CN114836368B

CN114836368B - Mitochondria purification kit

Info

Publication number: CN114836368B
Application number: CN202210522270.3A
Authority: CN
Inventors: 高传杰; 赵明红
Original assignee: Hangzhou Heavy Chain Technology Co ltd
Current assignee: Hangzhou Heavy Chain Technology Co ltd
Priority date: 2022-05-13
Filing date: 2022-05-13
Publication date: 2023-07-21
Anticipated expiration: 2042-05-13
Also published as: CN114836368A

Abstract

The invention discloses a mitochondrion purification kit, which comprises a biotin-marked anti-human mitochondrial outer membrane protein OMP25 monoclonal antibody HC01-R001, streptavidin coupled affinity material and related other materials. The heavy chain and light chain amino acid sequences of HC01-R001 are shown as SEQ ID No.1 and SEQ ID No. 2. The streptavidin coupled affinity material comprises a carrier matrix and streptavidin, and the related other materials comprise a buffer solution, an eluent and a disposable consumable. The kit can specifically identify and adsorb the complete human mitochondria, and can obtain the purified complete human mitochondria through the steps of cell disruption, mitochondrial adsorption, impurity washing, mitochondrial elution and the like. The kit has the advantages of high speed, high purity, simple operation, small damage to mitochondria and the like, and can be applied to various mitochondrial related researches.

Description

Mitochondria purification kit

Technical Field

The invention belongs to the technical field of biology, and relates to a mitochondrial purification kit.

Background

Mitochondria are an organelle that is coated with two membranes and found in most cells, where the structure of energy is produced, the main site where the cells breathe aerobically. Mitochondria possess their own genetic material and genetic system, but their genome size is limited, a semi-autonomous organelle. In addition to providing energy to cells, mitochondria are involved in processes such as cell differentiation, cell information transfer, and apoptosis, and possess the ability to regulate cell growth and cell cycle.

Mitochondria are one of the most sensitive organelles to various injuries. The most common pathological changes at the time of cell injury include alterations in mitochondrial number, size and structure. The human mitochondria are problematic in causing mitochondrial diseases, the mitochondrial diseases are a large class of inherited metabolic diseases, the inherited modes of the mitochondrial diseases are complex, the causes of the diseases are mainly caused by nuclear genes and mitochondrial genes, the clinical manifestations are complex, the diagnosis of exact causes is very difficult, and the causes are often determined by a double means of macromolecular enzymatic activity detection analysis and genetic gene analysis. The proteins within mitochondria are the basis for the normal function performed by mitochondria, and mitochondrial damage and alterations in function are often accompanied by alterations in proteomics within mitochondria. Mitochondrial proteins are divided into two parts, which are caused by the semi-autonomy of mitochondria. A large number of mitochondrial proteins are synthesized in the cytoplasm and transported targeted to the mitochondria. These proteins exist in an unfolded precursor form prior to transport, with the chaperones (belonging to the hsp70 family) to which they bind keeping the precursor protein in an unfolded state. Usually, a signal sequence at the N-terminal of a precursor protein is called a leader peptide, a leader peptide or a transit peptide, and the precursor protein is cleaved by a signal peptidase after the completion of the transit to form a mature protein, which is called post-translation. The protein synthesis capacity of mitochondria is limited, and about a few are encoded by the DNA of the mitochondria itself and translated, because it contains a complete set of RNA, DNA polymerase, RNA polymerase, tRNA, ribosome, amino acid activating enzyme, etc. for DNA replication, transcription and protein translation, indicating that mitochondria have independent genetic systems. The process is generally the same as that of nuclear genes.

Study of mitochondrial proteomics in different functional states is important for understanding the pathogenesis of mitochondrial function and related diseases. However, since most of the proteins in mitochondria are derived from cytoplasm, many proteins exist in mitochondria and also exist in cytoplasm in large quantity, and are not unique to mitochondria, which brings difficulty to analysis of mitochondrial proteomics, and it is necessary to obtain living mitochondria with high purity and complete envelope. At present, the separation and purification of mitochondria mainly depend on a sucrose gradient density separation method or a similar density separation method, and the mitochondria separated by the method has low purity, complex operation, expensive super-separation equipment is required, long time is consumed, and the used separation liquid and centrifugal force cause serious damage to the mitochondria and cannot meet the requirements of mitochondrial proteomics research. Although a small amount of immunomagnetic bead separation methods are adopted, most of the methods adopt a mode of knocking in a tag protein expression gene, are only applicable to cell lines, are not applicable to human body samples, and cannot separate purified mitochondria from magnetic beads. Therefore, there is an urgent need for a rapid purification method for mitochondria that is gentle, simple and easy to perform.

Disclosure of Invention

The invention aims to provide a mitochondria purification kit, which comprises a biotin-labeled anti-human mitochondrial outer membrane protein OMP25 monoclonal antibody HC01-R001, streptavidin coupled affinity materials and related other materials. The heavy chain amino acid sequence of the monoclonal antibody HC01-R001 is shown as SEQ ID No.1, and the light chain amino acid sequence of the monoclonal antibody HC01-R001 is shown as SEQ ID No. 2. The streptavidin coupled affinity material comprises a carrier matrix and streptavidin, wherein the carrier matrix is a magnetic microsphere or other materials with surface epoxy groups which can be combined with the streptavidin in a chemical bond. The related other materials include buffers, eluents, and disposable consumables.

The biotin-labeled anti-human mitochondrial outer membrane protein OMP25 monoclonal antibody HC01-R001 and streptavidin coupled affinity material can be combined with high affinity, can specifically adsorb human mitochondria, and can specifically combine with the extracellular region of the human mitochondrial outer membrane protein OMP 25.

It is another object of the present invention to provide the use of the mitochondrial purification kit in experiments for isolation and purification of isolated cells in human tissues, body fluids and cultured cell mitochondria in vitro.

The mitochondria purification kit provided by the invention can be applied to pathological diagnosis, biological research, biological mass spectrum and proteomics research.

The mitochondria purification kit provided by the invention can specifically identify and adsorb human mitochondria due to the specific biotin-marked anti-human mitochondrial outer membrane protein OMP25 monoclonal antibody HC01-R001 and streptavidin-coupled affinity material, and the human mitochondria can be obtained by separating and purifying through magnetic beads and eluting with eluent containing high-concentration biotin, so that the high-purity human mitochondria can be used for pathological diagnosis, biological research, biological mass spectrum, proteomics research and the like. Advantages of the present invention compared to other methods include: (1) The operation is quick and simple, the whole purification process can be completed within 1 hour, and expensive equipment is not needed; (2) Mild conditions and small chemical and physical damage to mitochondria; (3) high mitochondrial purity; (4) complete mitochondrial envelope and function; (5) separation of mitochondria and magnetic beads can be achieved; (6) Directly bind to the mitochondrial outer membrane protein without the aid of a tag protein.

Drawings

FIG. 1 is a schematic diagram of a step of rapid extraction of mitochondria from cultured cell lines or tissues.

FIG. 2 comparison of purified mitochondrial purity by different methods.

FIG. 3 comparison of the number of purified human mitochondrial proteins by different methods.

FIG. 4 various ratios of purified human mitochondrial proteins by different methods. Wherein A is the method of the invention and B is sucrose density gradient centrifugation.

Detailed Description

For a further understanding of the present invention, preferred embodiments of the invention are described below with reference to the drawings and examples, but it should be understood that these descriptions are intended only to illustrate the invention further and are not limiting to the claims of the invention.

EXAMPLE 1 Biotin labelling of anti-human mitochondrial outer membrane protein OMP25 monoclonal antibody HC01-R001

Antibody ultrafiltration treatment

1. Adding 200 mu L of marked reaction solution into an ultrafiltration column, adding 500 mu g of monoclonal antibody HC01-R001, uniformly mixing,

2. centrifuge at 4℃at 6000rpm for 2min. The filtrate was discarded and the residue was taken out,

3. 100. Mu.L of the labeled reaction solution was added to the ultrafiltration column and mixed well. At 4 ℃, max 14000 Xg, 2min,

4. the steps 2 6 to 7 times are repeated,

5. mixing the residual liquid in the ultrafiltration column, standing at room temperature for 1min,

6. inverting the ultrafiltration column in a new ultrafiltration tube at 4deg.C, 1000Xg, 2min, collecting filtrate,

7. mixing 50 μl PBS in ultrafiltration column, standing for 1min,

8. the ultrafiltration column was inverted, 4℃at 600 rpm,2min. And (3) collecting filtrate, combining the filtrate with the filtrate obtained in the step (6), and standing at 4 ℃ for standby.

(II) biotin-labeled antibody

1. The amount of biotin required for antibody labeling was calculated,

2. adding NHS-PEG4-Biotin solution into the ultrafiltered filtrate, reacting for 1h at room temperature,

3. sephadex separation and purification (removal of free biotin).

(III) gel column separation and purification of labeled antibody

1. Gel sterilization treatment: after washing the column with ultrapure water, the column was washed forward with 0.5mol/L NaOH at a flow rate of 3mL/min, 3 column volumes were washed,

2. balance: after the NaOH treatment, 2 column volumes were rinsed with ultra pure water, followed by 5 to 10 column volumes with a 7.0pH buffer containing 200mmol/L NaCl and 20mmol/L PB,

3. loading: after balancing, selecting a sample pump for loading, wherein the loading flow rate is 3mL/min, the loading volume is 1mL,

4. eluting: after the loading is finished, eluting by using an equilibrium buffer solution,

5. cleaning and preserving: after purification, back flushing 2 column volumes with 0.5mol/L NaOH for 30-60 min, after flushing, forward flushing 5 column volumes with ultrapure water, flushing 3 column volumes with 20% ethanol, then removing the column, sealing the two ends, and preserving at low temperature.

Example 2 streptavidin coupled magnetic microspheres

1. 10mg of the magnetic microsphere with the surface epoxy group activated is taken and placed in a 1.5mL EP tube with the surface with low adsorption, 1mL of 0.1M phosphate solution with pH of 7.4 is added, the mixture is mixed by slight oscillation, and placed on a magnetic frame, and the phosphate solution is sucked and discarded;

2. repeating the washing step for 2 times, and keeping the washed magnetic microspheres for later use;

3. 100. Mu.g of streptavidin was prepared at 1mg/mL with a pH7.4 solution of 0.1M phosphate, and the total volume was 100. Mu.L;

4. adding 3M ammonium sulfate solution dropwise into 100 μl of streptavidin solution, mixing well in time every time, preventing precipitation, and adding 100 μl;

5. centrifuging 200 μL of mixed solution of streptavidin and ammonium sulfate at high speed for 1min, taking supernatant, adding 10mg of magnetic microspheres, and mixing uniformly;

6.30 ℃ shaking incubation for 14h;

7. placing the incubated magnetic microspheres on a magnetic rack, and sucking the solution;

8. adding 1mL of 0.1M glycine solution with pH of 2.5, uniformly mixing, placing on a magnetic rack, and sucking the solution;

9. adding 1mL of 10mM Tris solution with pH of 8.8, uniformly mixing, placing on a magnetic rack, and sucking the solution;

10. adding 1mL of phosphate solution with pH of 7.4, uniformly mixing, and placing on a magnetic rack to suck the solution;

11. repeating the steps 102 times, adding 1mL of phosphate solution with pH of 7.4, uniformly mixing, and storing at 4 ℃ for standby.

EXAMPLE 3 mitochondrial purification in cultured HEK293 cells

According to the procedure shown in FIG. 1, the reagents used included PBS, buffer, and eluent, wherein the buffer was PBS containing 5% BSA and the eluent was PBS containing 2M biotin; disposable consumables used included low adsorption 1.5mL and 2mL specification EP tubes, 15mL centrifuge tubes, etc.

1. HEK293 cells cultured in 10cm dishes were collected, approximately 0.5-1X 10 ⁸ The individual cells were washed 3 times with 4℃pre-chilled PBS and resuspended in 1mL buffer;

2. adding the cells into a 2mL Dunn mill in an ice bath, and rapidly milling for 20-30 times;

3. centrifuging 700g of the ground cell suspension at 4 ℃ for 5min, placing the supernatant into a 15mL centrifuge tube, and diluting with 5mL precooled buffer solution;

4. dissolving 10 mug of biotin-labeled HC01-R001 in 1mL buffer solution, uniformly mixing with 10mg of streptavidin-coupled magnetic microspheres, and incubating for 2min at 4 ℃;

5. placing the magnetic microspheres on a magnetic rack, sucking the solution, and washing with 1mL of buffer solution for 3 times;

6. adding the magnetic microsphere into the diluted mitochondrial solution, and incubating at 4 ℃ for 5min in a rotating way;

7. placing the mixed solution on a magnetic rack, sucking the solution, and washing with 1mL buffer solution for 2 times;

8. adding 1mL of eluent containing 2M biotin, and rotating at 4 ℃ for incubation for 5min;

9. placing the mixed solution on a magnetic rack, and sucking the solution to a 1.5mL low-adsorption EP pipe;

centrifugation was performed at 10000g for 5min at 10.4℃and the supernatant was discarded, and the precipitated mitochondria were resuspended in 100. Mu.L PBS.

Example 4 comparison of purified mitochondrial purity by different methods

The purified mitochondria described in example 3 above were compared to the purified mitochondria by sucrose density gradient centrifugation, which was performed as follows:

1. HEK293 cells cultured in 10cm dishes were collected, approximately 0.5-1X 10 ⁸ Individual cells were washed 3 times with 4 ℃ pre-chilled PBS and resuspended in 1mL PBS.

2. The cells were added to a 2mL dunus mill in an ice bath and rapidly milled 20-30 times.

3. The above ground cell suspension was centrifuged at 700g for 5min at 4℃and the supernatant was diluted with 5mL of pre-chilled PBS.

4. A layer of 15ml of 1.0mol/L sucrose solution was carefully added to 15ml of 1.5mol/L sucrose solution in a Uitheraer centrifuge tube for Bockman SW28 rotor, creating a 30ml gradient.

5. Mitochondrial suspension (total volume 6 ml) was carefully added and centrifuged at 60000g (22000 r/min) at 4℃for 1h, and the mitochondria formed a thin layer at the 1mol/L and 1.5mol/L interface.

6. The mitochondria were aspirated gently on top of the 1.5mol/L layer with a Pasteur pipette.

7. The sucrose solution was diluted and centrifuged at 17000g for 15 min to pellet the mitochondria.

8. Washed once with 30ml buffer and resuspended in an appropriate volume of buffer suitable for subsequent work.

9. The mitochondria obtained by the two methods are added into a 2X loading buffer solution, boiled in boiling water for 5min, and then Werstern Blot experiments are carried out, and monoclonal antibodies for resisting mitochondrial outer membrane proteins, mitochondrial matrix proteins, cytoplasmic proteins and Golgi proteins are respectively used for detection, as shown in figure 2, the mitochondria sample purified by the method disclosed by the invention contains almost no cytoplasmic proteins, golgi proteins and other impurity proteins, so that the purity of the purified mitochondria is very high.

Example 5 human mitochondrial proteomic analysis

The mitochondrial sample purified by the method of the invention and the purified mitochondria by sucrose density gradient centrifugation are subjected to proteomic analysis, and the difference of proteomics of the samples obtained by the two methods is compared.

1. Adding 2X loading buffer solution into the mitochondrial sample, boiling for 5min, performing SDS-PAGE electrophoresis, and stopping electrophoresis after the electrophoresis front of the sample runs to about 1cm of the separation gel.

2. The separation gel was removed, rapidly stained with coomassie brilliant blue and destained with clear water.

3. The protein bands were cut with a cutter, placed in 500. Mu.L EP tube, added with 200/A1 decolorized solution (100 mmol/L sodium thiosulfate and 30mmol/L potassium ferricyanide solution mixed 1:1), left to stand for 30min, and the supernatant was discarded. Then adding 200/ul of water for cleaning, standing for 30min, and then discarding the supernatant. This step was repeated until the gel particles became colorless.

4. 200ul of acetonitrile is added into the colloidal particles, and the mixture is kept stand for 30min, and then the supernatant is discarded. This step was repeated until the gel was completely dehydrated and whitened. After the acetonitrile was sucked out, the gel was left at 37℃until it was completely dried.

5. After sequencing grade trypsin is prepared into a concentration of 12.5ng/ml by using 20mmol/L ammonium bicarbonate solution, a proper amount of enzyme solution is added into the colloidal particles, and 30rain is placed at 4-C to fully swell the colloidal particles. Excess enzyme solution was then aspirated for disposal to prevent excessive trypsin self-degrading peptide fragments during mass spectrometry detection.

6. Approximately 5ul of 25mmol/L ammonium bicarbonate solution was added to cover the colloidal particles to prevent the solution from drying out during the enzymatic hydrolysis. Placing the mixture in a temperature control box at 37 ℃ for heat preservation for 12-16 h.

7. The digested pellet was extracted 3 times with 60u1 of 0.1% TFA and 50% acetonitrile for 20min each, and the extracts were combined.

8. The solution was blow dried under a nitrogen stream and then LC-MS/MS identified.

9. As shown in FIG. 3, the sucrose density gradient centrifugation method for purifying mitochondria identified 151 proteins, and the method of the present invention identified 78 proteins, 57 of which were identified by both methods; as shown in FIG. 4, the mitochondrial proteins in the purified samples of the method of the invention account for 88% of the total protein identified, and the mitochondrial proteins in the sucrose density gradient centrifugation purified samples account for only 42% of the total protein identified, indicating that the purified mitochondria of the method of the invention have higher purity.

Sequence listing

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Claims

1. The kit is characterized by comprising a biotin-labeled anti-human mitochondrial outer membrane protein OMP25 monoclonal antibody HC01-R001 and streptavidin coupled affinity material, wherein the heavy chain amino acid sequence of the monoclonal antibody HC01-R001 is shown as SEQ ID No.1, and the light chain amino acid sequence is shown as SEQ ID No. 2.

2. The kit of claim 1, wherein the streptavidin-coupled affinity material comprises a carrier matrix and streptavidin, the carrier matrix being a surface epoxy-activated magnetic microsphere capable of binding with streptavidin in a chemical bond.

3. The kit according to claim 1 or 2, wherein the monoclonal antibody HC01-R001 binds with high affinity to streptavidin-coupled affinity material and specifically adsorbs human mitochondria.

4. The kit of claim 1, further comprising a buffer, an eluent, and a disposable surface low adsorption consumable.

5. Use of the kit according to any one of claims 1-4 in isolation and purification experiments of human tissues, cells isolated in body fluids and mitochondria cultured in vitro.

6. Use of a kit according to any one of claims 1-4 in biological research, in mass spectrometry and in proteomics research.