#956;m particle size, length 5cm &#215; i.d. 4.6mm (Supelco).</td>
		</tr>
		<tr>
			<td>Sodium orthovanadate (V)</td>
			<td>Macklin</td>
			<td>S817660-25G</td>
			<td>Sodium orthovanadate (Vanadate) is a general competitive inhibitor for protein phosphotyrosyl phosphatases. The inhibition by sodium orthovanadate is reversible upon the addition of EDTA or by dilution.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Macklin</td>
			<td>S824459-500G</td>
			<td>Vetec reagent grade, 99%</td>
		</tr>
		<tr>
			<td>Thiourea</td>
			<td>Sigma</td>
			<td>62-56-6</td>
			<td>ACS reagent, &#8805;99.0%</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma</td>
			<td>10708976001</td>
			<td>TRIS base is useful in the pH range of 7.0-9.0. It has a pKa of 8.1 at 25&#176;C.</td>
		</tr>
		<tr>
			<td>Trypsin (cell culture use)</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>This liquid formulation of trypsin contains EDTA and phenol red. Gibco Trypsin-EDTA is made from trypsin powder, an irradiated mixture of proteases derived from porcine pancreas. Due to its digestive strength, trypsin is widely used for cell dissociation, routine cell culture passaging, and primary tissue dissociation.</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma</td>
			<td>U5378-100G</td>
			<td>powder, BioReagent, for molecular biology, suitable for cell culture</td>
		</tr>
	</tbody>
</table>

preparation of mitochondria from ovarian cancer tissues and control ovarian tissues for quantitative proteomics analysis

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced stage, which seriously hampers therapy. Mitochondrial changes are a hallmark of human ovarian cancers, and mitochondria are the centers of energy metabolism, cell signaling, and oxidative stress. In-depth insights into the changes of the mitochondrial proteome in ovarian cancers compared to control ovarian tissue will benefit in-depth understanding of the molecular mechanisms of ovarian cancer, and the discovery of effective and reliable biomarkers and therapeutic targets. An effective mitochondrial preparation method coupled with an isobaric tag for relative and absolute quantification (iTRAQ) quantitative proteomics are presented here to analyze human ovarian cancer and control mitochondrial proteomes, including differential-speed centrifugation, density gradient centrifugation, quality assessment of mitochondrial samples, protein digestion with trypsin, iTRAQ labeling, strong cation exchange fractionation (SCX), liquid chromatography (LC), tandem mass spectrometry (MS/MS), database analysis, and quantitative analysis of mitochondrial proteins. Many proteins have been successfully identified to maximize the coverage of the human ovarian cancer mitochondrial proteome and to achieve the differentially expressed mitochondrial protein profile in human ovarian cancers.

There was a difference in the preparation of the mitochondria from ovarian cancer tissues and control ovarian tissues. This study found that it was much easier to prepare mitochondria from ovarian cancer tissues than from control ovarian tissues3,4. Some improvements had to be made to the protocol for the preparation of mitochondria from control ovarian tissues. First, prior to tissue homogenization, it was necessary to add 8 mL o...

Watch this Scientific Journal Video about Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis at JoVE.com

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis

This article presents a protocol of differential-speed centrifugation in combination with density gradient centrifugation to separate mitochondria from human ovarian cancer tissues and control ovarian tissues for quantitative proteomics analysis, resulting in a high-quality mitochondrial sample and high-throughput and high-reproducibility quantitative proteomics analysis of a human ovarian cancer mitochondrial proteome.

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced ...

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.

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Research

JoVE Journal

Cancer Research

5.7K visualizações.  Central South University. Mudanças mitocondriais desempenham papéis importantes nos cânceres de ovário humanos. Este protocolo estabelece uma plataforma eficaz para isolar e purificar mitocôndrias do câncer de ovário humano e os tecidos de controle para análise de proteômica em larga escala. Mitocôndrias, como centros de metabolismo energético, sinalização celular e estresse oxidativo, a visão sobre as mudanças mitocondriais proteome nos cânceres de ovário beneficiam nosso mecanismo molecular compreen...