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 mM mannitol, 70 mM sucrose, 100 mM potassium chloride (KCl), 50 mM Tris-HCl, 1 mM diamine tetraacetic acid (EDTA), 0.1 mM ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), 1 mM phenylmethanesulfonyl fluoride (PMSF) protease inhibitor, 2 mM sodium orthovanadate (V), and 0.2% bovine serum albumin (BSA), pH 7.4.</li>
	<li>Place ~1.5 g of ovarian cancer tissues in a clean glass dish.</li>
	<li>Add 2 mL of the pre-chilled mitochondrial isolation buffer to lightly wash the blood from the tissue surface 3x.</li>
	<li>Use clean ophthalmic scissors to fully mince the tissue into about 1 mm3 pieces, and transfer the minced tissues into a 50 mL centrifuge tube.</li>
	<li>Add 13.5 mL of mitochondrial isolation buffer containing 0.2 mg/mL nagarse, and then use an electric homogenizer to homogenize (use scale 2, 10 s 6x, interval 10 s) the minced tissues (2 min, 4 &#176;C).</li>
	<li>Add another 3 mL of mitochondrial isolation buffer into the tissue homogenates and mix them well by pipetting.</li>
	<li>Centrifuge the prepared tissue homogenate (1,300 x g, 10 min, 4 &#176;C). Remove the crude nuclear fraction (i.e., the pellet) and keep the supernatant.</li>
	<li>Recentrifuge the supernatant (10,000 x g, 10 min, 4 &#176;C). Remove the microsomes (i.e., the supernatant) and keep the pellet.</li>
	<li>Add 2 mL of mitochondrial isolation buffer and resuspend the pellet well by light pipetting.</li>
	<li>Centrifuge the pellet suspension (7,000 x g, 10 min, 4 &#176;C). Discard the supernatant and keep the crude mitochondria (i.e., the pellet).</li>
	<li>Add 12 mL of 25% density gradient medium (i.e., Nycodenz) to resuspend the extracted crude mitochondria.</li>
	<li>Make a discontinuous density gradient by filling a tube from bottom to top with 5 mL of 34%, 8 mL of 30%, 12 mL of 25% (containing the crude mitochondria from step 1.11), 8 mL of 23%, and 3 mL of 20% density gradient medium, and centrifuge it (52,000 x g, 90 min, 4 &#176;C).</li>
	<li>Use a long and blunt syringe to collect the purified mitochondria at the interface between the 25% and 30% density gradient medium into a clean tube.</li>
	<li>Add mitochondrial isolation buffer into the collected mitochondria to dilute it to a three-fold volume. Centrifuge (15,000 x g, 20 min, 4 &#176;C) and discard the supernatant.</li>
	<li>Add 2 mL of mitochondrial isolation buffer to resuspend the pellet, and centrifuge (15,000 x g, 20 min, 4 &#176;C). Discard the supernatant and keep the pellet.</li>
	<li>Collect the final pellet (i.e., the purified mitochondria) and store it at -20 &#176;C. 
	NOTE: Combine all purified mitochondria from the ovarian cancer tissue as the mitochondria sample for quantitative proteomics analysis.</li>
</ol>
2. Preparation of mitochondria from human control ovarian tissues
<ol>
	<li>Prepare 250 mL of the mitochondrial isolation buffer as described in step 1.1.</li>
	<li>Place ~1.5 g of the normal control ovarian tissues in a clean glass dish.</li>
	<li>Add 2 mL of pre-chilled mitochondrial isolation buffer to lightly wash the blood from the tissue surface 3x.</li>
	<li>Use clean ophthalmic scissors to fully mince the tissue into about 1 mm3 pieces, and transfer the minced tissues into a 50 mL centrifuge tube.</li>
	<li>Add 8 mL of 0.05% trypsin/20 mM EDTA in phosphate-buffered saline (PBS) to the minced control tissues, and digest (30 min, room temperature), which helps to lyse the tissues and cells and release mitochondria. Then centrifuge (200 x g, 5 min). Discard the supernatant, and keep the tissues and cells.</li>
	<li>Add 13.5 mL of mitochondrial isolation buffer containing 0.2 mg/mL nagarse, and then use an electric homogenizer to homogenize (use scale 2, 10 s 6x, interval 10 s) the minced tissues (2 min, 4 &#176;C).</li>
	<li>Add another 3 mL of mitochondrial isolation buffer into the tissue homogenates and mix them well by pipetting.</li>
	<li>Centrifuge the prepared tissue homogenate (1,300 x g, 10 min, 4&#176;C). Remove the crude nuclear fraction (i.e., the pellet) and keep the supernatant.</li>
	<li>Recentrifuge the supernatant (10,000 x g, 10 min, 4&#176;C). Remove the microsomes (i.e., the supernatant) and keep the pellet.</li>
	<li>Add 2 mL of mitochondrial isolation buffer to resuspend the pellet well by light pipetting.</li>
	<li>Centrifuge the pellet suspension (7,000 x g, 10 min, 4 &#176;C). Discard the supernatant and keep the crude mitochondria (i.e., the pellet).</li>
	<li>Add 12 mL of 25% density gradient medium to resuspend the extracted crude mitochondria.</li>
	<li>Make a discontinuous density gradient by filling a tube from bottom to top with 8 mL of 38%, 5 mL of 34%, 8 mL of 30%, 12 mL of 25% (containing the crude mitochondria from step 2.12), 8 mL of 23%, and 3 mL of 20% density gradient medium, and centrifuge it (52,000 x g, 90 min, 4 &#176;C).</li>
	<li>Use a long and blunt syringe to collect the purified mitochondria in the range from the interface between 25% and 30% to the interface between 34% and 38% into a clean tube.</li>
	<li>Add mitochondrial isolation buffer into the collected mitochondria to dilute it to a three-fold volume. Centrifuge (15,000 x g, 20 min, 4 &#176;C) and discard the supernatant.</li>
	<li>Add 2 mL of mitochondrial isolation buffer to resuspend the pellet, and centrifuge it (15,000 x g, 20 min, 4 &#176;C). Discard the supernatant and keep the pellet.</li>
	<li>Collect the final pellet (i.e., the purified mitochondria) and store it at -20 &#176;C. 
	NOTE: Combine all purified mitochondria from the control ovarian tissue as the mitochondrial samples for quantitative proteomics analysis.</li>
</ol>
3. Verification of the quality of purified tissue mitochondrial samples
<ol>
	<li>Take a tube of purified mitochondrial samples (i.e., the pellets from the ovarian cancer tissues from step 1.16 and the control ovarian tissues from step 2.17) for verification with electron microscopy (EM). 
	NOTE: The detailed protocol was described previously15,16.</li>
	<li>Prepare the mitochondrial protein extraction buffer by mixing 8 M urea, 2 M thiourea, 40 mM Tris, 1 mM EDTA, 130 mM dithiothreitol (DTT), and 4% (w:v) 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS). Adjust the pH to 8.52.</li>
	<li>Take a tube of purified mitochondrial samples (i.e., the pellets from the ovarian cancer tissues in step 1.16 and the control ovarian tissues in step 2.17) and add mitochondrial protein extraction buffer (mitochondrial samples to protein extraction buffer, 1:5) to resuspend the pellet. Freeze the samples in liquid nitrogen 3x and store at room temperature for 2 h.</li>
	<li>Centrifuge at 12,000 x g, 30 min, 4 &#176;C, collect the supernatant (i.e., the extracted mitochondrial protein sample), and measure the protein content with a bicinchoninic acid (BCA) protein assay kit.</li>
	<li>Prepare 50 mL of 1.5 M Tris-HCl (pH 8.8) by mixing 9.08 g of Tris and 40 mL of ddH2O, adjusting the pH to 8.8 using HCl, and adding ddH2O to a final volume of 50 mL. Store at 4 &#176;C.</li>
	<li>Prepare 50 mL of 1 M Tris-HCl (pH 6.8) by mixing 6.06 g of Tris and 40 mL of ddH2O, adjusting the pH to 6.8 using HCl, and adding ddH2O to a final volume of 50 mL. Store at 4 &#176;C.</li>
	<li>Prepare 100 mL of 30% acrylamide-bis solution by mixing 29 g of acrylamide, 1 g of bis-acrylamide, and 80 mL of ddH2O, and adding ddH2O to a final volume of 100 mL. Store it in a brown bottle at 4 &#176;C.</li>
	<li>Prepare 1,000 mL of 10x tris-glycine electrophoresis buffer by mixing 29 g of glycine, 58 g of Tris, 3.7 g of sodium dodecyl sulfate (SDS), and 800 mL of ddH2O, and then adding ddH2O to a final volume of 1,000 mL.</li>
	<li>Prepare 10 mL of loading buffer by mixing 2 mL of glycerin, 0.02 g of bromophenol blue, 0.4 g of SDS, 2 mL of Tris-HCl pH 6.8, and 7 mL of ddH2O, and then adding ddH2O to a final volume of 10 mL.</li>
	<li>Prepare 10 mL of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolving gel by mixing 4 mL of ddH2O, 3.3 mL of 30% acrylamide-Bis solution, 2.5 mL of 1.5 M Tris-HCl (pH 8.8), 0.1 mL of 10% SDS, 0.1 mL of 10% ammonium persulfate, and 0.004 mL of tetramethylethylenediamine (TEMED). Pour the SDS-PAGE resolving gel solution into the plate between the glass plates to the level of the bottom of the comb. Add 2 mL of isopropyl alcohol onto the top. Leave for 30 min.</li>
	<li>Remove the isopropyl alcohol and wash the top with ddH2O 3x. Pour the 5% concentration gel solution containing 4.1 mL of ddH2O, 1.0 mL of 30% acylamide-bis solution, 0.75 mL of 1 M Tris-HCl (pH 6.8), 0.06 mL of 10% SDS, 0.06 mL of 10% ammonium persulfate, and 0.006 mL of TEMED. Immediately insert the comb into the concentration gel solution, leave for 30 min, and then take out the comb.</li>
	<li>Prepare the 1x Tris-glycine electrophoresis buffer by diluting 100 mL of 10x Tris-glycine electrophoresis buffer with 900 mL of ddH2O and pour into the electrophoretic tank.</li>
	<li>Mix 30 &#181;g of the mitochondrial proteins from the ovarian cancer and control ovarian tissues from step 3.4 with loading buffer to reach a final volume of 20 &#181;L. Boil the mixture for 5 min, then separate it with the 10% SDS-PAGE resolving gel using a constant voltage of 100 V. When the bromphenol blue reaches the bottom, stop the electrophoresis.</li>
	<li>Prepare 1.5 L of electrophoretic transfer buffer by mixing 150 mL of 10x Tris-glycine electrophoresis buffer, 1,050 mL of ddH2O, and 300 mL of methanol.</li>
	<li>Soak the PVDF membrane in 100% methanol for 10 min and in electrophoretic transfer buffer for at least 5 min. Soak five sheets of filter paper in 1x elecrophoretic transfer buffer for at least 5 min.</li>
	<li>Take out the SDS-PAGE gel containing the proteins, and soak it in electrophoretic transfer buffer for 10 min.</li>
	<li>Make a transfer cassette by placing a wet sponge on the white plate, three sheets of wet filter paper, the PVDF membrane, the SDS-PAGE gel with the proteins, two sheets of wet filter paper, and the wet sponge from the bottom to top. Avoid any bubbles.</li>
	<li>Place the transfer cassette into the electrophoretic tank, pour in the electrophoretic transfer buffer, and then transfer for 2 h under a constant 200 mA current.</li>
	<li>Prepare 10x Tris-buffered saline (TBS) stock solution by mixing 12.114 g of Tris, 29.22 g of NaCl, and adding ddH2O to a final volume of 500 mL. Prepare 2 L of 1x TBST by mixing 200 mL of 10x TBS, 2 mL of Tween-20, and 1,798 mL of ddH2O. Prepare 100 mL of blocking solution by mixing 100 mL of TBST and 5 g of BSA.</li>
	<li>After transfer, take out the PVDF membrane, wash lightly in TBST for 5 min, then block it in blocking solution for 1 h at room temperature.</li>
	<li>Incubate the PVDF membrane (4 &#176;C overnight) with different primary antibodies specific to different subcellular organelles, including lamin B (cell nucleus; goat anti-human antibody 1: 1,000 blocking solution), flotillin-1 (cytomembrane; rabbit anti-human antibody 1:500 blocking solution), COX4I1 (mitochondrion; rabbit anti-human 1:1,000 blocking solution), GM130 (Golgi apparatus; mouse anti-human antibody 1:1,000 blocking solution), catalase (peroxisome; rabbit anti-human antibody 1:1,000 blocking solution), and cathepsin B (lysosome; rabbit anti-human antibody 1:1,000 blocking solution). Lightly wash in TBST for 5 min 3x.</li>
	<li>Incubate the PVDF membrane for 1 h at room temperature with the corresponding secondary antibodies (rabbit anti-goat, goat anti-rabbit, or goat anti-mouse). Lightly wash in TBST for 5 min 3x. Visualize it with electrochemiluminescence (ECL).</li>
</ol>
4. iTRAQ-SCX-LC-MS/MS analysis
NOTE: The detailed procedures for section 4 refer to the iTRAQ instructions (Table of Materials).
<ol>
	<li>Add SDT buffer (4% SDS, 100 mM Tris-HCl pH 7.6, and 100 mM DTT) to the purified mitochondrial samples (i.e., the pellets from the ovarian cancer tissues from step 1.16 and the control ovarian tissues from step 2.17). Vortex the sample until there is no visible precipitate. 
	NOTE: The mitochondrial sample to SDT buffer ratio should be 1:5.</li>
	<li>Boil the SDT-treated sample for 5 min, cool down the sample on ice, and then centrifuge (2,000 x g, 2 min).</li>
	<li>Collect the supernatant as the extracted mitochondrial protein samples and measure the protein content with a BCA protein assay kit.</li>
	<li>Take SDT-extracted mitochondrial proteins (200 &#181;g of each sample) for reduction, alkylation, digestion with trypsin, desalination, and lyophilization3,4. Do three replicates for each sample.</li>
	<li>Treat the tryptic peptides (100 &#181;g of each sample) from step 4.4 with 100 mM tetraethyl ammonium bromide solution (pH 8.5), and label the tryptic peptides with one of the 6-plex iTRAQ reagents according to its instructions3,4. Label each sample 3x.</li>
	<li>Mix equally 6 labeled tryptic peptide samples (three from ovarian cancer tissues and three from control ovarian tissues), and dry with a vacuum concentrator.</li>
	<li>Fractionate the mixed iTRAQ-labeled peptides with SCX chromatography into 60 fractions (one fraction per 1 min), then combine every two fractions as a SCX-fractionated sample (n = 30).</li>
	<li>Subject each SCX-fractionated sample to LC-MS/MS analysis on a mass spectrometer3,4 coupled with a nano LC system within a 60-min LC separation gradient to obtain MS/MS data.</li>
	<li>Search the MS/MS data to identify proteins with the search engine (Table of Materials).</li>
	<li>Use the iTRAQ reporter-ion intensities to quantify each protein and determine mitochondrial differentially expressed proteins (mtDEPs) with a change of &#62;1.5 or &#60;-1.5 fold, and p &#60; 0.05.</li>
</ol>

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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><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, &amp;ge;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, &amp;ge;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, &amp;ge;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, &amp;ge;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&amp;ndash;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, &amp;ge;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&amp;deg;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 &amp;micro;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-&amp;mu;m particle size, length 5cm &amp;times; 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, &amp;ge;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&amp;deg;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 ...

preparation-mitochondria-from-ovarian-cancer-tissues-control-ovarian

Research

JoVE Journal

Cancer Research

5.8K Views.  Central South University. Mitochondriale veranderingen spelen een belangrijke rol bij menselijke eierstokkanker. Dit protocol stelt een effectief platform op om mitochondriën te isoleren en te zuiveren van menselijke eierstokkanker en de controleweefsels voor grootschalige proteomics-analyse. Mitochondriën, als een centra van energiemetabolisme, celsignalering, en oxidatieve stress, inzicht in mitochondriale proteoom veranderingen in eierstokkanker ten goede komen aan ons begrip moleculair mechanisme, en is een ontd...

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

Characterization and Functional Prediction of Bacteria in Ovarian Tissues

The theory of a "sterile" female upper reproductive tract has been encountering increasing opposition due to advancements in bacterial detection. However, whether ovaries contain bacteria has not yet been confirmed yet. Herein, an experiment to detect bacteria in ovarian tissues was introduced. We chose ovarian cancer patients in the cancer group and noncancerous patients in the control group. 16S rRNA gene sequencing was used to differentiate bacteria in ovarian tissues from the cancer and control groups. Furthermore, we predicted the functional composition of the identified bacteria by using BugBase and PICRUSt. This method can also be used in other viscera and tissues since many organs have been proven to harbor bacteria in recent years. The presence of bacteria in viscera and tissues may help scientists evaluate cancerous and normal tissues and may be aid in the treatment of cancer.

Immunohistochemistry staining and 16S ribosomal RNA gene (16S rRNA gene) sequencing were performed in order to discover and distinguish bacteria in cancerous and noncancerous ovarian tissues in situ. The compositional and functional differences of the bacteria were predicted by using BugBase and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt).

The theory of a "sterile" female upper reproductive tract has been encountering increasing opposition due to advancements in bacterial detection. However, ...

Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule synthesis. Genetic and functional adaptations of mitochondria to the tumor environment drive proliferative and metastatic potential. The advent of DNA and RNA sequencing removed critical barriers to the evaluation of genetic mediators of tumorigenesis. However, to date, methodological approaches to evaluate tumor mitochondrial function remain elusive and require technical proficiency limiting the feasibility, ultimately diminishing diagnostic and prognostic value in both experimental and clinical settings. Here, we&#160;outline a simple and rapid method to quantify rates of oxidative phosphorylation (OXPHOS) and electron transfer (ET) capacity in freshly excised solid tumor homogenates using high-resolution respirometry. The protocol can be reproducibly applied across species and tumor types as well as adapted to evaluate a diversity of mitochondrial ET pathways. Using this protocol, we demonstrate that mice bearing a luminal B mammary cancer exhibit defective nicotinamide adenine dinucleotide-linked respiration and reliance on succinate to generate adenosine triphosphate via OXPHOS.

We developed a practical protocol and analytical approach to evaluate mitochondrial oxidative phosphorylation and electron transfer capacity in fresh tumor homogenates. This protocol can be easily adapted to survey various mitochondrial functions that contribute to cancer initiation, progression, and treatment response.

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Medicine

Establishing a 3D Ex Vivo Model for Ovarian Cancer-Omentum Interaction

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

Author Spotlight: Advanced Ex Vivo Model for Investigating Cancer-Adipose Microenvironment Interaction 

This protocol describes the establishment of a three-dimensional (3D) ex vivo model of cancer cell-omentum interaction. The model provides a platform for elucidating pro-tumor mechanisms within the adipose niche and for testing novel therapies.

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Biochemistry

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular mechanisms that maintain oocyte quality remain unclear, thus it is difficult to increase the birth rate of women over 35 years old at present. Oocytes contain more mitochondria than any type of cell in the body, and any mitochondrial dysfunction can lead to reduced oocyte quality. In the 1990s, oocyte cytoplasmic transfer resulted in great success in human reproduction but was accompanied by ethical controversies. Autologous mitochondrial transplantation is expected to be a useful technique to increase the quality of oocytes that have decreased due to age. In the present study, we used adipose-derived stem cells from aged mice as a mitochondria donor to increase the quality of oocytes of aged mice. Further development of autologous mitochondrial transfer technology will provide a new and effective treatment for infertility in aged women.

Here, we describe the isolation of mitochondria from mouse adipose-derived mesenchymal stem cells, and then transfer the mitochondria into aged mouse oocytes to improve the quality of the oocytes.

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular ...

Developmental Biology

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Biology

Improved Protein Quantification Using Two-Step Mitochondrial Isolation

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial proteome is composed of more than 1,000 proteins, and the abundance of each can vary dynamically in response to external stimuli or during disease progression. Here, we describe a protocol for isolating high-quality mitochondria from primary cells and tissues. The two-step procedure comprises (1) mechanical homogenization and differential centrifugation to isolate crude mitochondria, and (2) tag-free immune capture of mitochondria to isolate pure organelles and eliminate contaminants. Mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, and enrichment yields are calculated, allowing the discovery of novel mitochondrial proteins by subtractive proteomics. Our protocol provides a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.

Author Spotlight: Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification  

We present a two-step protocol for high-quality mitochondria isolation that is compatible with protein discovery and quantification at a proteome scale. Our protocol does not require genetic engineering and is thus suitable for studying mitochondria from any primary cells and tissues.

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial ...

Mitochondrial Isolation: A Technique to Isolate Mitochondria from Human Ovarian Cancer Tissue Sample

Source: Zhan, X. et al. <a href="https://www.jove.com/t/60435">Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis.</a> J. Vis. Exp. (2019)
This video describes the procedure for isolating mitochondria from human ovarian cancer tissues using differential-speed centrifugation in combination with density gradient centrifugation. The isolated mitochondria can be used for proteomic analysis of human ovarian cancer mitochondrial proteome.

Source: Zhan, X. et al.  Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis. J. Vis. ...

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

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