May 26, 2026
Mitochondria-specific Proteomic Signatures of Organ Aging: Mitoprotein Pertubation in Subcelluar Level
- Qing Gao1,
- Hao Deng1,
- kai Wang1,
- Guanwei Fan1
- 1Tianjin University of Traditional Chinese Medicine
- Qing
Protocol Citation: Qing Gao, Hao Deng, kai Wang, Guanwei Fan 2026. Mitochondria-specific Proteomic Signatures of Organ Aging: Mitoprotein Pertubation in Subcelluar Level. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3mrz7l25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 25, 2026
Last Modified: May 26, 2026
Protocol Integer ID: 317868
Keywords: specific proteomic signatures of organ aging, mitochondria protein, mitochondria, organ aging, mitochondria morphology, mitoprotein pertubation in subcelluar level, mitoprotein pertubation, universal mitochondria, outside mitochondria, specific proteomic signature, mitoprotein, aging indicator, atp level, others like atp level, mtdna, cytoplasmic protein, tissue respiratory capacity
Funders Acknowledgements:
National Natural Science Foundation of China
Grant ID: 82430121
National Natural Science Foundation of China
Grant ID: 82104689
Natural Science Foundation of Tianjin
Grant ID: 25JCLMJC00100
Abstract
The present study comprehensively analyzed the mitoprotein proteome perturbation inside and outside mitochondria between 3-month and 20-month-old mice. Mitochondria are extracted from nine different organs, and the mitochondria proteins and cytoplasmic proteins are sent to mass spectrum analysis separately. Meanwhile, mitochondria morphology and healthy status are evaluated. Results showed that tissue respiratory capacity, cristae density, and mtDNA damage out-stand others like ATP level and ROS production as more universal mitochondria aging indicators across nine organs.
Guidelines
Protein extraction: The extracted mitochondria sample and the left part were transferred to 1.5ml eppendorf tubes, added with sample lysis buffer, phosphatase inhibitor and PMSF (final concentration 1mM for each). Ultrasonication was performed on ice with the power set at 80 W, using a cycle of 1.0 s on and 1.0 s off, for a total duration of 2 minutes. Centrifuge the solution at 4°C and 12,000 rpm for 10 minutes. Collect the supernatant, then repeat the centrifugation step and collect the supernatant again. The resulting supernatant constitutes the total protein solution of the sample. The total protein concentrations were quantified by bicinchoninic acid assay.
SDS polyacrylamide gel electrophoresis: 10 µg protein was taken for SDS-PAGE (12%) separation. Then the gel was stained with Kaumas Brilliant Blue by eStain LG (GenScript, Nanjing). The analysis for gel was adopted by the automatic digital gel image analysis system (Tanon, Shanghai).
Protein digestion: According to the measured protein concentration, take the same quantity protein (50 µg) from each sample, and dilute different groups of samples to the same concentration and volume. Add prepared SP3 magnetic beads to the protein solution, followed by the corresponding volume of 100% acetonitrile (ACN), and incubate at room temperature for 20 minutes. After briefly centrifuging the samples, remove the supernatant. Wash the magnetic beads twice with 70% ethanol and twice with 70% ACN, respectively, removing the supernatant after each wash. Resuspend the magnetic beads by adding 50 mM ammonium bicarbonate (ABC) solution. Add DTT (25mM) and incubate at 55°C for 30 minutes. Add chloroacetamide, Trypsin, and LysC for enzymatic digestion (protein: enzyme = 50:1 (m/m), 100 µg of protein add 2 µg of enzyme), and incubate overnight at 37°C with shaking at 1500 rpm, enzymatic reaction was stopped by adjusting pH to 3 via adding phosphoric acid. The samples were desalted on SOLA™ SPE. After dried under vacuum, samples were resuspended and iRT peptides (Biognosys, ThermoFisher, 1:10) were added.
Liquid chromatography-mass spectrometry: The Proteomic data analysis was performed by Shanghai OE Biotech Co., Ltd. (Shanghai, China). All data were acquired using a Thermo Scientific Vanquish Neo UHPLC system coupled to an Orbitrap Astral mass spectrometer with a Thermo Scientific Easy-spray source. The separation was performed using a trap column (300 µm × 0.5 cm, 5 µm, Thermo Fisher Scientific) with a C18 analytical column of ES906 (PepMap TM Neo UHPLC 150 µm × 15 cm, 2 µm, Thermo Fisher Scientific). Mobile phases A and B were 0.1% formic acid in water and 0.1% formic acid in ACN-water (v/v=4:1), respectively. The separation was achieved using the following gradient elution program at 0.7 µl/min: 0 ~ 8 min, 7.5% B; 8 ~ 9 min, 35% B; 9 ~ 10 min, 100% B.
For DIA experiments on the Orbitrap Astral MS, the main settings are as follows: capillary was 1.6KV, the full first-stage MS scan range is 380 - 1500 m/z, the primary MS resolution is set to 240,000, the automatic gain control (AGC) is set to 500%, and the parent ion selection window is set to 2-Th, the number of DIA windows is set to 300, the normalized collision energy was set to 25%; and the secondary MS scan range is 150 - 2000 m/z, the RF lens is set to 50%, and the maximal injection time is 3 ms.
Database search: The LC-MS/MS raw data were imported in DIA-NN (Version 1.8.1) for analysis. The database we used was Mouse.MitoCarta3.0. The false positive rate of peptide identification was controlled below 1%. The main parameters of searching database were set as following:
| Item | Value |
|--------|--------|
| Enzyme | Trypsin |
| Max Missed Cleavages | 2 |
| Fixed modifications | Carbamidomethyl (C) |
| Variable modifications | Oxidation (M), Acetyl (Protein N-term) |
| Database pattern | Target-Reverse |
| PSM (Peptide-Spectral Matching) | 0.01 |
| FDR | 0.01 |
Colourimetric assays of mitochondrial enzyme activities: The activity of mitochondria electronic respiratory chain complex, including complex I (NADH-CoQ Reductase) , complex II (Succinic acid-coenzyme Q reductase), complex III (Coenzyme Q-Cytochrome C Reductase), complex IV (Cytochrome C Oxidase ) , complex V (F0F1-ATPase/ATP Synthase), and citrate synthase enzyme were detected by colourimetric assays according to the manufacturer's protocol using fresh extracted mitochondria. In brief, the enzymatic activities were derived from the linear slopes of the reporter dye absorbance changes. These slopes were exported to Microsoft Excel and converted using the appropriate molar extinction coefficient and dilution factor for each respective assay. Furthermore, the assay conditions, including the amount of both homogenate and substrates, were optimized to yield stable readings for all samples. Homogenized samples were used for respiratory chain complex and citrate synthase activity measurement with Complex I activity assay kit, Complex II activity assay kit, Complex III activity assay kit, Complex IV activity assay kit, Complex V activity assay kit, CS activity assay kit, respectively, following the manufacturer's instructions. All the activity assays were conducted in the same day, technical triplicates and an equal number of negative control wells were performed for each sample.
Complex I catalyzes the reaction between NADH and substrates to generate NAD+ and reduced ubiquinone. For a single 200 µL reaction system: Pipette 198 µL of Reagent IV and 2 µL of Reagent V, then mix thoroughly. Prior to the assay, incubate the working solution at 37°C for 5 minutes. In a UV-transparent 96-well plate, sequentially add 10 µL of sample, 200 µL of pre-warmed working solution, and 15 µL of Working Reagent VI. Mix immediately and thoroughly. The rate of decline in NADH absorbance at 340 nm can reflect its activity.
The activity of Complex II is determined by measuring the reduction rate of 2,6-dichloroindophenol (DCIP) at 600 nm. This reaction is driven by reduced coenzyme Q, the catalytic product of Complex II, and the decrease in absorbance of DCIP serves as the indicator for calculating activity.
In a 96-well plate or micro glass cuvette, sequentially add 10 µL of sample, 25 µL of Reagent VI, and 200 µL of Working Reagent IV. Tap the plate gently to ensure thorough mixing, then immediately measure the initial absorbance at 605 nm at 0 minutes (A_1) and after 2 minutes (A_2). Calculate ΔA = A_1 - A_2.
Complex III reduces cytochrome c (oxidized) to cytochrome c (reduced), leading to an increase in absorbance at 550 nm, its activity is calculated by measuring the rate of absorbance increase at 550 nm. To each well of a 96-well plate, sequentially add 25 µL of Reagent VI and 200 µL of the working solution. Mix thoroughly and incubate for exactly 2 minutes. Then, add 10 µL of the extracted mitochondria sample. Immediately after mixing the reaction system, measure the absorbance at 550 nm at time 0 minutes (A_1) and again at 2 minutes (A_2).
The activity of Complex IV is determined by measuring the oxidation rate of reduced cytochrome c, which is monitored as a decrease in absorbance at 550 nm. Sequentially add 200 µL of working solution and 10 µL of sample into the wells of a 96-well plate, and mix rapidly. Immediately after mixing the reaction system, measure the absorbance at 550 nm at 0 minutes (A_1) and at 1 minute (A_2).
Complex V hydrolyzes ATP to ADP. In an enzyme-coupled assay, the resulting ADP is converted, leading to the oxidation of reduced nicotinamide adenine dinucleotide (NADH) to NAD+. The activity of Complex V is calculated by measuring the rate of decrease in absorbance at 340 nm.
Citrate synthase (CS) catalyzes the reaction between acetyl-CoA and oxaloacetate to form citrate and free coenzyme A (CoA-SH). The resulting CoA-SH is then detected by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), generating a yellow-colored product, 5-thio-2-nitrobenzoic acid (TNB), which has a characteristic absorbance peak at 412 nm. The enzyme activity of CS is determined by measuring the rate of increase in absorbance at 412 nm.
Tissue respiratory capacity calculation method TRC=\(\sqrt{CI} + \sqrt{CII} + \sqrt{CIV} \)/3 was used as previously described.
_Mitochondrial membrane potential (MMP)_
Mitochondrial membrane potential was detected by Enhanced mitochondrial membrane potential assay kit with JC-1 according to manufacture’s instruction. At high mitochondrial membrane potential (ΔΨm), the lipophilic cationic dye JC-1 accumulates in the mitochondrial matrix, where it forms J-aggregates that emit red fluorescence. Conversely, at low ΔΨm, JC-1 cannot accumulate and remains as monomers in the cytoplasm, emitting green fluorescence. Following isolation, tissue cells were washed once with phosphate-buffered saline (PBS). The cell pellet was resuspended in 1 mL of complete cell culture medium. Then, 1 mL of JC-1 staining working solution (prepared by diluting 50 µL of JC-1 stock solution [200X] in 8 mL of pure water) was added. The mixture was inverted several times for thorough mixing and incubated at 37°C for 20 minutes in a cell culture incubator. Cells were centrifuged at 600 × g and 4°C for 4 minutes. The supernatant was carefully discarded. The cell pellet was washed twice with 1 mL of 1X JC-1 staining buffer. Cells were resuspended in 500 µL of 1X JC-1 staining buffer. Samples were subjected to analysis using a flow cytometer (BD Bioscience, America).
_Mitochondrial Permeability Transition Pore (MPTP)_
Calcein AM readily diffuses across the plasma membrane into the cytosol. Intracellular esterases then cleave the acetoxymethyl (AM) esters, yielding the fluorescent, hydrophilic dye calcein. This charged molecule is effectively trapped within the cell. Calcein AM was co-loaded with cobalt chloride (CoCl_2), Co^2+ is excluded from the mitochondrial matrix under normal conditions because the inner mitochondrial membrane is impermeable to it. Therefore, in healthy cells, the calcein signal within the mitochondria remains bright, while the cytosolic calcein fluorescence is quenched by Co^2+. The opening of the MPTP creates a large, non-selective pore in the inner mitochondrial membrane. This allows Co^2+ ions to enter the mitochondrial matrix. Upon entry, Co^2+ quenches the mitochondrial calcein fluorescence. Consequently, a decrease in intracellular calcein fluorescence is directly correlated with MPTP opening.
MPTP was detected by mitochondrial membrane potential (calcein AM) assay kit according to manufacture’s instruction. Following isolation, tissue cells were washed once with phosphate-buffered saline (PBS). Then, they were resuspended in an appropriate volume of a solution containing Calcein AM staining solution, fluorescence quenching solution, and Ionomycin control to achieve a cell density of approximately 1×10^6 cells/mL. The suspension was incubated at 37°C in the dark for 30-45 minutes. After incubation, the cells were collected by centrifugation at 300 × g for 5 minutes. Each pellet was resuspended in 1 mL of assay buffer and centrifuged again under the same conditions (300 × g for 5 minutes). The final cell pellet was resuspended in 400 µL of assay buffer and analyzed using a flow cytometer.
_Mitochondrial calcium concentration_
Rhod-2 AM is an acetoxymethyl ester derivative of Rhod-2 that is cell-permeant. Upon entering the cell, it is hydrolyzed by intracellular esterases to yield Rhod-2, a charged form that is trapped within the cell. As Rhod-2 shares the core structure of rhodamine-based mitochondria-specific probes, it accumulates within mitochondria where it chelates calcium ions. The Rhod-2-Ca^2+ complex exhibits strong fluorescence upon excitation at 552 nm, making Rhod-2 AM a widely used probe for monitoring changes in mitochondrial calcium concentration. Calcium concentration was detected by mitochondrial calcium assay kit with Rhod-2 AM according to manufacture’s instruction. In brief, isolated tissue cells were washed once with phosphate-buffered saline (PBS). Add 100 µl of Rhod-2 staining solution and incubate at 37°C for 10-30 minutes in a cell culture incubator. (The optimal incubation time varies with different cell types. A 20-minute incubation is recommended as a starting point, which was optimized for specific cells to achieve best results.) After incubation at 37°C, remove the supernatant and wash the cells twice with PBS. Resuspend the cells in 100 µl of PBS and analyze using a flow cytometer.
_Mitochondria Reactive Oxygen Species (ROS) production_
DCFH-DA (2',7'-Dichlorodihydrofluorescein diacetate) freely diffuses across the plasma membrane of living cells. Once inside the cell, it is hydrolyzed by intracellular esterases to form DCFH (2',7'-Dichlorodihydrofluorescein), which is non-fluorescent and membrane-impermeable. Subsequently, DCFH can be oxidized by intracellular reactive oxygen species (ROS) to generate highly fluorescent DCF (2',7'-Dichlorofluorescein). Here, ROS concentrations was detected by CheKine™ ROS content assay kit according to manufacture’s instruction. In brief, DCFH-DA was diluted 1:1000 in serum-free culture medium to achieve a final concentration of 10 µmol/L. After collection, resuspend the cells in the diluted DCFH-DA solution at a density of 1×10^6 to 2×10^7 cells/mL. Incubate the suspension at 37°C for 30 minutes in a cell culture incubator. Gently invert the tube every 3-5 minutes to ensure thorough contact between the probe and cells. Wash the cells three times with serum-free culture medium to completely remove any extracellular DCFH-DA. Using an excitation wavelength of 488 nm and an emission wavelength of 525 nm, measure the fluorescence intensity in real-time or at designated time points before and after stimulation. The fluorescence intensity of DCF was quantified using flow cytometry, serves as an indicator for analyzing intracellular ROS levels.
_mtDNA copy number and damage_
Mitochondria DNA copy number was detected by Mouse Mitochondrial Biogenesis Assay Kit by mtDNA Copy Number according to manufacture’s instruction. Mitochondria DNA damage was detected by Mouse Mitochondrial DNA Damage Quantification Kit according to manufacture’s instruction.
_Statistical analyses_
For proteomic data, a total of 7200 proteins expressed were identified as belonging to the proteome of mice this study. The thresholds of fold change (e1.2 or c0.83) and P-value c0.05 were used to identify differentially expressed proteins (DEPs). Annotation of all identified proteins was performed manually using Mouse.MitoCarta3.0 (https://www.broadinstitute.org/mitocarta/mitocarta30-inventory-mammalian-mitochondrial-proteins-and-pathways). Relocalization analysis was completed by relocalization analysis pepline we built, which is uploaded to github (https://github.com/gaoqing88/Mitoprotein-relocalization-pepline). Organheatmap was plotted by using R project (https://cran.rproject.org/web/packages/OrgHeatmap/index.html). For other data, each quantitative experiment was repeated three times. One-way analysis of variance was used to assess the differences among the groups. Statistical results were presented as means ± standard deviation (SD). P values of c 0.05 were denoted as statistically significant. All statistical analyses were calculated using GraphPad Prism 10.0 program (GraphPad Software, Inc., La Jolla, CA, USA).
Materials
Mitochondria extraction kit (Merck 26 Co., Inc., Guangzhou, China); Cell mitochondrial complex I, II, III, IV, V activity assay kits and Citrate Synthase (CS) activity assay Kit; Mouse Mitochondrial Biogenesis Assay Kit by mtDNA Copy Number (Elabscience Technology Co., Ltd, Wuhan, China); Mitochondrial membrane potential (MMP) assay kit, CheKine™ ROS content assay kit (Abbkine Scientific Co., Ltd, Wuhan, China); Enhanced mitochondrial membrane potential assay kit with JC-1 (Beyotime Biotech Inc., Shanghai, China); Mouse Mitochondrial DNA Damage Quantification Kit (RayBiotech, Inc., Shanghai, China); Sample lysis buffer (Beyotime Biotech Inc., Shanghai, China).
Animals
Twenty-four male C57BL/6J mice (twelve 3-month old mice and twelve 20-month old) were purchased from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All animal procedures received approval from the Institutional Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine, ensuring full compliance with institutional welfare guidelines. Prior to experimentation, the animals underwent a four-day acclimatization period under standard laboratory conditions with ad libitum access to food and water, and a controlled 12-hour light/dark cycle.
Mitochondria extraction
Fresh tissues (about 100 mg), including brain, liver, heart, spleen, lung, kidney, stomach, intestine, and muscle, were used for mitochondria extraction by Mitochondria extraction kit according to manufacturer’s instructions.
Protein extraction
The extracted mitochondria sample and the left part were transferred to 1.5ml eppendorf tubes, added with sample lysis buffer, phosphatase inhibitor and PMSF (final concentration 1mM for each). Ultrasonication was performed on ice with the power set at 80 W, using a cycle of 1.0 s on and 1.0 s off, for a total duration of 2 minutes. Centrifuge the solution at 4°C and 12,000 rpm for 10 minutes. Collect the supernatant, then repeat the centrifugation step and collect the supernatant again. The resulting supernatant constitutes the total protein solution of the sample. The total protein concentrations were quantified by bicinchoninic acid assay.
SDS polyacrylamide gel electrophoresis
10 µg protein was taken for SDS-PAGE (12%) separation. Then the gel was stained with Kaumas Brilliant Blue by eStain LG (GenScript, Nanjing). The analysis for gel was adopted by the automatic digital gel image analysis system (Tanon, Shanghai).
Protein digestion
According to the measured protein concentration, take the same quantity protein (50 µg) from each sample, and dilute different groups of samples to the same concentration and volume. Add prepared SP3 magnetic beads to the protein solution, followed by the corresponding volume of 100% acetonitrile (ACN), and incubate at room temperature for 20 minutes. After briefly centrifuging the samples, remove the supernatant. Wash the magnetic beads twice with 70% ethanol and twice with 70% ACN, respectively, removing the supernatant after each wash.
Resuspend the magnetic beads by adding 50 mM ammonium bicarbonate (ABC) solution. Add DTT (25mM) and incubate at 55°C for 30 minutes. Add chloroacetamide, Trypsin, and LysC for enzymatic digestion (protein: enzyme = 50:1 (m/m), 100 µg of protein add 2 µg of enzyme), and incubate overnight at 37°C with shaking at 1500 rpm, enzymatic reaction was stopped by adjusting pH to 3 via adding phosphoric acid. The samples were desalted on SOLA™ SPE. After dried under vacuum, samples were resuspended and iRT peptides (Biognosys, ThermoFisher, 1:10) were added.
Liquid chromatography-mass spectrometry
The Proteomic data analysis was performed by Shanghai OE Biotech Co., Ltd. (Shanghai, China). All data were acquired using a Thermo Scientific Vanquish Neo UHPLC system coupled to an Orbitrap Astral mass spectrometer with a Thermo Scientific Easy-spray source. The separation was performed using a trap column (300 µm × 0.5 cm, 5 µm, Thermo Fisher Scientific) with a C18 analytical column of ES906 (PepMap TM Neo UHPLC 150 µm × 15 cm, 2 µm, Thermo Fisher Scientific). Mobile phases A and B were 0.1% formic acid in water and 0.1% formic acid in ACN-water (v/v=4:1), respectively. The separation was achieved using the following gradient elution program at 0.7 µl/min: 0 ~ 8 min, 7.5% B; 8 ~ 9 min, 35% B; 9 ~ 10 min, 100% B.
For DIA experiments on the Orbitrap Astral MS, the main settings are as follows: capillary was 1.6KV, the full first-stage MS scan range is 380 - 1500 m/z, the primary MS resolution is set to 240,000, the automatic gain control (AGC) is set to 500%, and the parent ion selection window is set to 2-Th, the number of DIA windows is set to 300, the normalized collision energy was set to 25%; and the secondary MS scan range is 150 - 2000 m/z, the RF lens is set to 50%, and the maximal injection time is 3 ms.
Database search
The LC-MS/MS raw data were imported in DIA-NN (Version 1.8.1) for analysis. The database we used was Mouse.MitoCarta3.0. The false positive rate of peptide identification was controlled below 1%. The main parameters of searching database were set as following:
| Item | Value |
|--------|--------|
| Enzyme | Trypsin |
| Max Missed Cleavages | 2 |
| Fixed modifications | Carbamidomethyl (C) |
| Variable modifications | Oxidation (M), Acetyl (Protein N-term) |
| Database pattern | Target-Reverse |
| PSM (Peptide-Spectral Matching) | 0.01 |
| FDR | 0.01 |
Colourimetric assays of mitochondrial enzyme activities
The activity of mitochondria electronic respiratory chain complex, including complex I (NADH-CoQ Reductase), complex II (Succinic acid-coenzyme Q reductase), complex III (Coenzyme Q-Cytochrome C Reductase), complex IV (Cytochrome C Oxidase), complex V (F0F1-ATPase/ATP Synthase), and citrate synthase enzyme were detected by colourimetric assays according to the manufacturer's protocol using fresh extracted mitochondria. In brief, the enzymatic activities were derived from the linear slopes of the reporter dye absorbance changes. These slopes were exported to Microsoft Excel and converted using the appropriate molar extinction coefficient and dilution factor for each respective assay. Furthermore, the assay conditions, including the amount of both homogenate and substrates, were optimized to yield stable readings for all samples. Homogenized samples were used for respiratory chain complex and citrate synthase activity measurement with Complex I activity assay kit, Complex II activity assay kit, Complex III activity assay kit, Complex IV activity assay kit, Complex V activity assay kit, CS activity assay kit, respectively, following the manufacturer's instructions. All the activity assays were conducted in the same day, technical triplicates and an equal number of negative control wells were performed for each sample.
Complex I catalyzes the reaction between NADH and substrates to generate NAD+ and reduced ubiquinone. For a single 200 µL reaction system: Pipette 198 µL of Reagent IV and 2 µL of Reagent V, then mix thoroughly. Prior to the assay, incubate the working solution at 37°C for 5 minutes. In a UV-transparent 96-well plate, sequentially add 10 µL of sample, 200 µL of pre-warmed working solution, and 15 µL of Working Reagent VI. Mix immediately and thoroughly. The rate of decline in NADH absorbance at 340 nm can reflect its activity.
The activity of Complex II is determined by measuring the reduction rate of 2,6-dichloroindophenol (DCIP) at 600 nm. This reaction is driven by reduced coenzyme Q, the catalytic product of Complex II, and the decrease in absorbance of DCIP serves as the indicator for calculating activity.
In a 96-well plate or micro glass cuvette, sequentially add 10 µL of sample, 25 µL of Reagent VI, and 200 µL of Working Reagent IV. Tap the plate gently to ensure thorough mixing, then immediately measure the initial absorbance at 605 nm at 0 minutes (A_1) and after 2 minutes (A_2). Calculate ΔA = A_1 - A_2.
Complex III reduces cytochrome c (oxidized) to cytochrome c (reduced), leading to an increase in absorbance at 550 nm, its activity is calculated by measuring the rate of absorbance increase at 550 nm. To each well of a 96-well plate, sequentially add 25 µL of Reagent VI and 200 µL of the working solution. Mix thoroughly and incubate for exactly 2 minutes. Then, add 10 µL of the extracted mitochondria sample. Immediately after mixing the reaction system, measure the absorbance at 550 nm at time 0 minutes (A_1) and again at 2 minutes (A_2).
The activity of Complex IV is determined by measuring the oxidation rate of reduced cytochrome c, which is monitored as a decrease in absorbance at 550 nm. Sequentially add 200 µL of working solution and 10 µL of sample into the wells of a 96-well plate, and mix rapidly. Immediately after mixing the reaction system, measure the absorbance at 550 nm at 0 minutes (A_1) and at 1 minute (A_2).
Complex V hydrolyzes ATP to ADP. In an enzyme-coupled assay, the resulting ADP is converted, leading to the oxidation of reduced nicotinamide adenine dinucleotide (NADH) to NAD+. The activity of Complex V is calculated by measuring the rate of decrease in absorbance at 340 nm.
Citrate synthase (CS) catalyzes the reaction between acetyl-CoA and oxaloacetate to form citrate and free coenzyme A (CoA-SH). The resulting CoA-SH is then detected by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), generating a yellow-colored product, 5-thio-2-nitrobenzoic acid (TNB), which has a characteristic absorbance peak at 412 nm. The enzyme activity of CS is determined by measuring the rate of increase in absorbance at 412 nm.
Tissue respiratory capacity calculation method TRC=\(\sqrt{CI} + \sqrt{CII} + \sqrt{CIV} \)/3 was used as previously described.
Mitochondrial membrane potential (MMP)
Mitochondrial membrane potential was detected by Enhanced mitochondrial membrane potential assay kit with JC-1 according to manufacture’s instruction. At high mitochondrial membrane potential (ΔΨm), the lipophilic cationic dye JC-1 accumulates in the mitochondrial matrix, where it forms J-aggregates that emit red fluorescence. Conversely, at low ΔΨm, JC-1 cannot accumulate and remains as monomers in the cytoplasm, emitting green fluorescence. Following isolation, tissue cells were washed once with phosphate-buffered saline (PBS). The cell pellet was resuspended in 1 mL of complete cell culture medium. Then, 1 mL of JC-1 staining working solution (prepared by diluting 50 µL of JC-1 stock solution [200X] in 8 mL of pure water) was added. The mixture was inverted several times for thorough mixing and incubated at 37°C for 20 minutes in a cell culture incubator. Cells were centrifuged at 600 × g and 4°C for 4 minutes. The supernatant was carefully discarded. The cell pellet was washed twice with 1 mL of 1X JC-1 staining buffer. Cells were resuspended in 500 µL of 1X JC-1 staining buffer. Samples were subjected to analysis using a flow cytometer (BD Bioscience, America).
Mitochondrial Permeability Transition Pore (MPTP)
Calcein AM readily diffuses across the plasma membrane into the cytosol. Intracellular esterases then cleave the acetoxymethyl (AM) esters, yielding the fluorescent, hydrophilic dye calcein. This charged molecule is effectively trapped within the cell. Calcein AM was co-loaded with cobalt chloride (CoCl_2), Co^^2+ is excluded from the mitochondrial matrix under normal conditions because the inner mitochondrial membrane is impermeable to it. Therefore, in healthy cells, the calcein signal within the mitochondria remains bright, while the cytosolic calcein fluorescence is quenched by Co^^2+^^. The opening of the MPTP creates a large, non-selective pore in the inner mitochondrial membrane. This allows Co^^2+ ions to enter the mitochondrial matrix. Upon entry, Co^^2+ quenches the mitochondrial calcein fluorescence. Consequently, a decrease in intracellular calcein fluorescence is directly correlated with MPTP opening.
MPTP was detected by mitochondrial membrane potential (calcein AM) assay kit according to manufacture’s instruction. Following isolation, tissue cells were washed once with phosphate-buffered saline (PBS). Then, they were resuspended in an appropriate volume of a solution containing Calcein AM staining solution, fluorescence quenching solution, and Ionomycin control to achieve a cell density of approximately 1×10^6 cells/mL. The suspension was incubated at 37°C in the dark for 30-45 minutes. After incubation, the cells were collected by centrifugation at 300 × g for 5 minutes. Each pellet was resuspended in 1 mL of assay buffer and centrifuged again under the same conditions (300 × g for 5 minutes). The final cell pellet was resuspended in 400 µL of assay buffer and analyzed using a flow cytometer.
Mitochondrial calcium concentration
Rhod-2 AM is an acetoxymethyl ester derivative of Rhod-2 that is cell-permeant. Upon entering the cell, it is hydrolyzed by intracellular esterases to yield Rhod-2, a charged form that is trapped within the cell. As Rhod-2 shares the core structure of rhodamine-based mitochondria-specific probes, it accumulates within mitochondria where it chelates calcium ions. The Rhod-2-Ca^^2+ complex exhibits strong fluorescence upon excitation at 552 nm, making Rhod-2 AM a widely used probe for monitoring changes in mitochondrial calcium concentration.
Calcium concentration was detected by mitochondrial calcium assay kit with Rhod-2 AM according to manufacture’s instruction. In brief, isolated tissue cells were washed once with phosphate-buffered saline (PBS). Add 100 µl of Rhod-2 staining solution and incubate at 37°C for 10-30 minutes in a cell culture incubator. (The optimal incubation time varies with different cell types. A 20-minute incubation is recommended as a starting point, which was optimized for specific cells to achieve best results.) After incubation at 37°C, remove the supernatant and wash the cells twice with PBS. Resuspend the cells in 100 µl of PBS and analyze using a flow cytometer.
Mitochondria Reactive Oxygen Species (ROS) production
DCFH-DA (2',7'-Dichlorodihydrofluorescein diacetate) freely diffuses across the plasma membrane of living cells. Once inside the cell, it is hydrolyzed by intracellular esterases to form DCFH (2',7'-Dichlorodihydrofluorescein), which is non-fluorescent and membrane-impermeable. Subsequently, DCFH can be oxidized by intracellular reactive oxygen species (ROS) to generate highly fluorescent DCF (2',7'-Dichlorofluorescein). Here, ROS concentrations was detected by CheKine™ ROS content assay kit according to manufacture’s instruction. In brief, DCFH-DA was diluted 1:1000 in serum-free culture medium to achieve a final concentration of 10 µmol/L. After collection, resuspend the cells in the diluted DCFH-DA solution at a density of 1×10^6 to 2×10^7 cells/mL. Incubate the suspension at 37°C for 30 minutes in a cell culture incubator. Gently invert the tube every 3-5 minutes to ensure thorough contact between the probe and cells. Wash the cells three times with serum-free culture medium to completely remove any extracellular DCFH-DA. Using an excitation wavelength of 488 nm and an emission wavelength of 525 nm, measure the fluorescence intensity in real-time or at designated time points before and after stimulation. The fluorescence intensity of DCF was quantified using flow cytometry, serves as an indicator for analyzing intracellular ROS levels.
mtDNA copy number and damage
Mitochondria DNA copy number was detected by Mouse Mitochondrial Biogenesis Assay Kit by mtDNA Copy Number according to manufacture’s instruction. Mitochondria DNA damage was detected by Mouse Mitochondrial DNA Damage Quantification Kit according to manufacture’s instruction.
Statistical analyses
For proteomic data, a total of 7200 proteins expressed were identified as belonging to the proteome of mice this study. The thresholds of fold change (>1.2 or <0.83) and P-value <0.05 were used to identify differentially expressed proteins (DEPs). Annotation of all identified proteins was performed manually using Mouse.MitoCarta3.0 (https://www.broadinstitute.org/mitocarta/mitocarta30-inventory-mammalian-mitochondrial-proteins-and-pathways). Relocalization analysis was completed by relocalization analysis pepline we built, which is uploaded to github (https://github.com/gaoqing88/Mitoprotein-relocalization-pepline). Organheatmap was plotted by using R project (https://cran.rproject.org/web/packages/OrgHeatmap/index.html). For other data, each quantitative experiment was repeated three times. One-way analysis of variance was used to assess the differences among the groups. Statistical results were presented as means ± standard deviation (SD). P values of < 0.05 were denoted as statistically significant. All statistical analyses were calculated using GraphPad Prism 10.0 program (GraphPad Software, Inc., La Jolla, CA, USA).