CN114836368A

CN114836368A - Mitochondria purification kit

Info

Publication number: CN114836368A
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: 2022-08-02
Anticipated expiration: 2042-05-13
Also published as: CN114836368B

Abstract

The invention discloses a mitochondrial purification kit, which comprises a biotin-labeled anti-human mitochondrial outer membrane protein OMP25 monoclonal antibody HC01-R001, streptavidin-coupled affinity material and other related 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 relevant other materials comprise a buffer, an eluent and a disposable consumable. The kit can specifically identify and adsorb complete human mitochondria, and can obtain 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, simplicity in operation, small damage to mitochondria and the like, and can be applied to related researches on a plurality of mitochondria.

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 organelles present in most cells, which are two-layer membrane-coated organelles, structures that produce energy in the cell, and major sites where the cell undergoes aerobic respiration. Mitochondria possess their own genetic material and genetic system, but have a limited genomic size and are a semi-autonomous organelle. In addition to providing energy to cells, mitochondria are involved in processes such as cell differentiation, cell information transmission, and apoptosis, and possess the ability to regulate cell growth and cell cycle.

Mitochondria are one of the most susceptible organelles to various insults. The most common pathological changes in cell damage include changes in mitochondrial number, size and structure. The mitochondrial diseases can be caused by the occurrence of problems of human mitochondria, the mitochondrial diseases are a large group of inherited metabolic diseases, the inheritance mode of the mitochondrial diseases is complex, the causes of the diseases are mainly caused by nuclear genes and mitochondrial genes, the clinical manifestation is complex, the diagnosis of exact causes is very difficult, and the causes are often determined by the double means of macromolecular enzymatic activity detection and analysis and genetic gene analysis. Proteins within the mitochondria are the basis for normal mitochondrial function, and mitochondrial damage and altered function are often accompanied by altered proteomics within the 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 directionally to the mitochondria. These proteins are present in unfolded precursor form prior to transport, and chaperones (of the hsp70 family) bound to them maintain the precursor proteins in an unfolded state. Usually, the N-terminal signal sequence of the precursor protein is called leader, leader or transit peptide, and after completion of the transit, it is cleaved by a signal peptidase to obtain the mature protein, which is called posttranslation. Mitochondria have limited protein synthesis capacity, and a very small fraction are encoded and translated by their own DNA, since they contain a complete set of equipment for DNA replication, transcription, and protein translation, including RNA, DNA polymerase, RNA polymerase, tRNA, ribosomes, amino acid activating enzymes, etc., indicating that mitochondria have an independent genetic system. The process is substantially the same as for nuclear genes.

The study of mitochondrial proteomics under different functional states is of great significance for understanding mitochondrial function and pathogenesis of related diseases. However, since most of the proteins in mitochondria are from the cytoplasm, many proteins exist in both the mitochondria and the cytoplasm, and are not specific to the mitochondria, which brings difficulty to the analysis of mitochondrial proteomics, and the analysis can be realized only by obtaining live 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, the purity of the mitochondria separated by the method is very low, the operation is complex, expensive ultra-separation equipment is needed, the time consumption is long, and the separation liquid and the centrifugal force cause serious damage to the mitochondria, so that the requirement of research on mitochondrial proteomics can not be met. Although there are a few methods using immunomagnetic bead separation, most methods using knock-in of the gene expressed by the tag protein are only suitable for cell lines, are not suitable for human samples, and cannot separate purified mitochondria from magnetic beads. Therefore, there is an urgent need for a method for rapid purification of mitochondria that is mild, 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 material and other related 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 material which can be chemically bonded with the streptavidin and is activated by surface epoxy groups. The relevant 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, specifically adsorb human mitochondria, and can be specifically combined with the extracellular region of human mitochondrial outer membrane protein OMP 25.

The invention also aims to provide the application of the mitochondria purification kit in the separation and purification experiments of mitochondria of human tissues, isolated cells in body fluid and cultured cells in vitro.

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

According to the mitochondrial purification kit provided by the invention, due to the affinity material which is provided with the coupling of the specific biotin-labeled anti-human mitochondrial outer membrane protein OMP25 monoclonal antibody HC01-R001 and streptavidin, the kit can specifically identify and adsorb human mitochondria, is separated and purified by magnetic beads, and is eluted by eluent containing high-concentration biotin to obtain high-purity human mitochondria, and can be used for pathological diagnosis, biological research, biological mass spectrometry, proteomic 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) the conditions are mild, and the chemical and physical damage to mitochondria is small; (3) the purity of mitochondria is high; (4) the mitochondria have complete envelope and function; (5) the separation of mitochondria and magnetic beads can be realized; (6) directly bind to mitochondrial outer membrane proteins without the aid of tag proteins.

Drawings

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

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

FIG. 3 comparison of the amounts of human mitochondrial protein species purified by different methods.

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

Detailed Description

For a further understanding of the invention, reference is made to the following description of the preferred embodiment of the invention taken in conjunction with the accompanying drawings and examples, but it is to be understood that the description is intended to illustrate the invention further and not to limit the scope of the invention as claimed.

Example 1 Biotin labelling of anti-human mitochondrial outer Membrane protein OMP25 monoclonal antibody HC01-R001

(II) antibody ultrafiltration treatment

1. Adding 200 μ L of labeled reaction solution into ultrafiltration column, adding 500 μ g of monoclonal antibody HC01-R001, mixing,

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

3. add 100. mu.L of labeled reaction solution to the ultrafiltration column and mix well. 4 ℃, Max 14000 Xg, 2min,

4. the steps 26 to 7 times are repeated,

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

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

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

8. the column was inverted and filtered at 4 ℃ and 6000rpm for 2 min. Collecting filtrate, combining with the filtrate in the step 6, and standing at 4 ℃ for later use.

(II) Biotin-labeled antibody

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

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

3. and separating and purifying the glucan gel (removing free biotin).

(III) separating and purifying the labeled antibody by gel column chromatography

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

2. balancing: after the NaOH treatment is finished, washing the column volume by using ultrapure water for 2 times, then washing the column volume by using a buffer solution containing 200mmol/L NaCl and 20mmol/L PB and having a pH of 7.0 for 5-10 times,

3. loading: after the balance is finished, a sample pump is selected for sample loading, the sample loading flow rate is 3mL/min, the sample loading volume is 1mL,

4. and (3) elution: after the sample loading is finished, eluting by using an equilibrium buffer solution,

5. cleaning and storing: and after purification, reversely washing the column by using 0.5mol/L NaOH for 2 volumes for 30-60 min, after washing, positively washing the column by using ultrapure water for 5 volumes, then washing the column by using 20% ethanol for 3 volumes, then detaching the column, sealing two ends, and storing at low temperature.

Example 2 streptavidin-coupled magnetic microspheres

1. Placing 10mg of magnetic microspheres with activated surface epoxy groups into a 1.5mL EP tube with low adsorption on the surface, adding 1mL of 0.1M phosphate solution with pH of 7.4, slightly shaking and uniformly mixing, placing on a magnetic frame, and completely absorbing and discarding the phosphate solution;

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

3. taking 100 mu g of streptavidin, preparing the streptavidin into 1mg/mL by 0.1M phosphate solution with pH7.4, and making the total volume of the streptavidin be 100 mu L;

4. dropwise adding 3M ammonium sulfate solution into 100 mu L of streptavidin solution, and uniformly mixing each drop in time to prevent precipitation, and adding 100 mu L;

5. centrifuging 200 μ L mixed solution of streptavidin and ammonium sulfate at high speed for 1min, collecting supernatant, adding 10mg magnetic microsphere, and mixing;

shaking and incubating for 14h at 6.30 ℃;

7. placing the incubated magnetic microspheres on a magnetic frame, and completely absorbing the solution;

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

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

10. adding 1mL of phosphate solution with the pH value of 7.4, uniformly mixing, placing on a magnetic frame, and completely sucking the solution;

11. repeating the step 102 times, adding 1mL of phosphate solution with pH of 7.4, mixing, and storing at 4 deg.C for use.

Example 3 mitochondrial purification in cultured HEK293 cells

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

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

2. adding the cells into a 2mL Dunn grinder in an ice bath, and quickly grinding for 20-30 times;

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

4. dissolving 10 mu g of biotin-labeled HC01-R001 in 1mL of 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 frame, completely sucking the solution, and washing for 3 times by using 1mL of buffer solution;

6. adding magnetic microspheres into the diluted mitochondrial solution, and performing rotary incubation at 4 ℃ for 5 min;

7. placing the mixed solution on a magnetic frame, completely sucking the solution, and washing for 2 times by using 1mL of buffer solution;

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

9. placing the mixed solution on a magnetic frame, and sucking the solution to a low-adsorption EP (EP) tube of 1.5 mL;

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

Example 4 comparison of purity of mitochondria purified by different methods

Comparing the purity of the purified mitochondria described in example 3 above with the purity of mitochondria purified by sucrose density gradient centrifugation, the purification of mitochondria by sucrose density gradient centrifugation is as follows:

1. HEK293 cells cultured in 10cm dishes were harvested at about 0.5-1X 10 ⁸ The cells were washed 3 times with 4 ℃ pre-cooled PBS and resuspended in 1mL PBS.

2. The cells were added to a 2mL dounce grinder in an ice bath and quickly ground 20-30 times.

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

4. Carefully add a layer of 15ml of 1.0mol/L sucrose solution to 15ml of 1.5mol/L sucrose solution in a Uitradear centrifuge tube for a Bockman SW28 rotor to form a 30ml gradient.

5. The mitochondrial suspension (total volume 6ml) was carefully added and centrifuged at 60000g (22000r/min) at 4 ℃ for 1h, resulting in a thin layer of mitochondria at the 1mol/L and 1.5mol/L interfaces.

6. Mitochondria were gently aspirated off the top of the 1.5mol/L layer using a Pasteur pipette.

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

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

9. The mitochondria obtained by the two methods are added into 2X loading buffer solution, boiled for 5min in boiling water, and then subjected to Werstern Blot experiment, and are respectively detected by monoclonal antibodies resisting mitochondrial outer membrane protein, mitochondrial matrix protein, cytoplasmic protein and Golgi protein, as shown in figure 2, the mitochondria sample purified by the method disclosed by the invention hardly contains hybrid proteins such as cytoplasmic protein and Golgi protein, and the purified mitochondria has high purity.

Example 5 human mitochondrial proteomics analysis

The mitochondria sample purified by the method and the mitochondria purified by the sucrose density gradient centrifugation method are analyzed by proteomics, and the difference of the proteomics of the samples obtained by the two methods is compared.

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

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

3. Cutting protein bands with gel cutter, placing in 500 μ L EP tube, adding 200/A1 decolorized solution (prepared by mixing 100mmol/L sodium thiosulfate and 30mmol/L potassium ferricyanide solution at a ratio of 1: 1), standing for 30min, and discarding supernatant. Then 200/ul water is added for cleaning, standing for 30min, and then supernatant is discarded. This procedure was repeated until the crumb became colorless.

4. 200ul acetonitrile was added to the micelle and left to stand for 30min, and then the supernatant was discarded. This procedure was repeated until the gel was completely dehydrated to white. After the acetonitrile was removed by suction, the gel particles were left to dry completely at 37 ℃.

5. Sequencing-grade trypsin is prepared into 12.5ng/ml by using 20mmol/L ammonium bicarbonate solution, then a proper amount of enzyme solution is added into the colloidal particles, and the colloidal particles are fully swelled by placing 30rain at 4-C. Then sucking out excessive enzyme solution and discarding to prevent excessive peptide fragments degraded by trypsin during mass spectrometry detection.

6. Approximately 5ul of 25mmol/L ammonium bicarbonate solution was added to cover the gel to prevent the solution from drying during the enzymatic hydrolysis. And (5) placing the mixture in a 37 ℃ temperature control box for heat preservation for 12-16 h.

7. The enzymolyzed micelle is extracted with 60u1 of 0.1% TFA and 50% acetonitrile for 3 times, each for 20min, and the extractive solutions are combined.

8. The solution was blown dry in a nitrogen stream and then subjected to LC-MS/MS identification.

9. As shown in FIG. 3, the purification of mitochondria by sucrose density gradient centrifugation identified 151 proteins, and the method of the invention identified 78 proteins, 57 of which were identified by both methods; as shown in fig. 4, the mitochondrial protein in the sample purified by the method of the present invention accounted for 88% of the total protein identified, and the mitochondrial protein in the sample purified by sucrose density gradient centrifugation accounted for only 42% of the total protein identified, indicating that the mitochondria purified by the method of the present invention are more pure.

Sequence listing

<110> Hangzhou heavy chain science and technology Co., Ltd

<120> mitochondrial purification kit

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Val Ser Ser Gly Lys Pro Lys Asp Lys Thr Val Ala Pro Thr Leu Met

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Claims

1. A mitochondrial purification kit comprising a biotin-labeled anti-human mitochondrial outer membrane protein OMP25 monoclonal antibody HC01-R001, streptavidin-conjugated affinity material, and related other materials.

2. The kit according to claim 1, 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.

3. The kit of claim 1, wherein the streptavidin-coupled affinity material comprises a support matrix and streptavidin, wherein the support matrix is a magnetic microsphere or other material activated by surface epoxy groups that can be chemically bonded to streptavidin.

4. The kit of claims 1-3, wherein the monoclonal antibody HC01-R001 and streptavidin-conjugated affinity material are capable of high affinity binding and specific adsorption to human mitochondria.

5. The kit of claim 1, wherein the associated other materials include buffers, eluents, disposable surface low adsorption consumables.

6. The kit according to claims 1-5, for use in experiments for the isolation and purification of mitochondria in human tissues, in vivo isolated cells and in vitro cultured cells.

7. There is provided a use of the kit of claims 1-5 in pathological diagnosis, biological research, biomolecular mass spectrometry and proteomics research.