CN110551687A - Method for separating exosomes in blood plasma based on solid-phase metal affinity chromatography - Google Patents

Abstract

The present invention provides a simple and rapid method for separating exosomes or exosome proteins in plasma based on solid-phase metal affinity chromatography, comprising at least the following steps: 1) using monodisperse immobilized affinity chromatography microspheres CAE-Ti ^{4 +} ‑IMAC adsorbs exosomes; 2) uses ammonia water to elute exosomes; 3) immediately replaces ammonia water with phosphate buffer to reduce the damage of ammonia water to exosomes. The collection method of the present invention has the characteristics of low cost, simple and rapid operation, time-saving and labor-saving, and high collection efficiency, and the method allows various subsequent molecular analysis of exosomes, including analysis of genome and proteome. The present invention can effectively solve the problems of long time, high cost and low purity of the current exosome separation and purification, provides a feasible technical solution for the study of disease plasma exosomes, and has good advantages in discovering new exosome markers and the like. application prospects.

Description

A method for separating exosomes from plasma based on solid-phase metal affinity chromatography

technical field

The invention relates to the field of biotechnology, in particular to a method for simple and rapid separation of exosomes in plasma by solid-phase metal affinity chromatography.

Background technique

Exosomes are small membrane vesicles (30-200 nm) that contain complex RNAs and proteins. The membrane structure of exosomes is very similar to that of cell membranes. Both are composed of phospholipid bilayers with a thickness of about 5 nm. The main components are ceramides, cholesterol, sphingolipids, and glycerophospholipids containing long saturated fatty chains. The surface and interior of exosomes are rich in various proteins and contain important biological macromolecules such as multiple nucleic acids. Different cells may secrete specific proteins and RNAs whose main function is to transmit information between cells and play important physiopathological functions in a variety of biological states and diseases, including tumorigenesis, angiogenesis, and immunosuppression. , transfer, etc. Therefore, the study of biomolecules in exosomes can help to discover biomarkers in the process of disease occurrence and development, and realize early diagnosis, prognosis evaluation, drug resistance detection, drug delivery treatment, etc. of minimally invasive or non-invasive disease biological target signals.

However, the primary problem facing basic research and clinical applications is how to isolate and purify exosomes from clinical samples in a simple, fast, time-saving and labor-saving way, especially serum or plasma samples that are commonly used for clinical detection.

At present, exosome isolation methods mainly include ultracentrifugation, filtration, immunoaffinity, and polyethylene glycol precipitation. Each of these methods has pros and cons. Ultracentrifugation is the most commonly used method for exosome extraction at present. The amount of exosomes obtained is large, but the ultracentrifuge and consumables are expensive, which is not suitable for large-scale application and promotion, and the obtained exosomes contain a large amount of protein contamination. , In addition, due to the centrifugal force during the centrifugation process, it will also cause the rupture of exosomes. The exosomes obtained by density gradient centrifugation are of high purity, but the steps are cumbersome and time-consuming. Filtration methods include membrane filtration or size exclusion chromatography. Membrane filtration will cause a series of problems such as protein blocking of membrane pores and exosome rupture. Size exclusion chromatography requires specialized equipment and is extremely time-consuming. The immunoaffinity method has the advantages of high specificity, simple operation, and does not affect the integrity of exosome morphology. However, the efficiency is low, and the biological activity of exosomes is easily affected by pH and salt concentration, which is not conducive to downstream experiments and is difficult to be widely popularized. The polyethylene glycol precipitation method is currently the lowest cost and the simplest method to isolate exosomes. Polyethylene glycol can be combined with hydrophobic proteins and lipid molecules to co-precipitate. It was used to collect viruses from serum and other samples in the early stage. It has also been used to precipitate exosomes, and the principle may be related to the competitive binding of free water molecules. However, there are also many problems, such as low purity and recovery rate, long precipitation time, many impurities in the precipitation, non-uniform particle size, and generation of polymers that are difficult to remove.

Therefore, the existing exosome isolation and purification methods cannot meet the research needs in this field. The development of new methods for exosome isolation that is simple to operate, saves time and effort will promote clinical applications and basic research.

At present, a method for separating and purifying exosomes has been reported by means of the affinity between TiO ₂ and phosphate groups (Anovel strategy for facile serum exosome isolation based on specific interactions between phospholipid bilayers and TiO ₂ . Chem. Sci., 2019, 10, 1579), this method is simpler and faster than traditional methods, but the purification efficiency is low.

Immobilized metal ion affinity chromatography (IMAC), also known as solid-phase metal affinity chromatography, is a technique for protein separation based on the affinity of proteins to metal ions immobilized on a matrix.

Immobilized Affinity Chromatography Microspheres CAE-Ti ⁴⁺ -IMAC is an IMAC technology tool, which is used to enrich phosphorylated peptides of proteins in previous research applications, but has not been used for exosome separation and enrichment. Application coverage.

SUMMARY OF THE INVENTION

In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a simple and rapid method for separating exosomes in serum or plasma by solid-phase metal affinity chromatography. The present invention utilizes monodisperse immobilized affinity chromatography microspheres, CAE-Ti ⁴⁺ -IMAC, that is, magnetic microspheres with a diameter of about 10 μm immobilized with tetravalent titanium ions (product number: 2749380, Bailingwei Technology) to enrich plasma of exosomes. When plasma samples with exosomes are added, the phosphate groups on the cell membrane of exosomes bind to the metal Ti ions on the CAE-Ti ⁴⁺ -IMAC magnetic spheres due to their high affinity, thereby realizing the capture of exosomes. Pick. Rinse with buffer PBS after grabbing to remove other impurities. Finally, exosome collection and characterization are performed through an elution step.

The technical scheme of the present invention includes:

A method for separating exosomes in plasma, which comprises the following steps:

1) Use monodisperse immobilized affinity chromatography microspheres CAE-Ti ⁴⁺ -IMAC to adsorb exosomes;

2) Use ammonia water to elute exosomes;

3) Use phosphate buffer instead of ammonia.

As in the aforementioned separation method, step 1) is carried out at 4-8°C; and or, the adsorption time is 5-20 min.

As in the aforementioned separation method, the plasma needs to be filtered before step 1) to remove cell debris, dead bodies and large vesicles (diameter greater than 200 nm);

Preferably, filtration is performed using a 0.22 μm filter.

As in the aforementioned separation method, 1-5 mg of monodisperse immobilized affinity chromatographic microspheres are used per 1-200 μL of plasma for exosome separation; ball.

As in the aforementioned separation method, the ammonia concentration in step 2) is 10% (w/v).

As in the aforementioned separation method, the elution method in step 2) is: after rotating at 4-8° C. for 3-20 min, centrifuging to collect the supernatant.

As in the aforementioned separation method, the method of replacing the ammonia water in step 3) is as follows: centrifugally remove the ammonia water in an ultrafiltration tube, add phosphate buffer for centrifugation 2-5 times, and finally resuspend with phosphate buffer.

A method for separating exosome proteins in plasma, comprising the following steps:

1) Use monodisperse immobilized affinity chromatography microspheres CAE-Ti ⁴⁺ -IMAC to adsorb exosomes;

2) Use cell lysate to extract exosomal proteins.

According to the aforementioned method for separating exosomal proteins, the cell lysate is composed of the following components:

2% (w/v) SDS, 0.1M Dithiothreitol, 0.1M Tris-HCl.

As mentioned above, the separation method of exosome protein, the method of using lysate to extract is:

Add the lysate to a boiling water bath for 3-10 min, ultrasonically treat for 3-20 min, and then centrifuge for 5-10 min to collect the supernatant.

The present invention has the following beneficial effects:

The present invention adopts monodisperse immobilized affinity chromatography microspheres - CAE-Ti ⁴⁺ -IMAC to separate and purify exosomes in plasma. The principle is that the phosphate group in the hydrophilic head on the exosome cell membrane Due to the high affinity between the phosphate group and the metal Ti ions on the CAE-Ti ⁴⁺ -IMAC magnetic spheres and the small steric hindrance on the surface of the magnetic beads, exosomes can be separated and purified through the steps of binding, washing, and elution. The method is simple, rapid, low-cost, time-saving and labor-saving, and is suitable for the separation and purification of exosomes from large-scale clinical plasma samples and subsequent research.

Compared with the similar material TiO ₂ , the CAE-Ti ⁴⁺ -IMAC of the present invention has a higher separation efficiency of exosomes, which is about 3 times that of TiO ₂ .

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through specific embodiments. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention only to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but is not intended to limit the present invention. Any equivalent replacement in the art made according to the present disclosure shall fall within the protection scope of the present invention.

Description of drawings

Figure 1 shows the analysis results of protein SDS-PAGE (Coomassie brilliant blue staining) before and after the isolation of plasma exosomes.

Figure 2 is a Western blot analysis of exosome marker proteins in plasma.

Figure 3 is a transmission electron microscope image of the eluted purified exosomes.

Figure 4 is a fluorescence imaging image of CAE-Ti ⁴⁺ -IMAC binding to plasma exosomes.

Figure 5 is a comparison diagram of the effect of equal amount of CAE-Ti ⁴⁺ -IMAC and TiO ₂ in isolating equal amount of plasma exosomes.

Detailed ways

Example 1 Isolation and characterization of plasma exosomes

1. Method

(1) Weigh 4 mg of solid-phase metal affinity chromatography as monodisperse immobilized affinity chromatography microspheres, (CAE-Ti ⁴⁺ -IMAC), and wash the microspheres with 500 μL of phosphate buffered saline (PBS, pH 7.5) 3 times, centrifuged at 500g for 1min.

(2) The collected human plasma was filtered through a 0.22-micron filter to remove cell debris, dead bodies and large vesicles, etc., then 100 μl was mixed with 100 μl PBS, and then added to a 1.5 ml centrifuge containing CAE-Ti ⁴⁺ -IMAC. in the tube;

(3) rotating and mixing the centrifuge tube containing the mixture for 10 minutes under the condition of 4 degrees;

(4) Wash the microspheres three times with 500 μl of PBS, and centrifuge at 500 g for 3 min to remove non-specifically bound proteins and molecules;

(5) To extract exosome protein, add 100 μl of SDT solution (2% SDS, 0.1M DTT, 0.1M Tris/HCl, pH7.6) to a centrifuge tube, boil in boiling water for 5 min, and sonicate in the water bath The exosome lysate was collected by centrifugation at 12,000 g for 10 min at 4 °C for 5 min.

(6) To elute intact exosomes, add 100 μl of 10% ammonia water. After spinning at 4 °C for 10 min, the supernatant containing exosomes was collected by centrifugation at 10,000 g for 5 min, and quickly transferred to a 30KD ultrafiltration tube. The ammonia water was replaced with PBS three times, and 300 μl of PBS was added each time, 10,000 g at 4 °C. Centrifuge for 5 min. Finally, invert the ultrafiltration tube and centrifuge at 10,000 g for 5 min to collect intact exosomes.

(7) Use a transmission electron microscope (Hitachi H-600 transmission electron microscope) to take pictures of intact exosomes to observe their morphology and size. The anti-TSG101 antibody labeled with orange fluorescent TRITC (Rhodamine Tetramethylisothiocyanate) was used for fluorescence imaging to observe the binding state of CAE-Ti ⁴⁺ -IMAC and its exosomes.

2. Results

1) Specificity

Figure 1 shows the analysis result of plasma exosome protein SDS-PAGE (Coomassie brilliant blue staining) obtained in step (5) in this example. It can be seen from the figure that after three washings with PBS buffer, non-specifically bound plasma proteins are removed, and exosomes are eluted.

The results in Figure 1 show that the method of the present invention is highly specific.

2) Separation efficiency

Figure 2 shows the step (5) Western blot analysis of exosome marker proteins in plasma in this example. It can be seen from the figure that the vesicles isolated from plasma by monodisperse immobilized affinity chromatography microspheres are exosomes with marker proteins TSG101 and CD9. The plasma contains both exosomes and plasma proteins. The washed supernatant is Exosome-free plasma protein.

The results in Fig. 2 show that the present invention has high efficiency in isolating exosomes and can enable most of the exosomes to be isolated.

3) Exosome identification

Figure 3 shows the TEM image of the exosomes eluted in step (6) in this example. It can be seen from the figure that the exosomes are about 100 nm in diameter, have a complete structure, retain the phospholipid bilayer, and have a typical saucer-like morphology.

Figure 4 shows the fluorescence imaging image of the binding of CAE-Ti ⁴⁺ -IMAC to exosomes in step (4) in this example. Compared with the CAE-Ti ⁴⁺ -IMAC blank control, after incubation with anti-TSG101 antibody, CAE-Ti ⁴⁺ -IMAC had little background fluorescence, while CAE-Ti ⁴⁺ -IMAC bound to exosomes, It shows that the fluorescence is significantly enhanced, and it can be observed that the exosomes are bound to the surface of the microspheres.

Figures 3 and 4 show that the isolated objects are indeed exosomes.

4) Semi-quantitative comparison of enrichment effects

Figure 5 shows the step (5) Western blot analysis of exosome marker proteins in plasma in this example. Using an equal amount of CAE-Ti ⁴⁺ -IMAC or TiO ₂ mixed with an equal amount of plasma (100 μl) to enrich for exosomes, and then taking the same exosome protein for Western blot analysis, it was found that 1 mg or 2 mg of CAE -Ti ⁴⁺ -IMAC was able to enrich more exosomal marker proteins TSG101 and CD9 (~3-fold) than an equivalent amount of TiO ₂ .

Figure 5 shows that CAE-Ti ⁴⁺ -IMAC can isolate more plasma exosomes.

In summary, the present invention can efficiently enrich exosomes in plasma through solid-phase metal affinity chromatography with high affinity for phosphate groups on the surface of exosomes and small steric hindrance on the surface, which can achieve simple, fast, time-saving and labor-saving. , Low-cost plasma exosome isolation. This method can be applied to large-scale clinical sample exosome research, which is helpful for the discovery of exosome-related biomarkers, and provides technical support for research on the mechanism of disease occurrence and development.

Claims (10)

1. a separation method of exosome in plasma, is characterized in that, it comprises the steps: 1) Use monodisperse immobilized affinity chromatography microspheres CAE-Ti ⁴⁺ -IMAC to adsorb exosomes; 2) Use ammonia water to elute exosomes; 3) Use phosphate buffer instead of ammonia. 2. The separation method of claim 1, wherein step 1) is carried out at 4-8°C; and or, the adsorption time is 5-20 min. 3. The separation method of claim 1, wherein the plasma needs to be filtered before step 1) to remove cell debris, dead bodies and vesicles with a diameter greater than 200 nm; Preferably, filtration is performed using a 0.22 μm filter. 4. The separation method according to claim 1, characterized in that, 1-5 mg of monodisperse immobilized affinity chromatography microspheres are used per 1-200 μL of plasma to separate exosomes; preferably, per 100 μL of plasma Use 1 mg of monodisperse immobilized affinity chromatography microspheres. 5. The separation method according to any one of claims 1 to 4, wherein the ammonia concentration in step 2) is 10% (w/v). 6 . The separation method according to claim 5 , wherein the elution method in step 2) is: after rotating at 4-8° C. for 3-20 min, centrifuging to collect the supernatant. 7 . 7. separation method as claimed in claim 1～4, is characterized in that, the mode that replaces ammoniacal liquor in step 3) is: centrifugally remove ammoniacal liquor in ultrafiltration tube, then add phosphate buffer solution and centrifuge 2-5 times, finally Resuspend in phosphate buffered saline. 8. A method for separating exosomal proteins in plasma, comprising the steps of: 1) Use monodisperse immobilized affinity chromatography microspheres CAE-Ti ⁴⁺ -IMAC to adsorb exosomes; 2) Use cell lysate to extract exosomal proteins. 9. the separation method of exosome protein as claimed in claim 8, is characterized in that, described cell lysate is made up of following components: 2% (w/v) SDS, 0.1M Dithiothreitol, 0.1M Tris-HCl. 10. exosome protein separation method as claimed in claim 8 or 9, is characterized in that, the method that uses lysate to extract is: Add the lysate to a boiling water bath for 3-10 min, ultrasonically treat for 3-20 min, and then centrifuge for 5-10 min to collect the supernatant.