Method for absolute quantification of exosome surface protein and use thereof

Abstract

A method for the absolute quantification of an exosome surface protein and the use thereof. The method for the absolute quantification of the exosome surface protein comprises: (1) extracting an exosome containing a target surface protein using a solid-phase support material coupled with an antibody which identifies the target surface protein, sequentially performing exosome lysis and desalting, and preparing the desalted protein into a sample to be detected; (2) analyzing the sample to be detected using HPLC-PRM / MS; selecting a characteristic peptide fragment in the target surface protein as an internal standard, labeling the characteristic peptide fragment with a heavy isotope to obtain a heavy peptide, using the characteristic peptide fragment without isotope labeling as a light peptide, diluting the synthesized light peptide into different concentration gradients, diluting the synthesized heavy peptide into a fixed concentration, and mixing the two at a volume ratio of 1:1 to obtain a standard; and (3) establishing a standard curve of the internal standard, and performing the absolute quantification of the exosome surface protein according to the standard curve. The method for the absolute quantification of the exosome surface protein has high sensitivity, good reproducibility and high accuracy, and the quantitative range can reach 5 orders of magnitude.

Description

A method for absolute quantification of exosome surface proteins and its application Technical Field

[0001] The present application belongs to the field of analysis and detection technology, and specifically relates to a method for absolute quantification of exosome surface proteins and its application. Background Art

[0002] Exosome surface protein analysis and detection include traditional biological methods and photoelectric detection technologies. Traditional biological methods include enzyme-linked immunosorbent assay (ELISA), western blotting (WB), polymerase chain reaction (PCR), etc. Photoelectric detection technologies include electrochemical methods, chemiluminescence, surface plasmon resonance, etc. These biological technologies play a very important role in the analysis and characterization of exosome proteins.

[0003] The Exosome ELISA Kit is an enzyme-linked immunosorbent assay (ELISA) kit for the qualitative and quantitative analysis of extracellular vesicles (EVs) in cell culture supernatants and body fluid samples. A protein that specifically binds to phosphatidylserine (PS) on the surface of EVs is pre-coated onto the plate. After the EVs react with the protein and become immobilized, a biotinylated antibody targeting the target EV surface marker protein is used as the primary antibody for detection, and the HRP-conjugated streptavidin provided in the kit is used as the secondary antibody. This allows for highly sensitive detection of EVs bearing the target marker protein on their surface.

[0004] The Exosome ELISA Kit is a quantitative exosome detection method based on ELISA technology. Exosome concentration results are obtained using a standard curve with known exosome counts. The kit specifically binds to proteins such as CD63, CD9, and CD81, and can be used to detect human exosomes. Because different body fluids and cell lines express different proteins, multiple samples must be tested, resulting in large sample volumes and variability between test batches.

[0005] Flow cytometry analysis is also a method for detecting exosomal proteins. Flow cytometry analysis is a cell-based analysis method that mainly uses specific antibodies to label specific proteins and uses a cell analyzer to analyze the number of labeled proteins in the cells to achieve protein quantification.

[0006] The application principle of flow cytometry is mainly based on the antigen and antibody reaction, which is essentially the same as that of protein immunoblotting and immunohistochemistry. However, since both immunohistochemistry and protein immunoblotting have enzyme-catalyzed substrate amplification systems, flow cytometry is not suitable for detecting low-expression proteins.

[0007] In proteomics research, antibody-based detection methods such as flow cytometry and ELISA kits place extremely high demands on antibody quality. Furthermore, gene expression levels are poorly correlated with changes in protein content. These traditional methods present significant challenges in analyzing protein levels. Therefore, developing a novel protein analysis method with high sensitivity, reproducibility, and accuracy is of great value.

[0008] Summary of the Invention

[0009] This application provides a method for absolute quantification of exosome surface proteins and its application. This method boasts high sensitivity, excellent reproducibility, and high accuracy, with a quantification range of up to five orders of magnitude, a quantitative reproducibility CV <10%, and a relative error <15%. Compared to classic flow cytometry and ELISA techniques, this method can achieve more accurate and reproducible quantitative verification results. It can simultaneously quantify 50 proteins within an hour, achieving higher detection efficiency.

[0010] In a first aspect, the present application provides a method for absolute quantification of exosome surface proteins, the method comprising:

[0011] (1) extracting exosomes containing the target surface protein using a solid support material coupled to an antibody that recognizes the target surface protein, then sequentially lysing the exosomes and performing a desalting treatment, and preparing the desalted protein into a test sample;

[0012] (2) Analyze the sample using HPLC-PRM / MS; select the characteristic peptide in the target surface protein as the internal standard, use the heavy isotope to label the characteristic peptide to obtain the heavy peptide, and the unlabeled isotope characteristic peptide as the light peptide. Dilute the synthesized light peptide into different gradient concentrations, dilute the synthesized heavy peptide into a fixed concentration, and mix them into a standard at a volume ratio of 1:1;

[0013] (3) Establish a standard curve for the internal standard and perform absolute quantification of exosome surface proteins based on the standard curve.

[0014] This application provides a method for absolute quantification of exosome surface proteins. Exosomes in different body fluids are captured using a solid support material coupled with an antibody that recognizes the target surface protein. Light and heavy peptides are synthesized using a unique peptide fragment from the target surface protein as an internal standard. The exosomes are lysed with a lysis buffer and enzymatically digested. The sample is then separated using high-performance liquid chromatography. High-resolution mass spectrometry in parallel reaction monitoring mode is then used for targeted detection of exosome surface proteins, and secondary fragment ions are used for quantification. The method has high sensitivity, good reproducibility, and high accuracy, with a quantitative range of up to 5 orders of magnitude, a quantitative reproducibility CV of <10%, and a relative error of <15%. Compared with classic flow cytometry and ELISA techniques, the method can obtain more accurate and reproducible quantitative verification results.

[0015] In this application, PRM / MS can directly detect all secondary ions, reducing the selection and optimization process of product ions.

[0016] Preferably, the solid support material is selected from magnetic beads or agarose medium.

[0017] Preferably, the antibody that recognizes the target surface protein is selected from monoclonal antibodies and / or nanobodies.

[0018] In the present application, in addition to antibodies that recognize target surface proteins, affinity peptides and other peptide segments that can recognize target surface proteins can also be coupled to the solid support material.

[0019] Preferably, when the solid support material is magnetic beads, the coupling method of the magnetic beads and the antibody is a coupling method of carboxyl magnetic beads.

[0020] In this application, the steps of coupling magnetic beads and antibodies using the coupling method of carboxyl magnetic beads include:

[0021] a. The magnetic beads were washed with MES buffer, the MES buffer was mixed with the magnetic beads and ice-bathed for 0.5-1 h, and EDC and NHS were added after ice-bath and mixed for 30-40 min to obtain activated magnetic beads; the final concentration of the EDC was 0.8-1.2 mg / mL, and the final concentration of the NHS was 0.8-1.2 mg / mL;

[0022] b. washing the activated magnetic beads with MES buffer;

[0023] c. Mix the antibody that recognizes the target surface protein with the activated magnetic beads. The mass ratio of the antibody that recognizes the target surface protein to the activated magnetic beads is 250 μg antibody / (0.8-1.2) mg magnetic beads. Let the reaction stand for 4-6 hours. Wash the reaction product with MES buffer and perform blocking treatment.

[0024] d. The blocked magnetic beads coupled with antibodies that recognize the target surface protein are placed in a preservation solution, wherein the preservation solution comprises, by concentration, PBS + (1-1.5‰) p-300 + (1-1.5%) Tween 20, pH 7.2-7.6.

[0025] In the present application, in the coupling method for carboxyl magnetic beads, EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) is a water-soluble carbodiimide used as a carboxyl group activation reagent in amide synthesis. It is also used to activate phosphate groups, cross-link proteins and nucleic acids, and prepare immunoconjugates. The pH range for use is 4.0-6.0, and it is often used in combination with N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide to improve coupling efficiency.

[0026] In the present application, when the solid phase support material is an agarose medium, the agarose medium is selected from: hydrogen bromide activated cross-linked agarose matrix, NHS agarose microspheres or amino functionalized modified agarose beads.

[0027] In the present application, the step of activating the cross-linked agarose matrix to couple the antibody with hydrogen bromide comprises:

[0028] a. Dissolve the CD9 antibody in a coupling buffer containing 0.5 M NaCl, 0.1 M NaHCO3, pH 8.3. The concentration of the CD9 antibody is 5 mg / mL.

[0029] b. The hydrogen bromide cross-linked agarose matrix was rinsed three times with a coupling buffer containing 0.5 M NaCl, 0.1 M NaHCO3, pH 8.3, and then dried.

[0030] c. Add CD9 antibody to the pretreated hydrogen bromide cross-linked agarose matrix so that the mass ratio of CD9 antibody to activated cross-linked agarose matrix is ​​250 μg antibody / 1.2 mg cross-linked agarose matrix, and mix by inversion overnight at 4°C.

[0031] d. After the cross-linking reaction is completed, the protein concentration is collected and the cross-linking value is calculated.

[0032] e. Wash the free protein with 5 volumes of cross-linking buffer and then with 10 volumes of distilled water.

[0033] f. The washed gel was suspended in water, and ethanolamine (1 mol / L) or Tris-HCl buffer (pH 8.0) was added. The reaction was stirred at room temperature for at least 2 h to eliminate residual active groups.

[0034] g. Wash the column alternately with a buffer solution containing 0.5 mol / L NaCl and 0.1 mol / L acetate (pH 4.0) and a buffer solution containing 0.5 mol / L NaCl and 0.1 mol / L Tris-HCl (pH 8). Repeat the above washing cycle at least three times, and then wash with 10 column volumes of PBS.

[0035] h. Use 20% ethanol to store the coupling product.

[0036] In this application, the steps of coupling the NHS agarose microspheres with the antibody are as follows:

[0037] a. Dissolve an appropriate amount of antibody in 100mM 2-morpholinoethanesulfonic acid (MES) to a concentration of 3.0mg / mL. Antibodies already stored in buffer should be dialyzed or desalted to remove any primary amine groups in the buffer. Then, dissolve the antibody in 100mM 2-morpholinoethanesulfonic acid (MES) to a concentration of 3.0mg / mL. Store the prepared antibody solution at 4°C until ready for use.

[0038] b. Quickly wash the agarose beads with pre-cooled 1 mM hydrochloric acid solution to prevent hydrolysis of the NHS group during the washing process.

[0039] c. Immediately after washing the agarose beads, add the antibody solution at a mass ratio of 250 μg of CD9 antibody to 1.2 mg of agarose beads. Place the solution on a vertical mixer and mix at room temperature for 2-4 hours. If mixing is uneven, remove the EP tube and vortex for 15 seconds every 15 minutes.

[0040] d. Add 1 mL of 3 M ethanolamine, vortex for 30 seconds, place the EP tube in a vertical mixer and react at room temperature for 2 hours, centrifuge and discard the supernatant.

[0041] e. Add 1 mL of PBS buffer containing 0.05% sodium azide to the EP tube, mix thoroughly, centrifuge and discard the supernatant, and repeat twice.

[0042] f. Add 400 μL of PBS buffer containing 0.05% sodium azide to the EP tube, mix thoroughly, and store at 4°C until use.

[0043] In the present application, the steps of coupling the amino-functionalized agarose beads to the antibody include:

[0044] a. Remove the protective agent, wash the amino agarose beads with MES buffer, and shake on a mixer.

[0045] b. Wash three times and discard the MES buffer with a pipette.

[0046] c. MES buffer was mixed with the amino-functionalized agarose matrix and placed on ice for 0.5 h. After the ice bath, EDC and NHS were added and mixed for 30 min. The final concentration of the EDC was 1.2 mg / mL, and the final concentration of the NHS was 1.2 mg / mL to obtain an activated solid support material.

[0047] d. and add ligand (ligand is CD9 antibody).

[0048] e. If the ligand is unstable at room temperature, perform the following steps in a refrigerator:

[0049] Add 1 mL of activated amino-agarose beads to the previous CD9 antibody solution so that the mass ratio of CD9 antibody to amino-functionalized agarose beads is 250 μg antibody / 1.2 mg agarose beads. Stir gently, remove an aliquot of the suspension, and test for activity and / or absorbance at 280 nm. Continue gentle stirring for 1-3 hours or until the activity measurement remains constant, indicating complete immobilization. Avoid magnetic stirring. (Stirring time should not exceed 3 hours because CDI (N,N'-carbonyldiimidazole) will decompose. However, if immobilization must be performed in a cold room, stirring time may be longer due to the low stability of the ligand). Wash the suspension with distilled water to eliminate excess reagent, then wash with 1.0 M NaCl solution, and finally wash with distilled water.

[0050] f. The ligand is bound to the amino matrix and can be stored in a 0.03% sodium azide solution at 4-10°C.

[0051] In this application, the diameter of the magnetic beads used is only 1-4 μm, which is significantly smaller than that of agarose beads. The magnetic beads do not have a porous structure, but the number of magnetic beads per volume is greater than that of agarose beads, so that the magnetic beads have sufficient antibody binding surface area to meet high-capacity antibody binding; at the same time, it avoids the disadvantages brought by the porous structure and combines with the magnetic frame to quickly separate the precipitate and supernatant, which not only shortens the operation time but also makes the operation more automated.

[0052] In this application, the agarose matrix is ​​sponge-like, with a diameter of 50-150 μm. It binds to the target protein by binding to the antibody. It can directly, efficiently, and quickly bind to the antibody without the need for specialized equipment. Because the agarose matrix is ​​porous, it has a larger surface area for contact with the protein, theoretically resulting in a higher binding capacity.

[0053] Preferably, in step (1), the lysis of exosomes and desalination treatment are performed by a method comprising the following steps:

[0054] Exosomes were lysed using an exosome lysis buffer, heated, cooled, diluted 4-5 times with tetraethylammonium bromide solution, digested with protease, acidified with 10-15% TFA to a final concentration of 0.5-5% TFA, and vortexed with ethyl acetate to precipitate the protein. The protein was passed through a desalting column, and the eluate was collected and lyophilized and concentrated to obtain the purified protein.

[0055] Preferably, the temperature of the heating treatment is 90-95°C (for example, it can be 90°C, 91°C, 92°C, 93°C, 94°C or 95°C, etc.), and the time of the heating treatment is 10-15min (for example, it can be 10min, 11min, 12min, 13min, 14min or 15min, etc.).

[0056] Preferably, the concentration of the tetraethylammonium bromide solution is 40-60 mM (eg, 40 mM, 45 mM, 50 mM, 55 mM or 60 mM, etc.).

[0057] Preferably, the protease is a combination of recombinant trypsin and lysyl endonuclease, wherein the mass ratio of the recombinant trypsin to the substrate is 1 μg:(20-50 μg), for example, it can be 1 μg:20 μg, 1 μg:30 μg, 1 μg:40 μg or 1 μg:50 μg, etc., and the mass ratio of the lysyl endonuclease to the substrate is 1 μg:(50-100 μg), for example, it can be 1 μg:50 μg, 1 μg:60 μg, 1 μg:70 μg, 1 μg:80 μg, 1 μg:90 μg or 1 μg:100 μg, etc.

[0058] Preferably, the digestion time is 14-16 hours, for example, 14 hours, 15 hours or 16 hours.

[0059] Preferably, in step (1), the sample to be tested is prepared by a method comprising the following steps:

[0060] The protein was dissolved in 0.1-0.2% (for example, 0.1% or 0.2%) formic acid aqueous solution, vortexed and centrifuged at 20000-21000g (for example, 20000g or 21000g, etc.) for 8-12min (for example, 8min, 9min, 10min, 11min or 12min, etc.), and an equal volume was loaded. The sample and heavy peptide were mixed in a volume ratio of 1:1 and then subjected to mass spectrometry.

[0061] Preferably, in step (2), the HPLC chromatographic conditions are:

[0062] The chromatographic column was a C18 capillary column 1.9 μm, 75 μm × 20 cm; the injection volume was 2 ± 0.5 μL;

[0063] The mobile phase consisted of mobile phase A and mobile phase B, where mobile phase A was a 0.1% formic acid aqueous solution and mobile phase B was an 80% acetonitrile aqueous solution containing 0.1% formic acid;

[0064] The elution program is as follows:

[0065] Preferably, in step (2), the mass spectrometry conditions of the MS are:

[0066] Primary mass spectrometry, scan range: 400-1200 m / z, resolution: 60000, AGC target: 1e5, Maximum IT: 50 ms;

[0067] Secondary PRM scan, isolation window: 2.0 m / z, resolution: 60000, AGC target: 1e6, maximum IT: 100 ms.

[0068] As a preferred technical solution of the present application, the method for absolute quantification of exosome surface proteins comprises the following steps:

[0069] (1) using a solid support material coupled to an antibody that recognizes the target surface protein to extract exosomes containing the target surface protein, then sequentially lysing the exosomes and desalting the exosomes, and preparing the desalted protein into a test sample; wherein the solid support material is selected from magnetic beads or agarose medium.

[0070] The exosome lysis and desalination treatment is performed using a method comprising the following steps:

[0071] Exosomes were lysed using an exosome lysis buffer, heated to 90-95°C, cooled, diluted 4-5 times with 40-60 mM tetraethylammonium bromide solution, and digested for 14-16 hours using recombinant trypsin and lysyl endonuclease at a ratio of 1 μg:(20-50 μg) of recombinant trypsin to substrate and 1 μg:(50-100 μg) of lysyl endonuclease to substrate. The protein was acidified with 10-15% TFA to a final concentration of 0.5-5%. The protein was vortex-precipitated with ethyl acetate, passed through a desalting column, and the eluate was collected and lyophilized to obtain the purified protein.

[0072] The sample to be tested is prepared by a method comprising the following steps:

[0073] The protein was dissolved in 0.1-0.2% formic acid aqueous solution, vortexed and centrifuged at 20000-21000g for 8-12 minutes, the supernatant was collected, and the sample and heavy peptide were mixed at a volume ratio of 1:1 and then detected by liquid chromatography-mass spectrometry.

[0074] (2) HPLC-PRM / MS was used to analyze the sample to be tested; the characteristic peptide fragment in the target surface protein was selected as the internal standard, and the characteristic peptide fragment was labeled with a heavy isotope to obtain a heavy peptide, and the characteristic peptide fragment without an isotope was used as a light peptide. The synthesized light peptide was diluted into different gradient concentrations, and the synthesized heavy peptide was diluted into a fixed concentration, and then mixed into a standard in a volume ratio of 1:1.

[0075] The chromatographic conditions of the HPLC are:

[0076] The chromatographic column was a C18 capillary column 1.9 μm, 75 μm × 20 cm; the injection volume was 2 ± 0.5 μL;

[0077] The mobile phase consisted of mobile phase A and mobile phase B, where mobile phase A was a 0.1% formic acid aqueous solution and mobile phase B was an 80% acetonitrile aqueous solution containing 0.1% formic acid;

[0078] The elution program is as follows:

[0079] The mass spectrometry conditions of the MS are:

[0080] Primary mass spectrometry, scan range: 400-1200 m / z, resolution: 60000, AGC target: 1e5, Maximum IT: 50 ms;

[0081] Secondary PRM scan, isolation window: 2.0 m / z, resolution: 60000, AGC target: 1e6, maximum IT: 100 ms.

[0082] (3) Establish a standard curve for the internal standard and perform absolute quantification of exosome surface proteins based on the standard curve.

[0083] In a second aspect, the present application provides the application of the method for absolute quantification of exosome surface proteins as described in the first aspect in the detection of exosome quality.

[0084] Preferably, in the application of exosome quality detection, when absolutely quantifying CD9 protein in exosomes, the characteristic peptide segment used is selected from any one of (1), (2) or (3) or a combination of at least two of the following:

[0085] (1) DVLETFTVK (SEQ ID NO: 1);

[0086] (2) KDVLETFTVK (SEQ ID NO: 2);

[0087] (3)EVQEFYK (SEQ ID NO: 3).

[0088] Preferably, in the application of exosome quality detection, when absolutely quantifying CD9 protein in exosomes, the characteristic peptide segment used is KDVLETFTVK (SEQ ID NO: 2).

[0089] Preferably, the characteristic peptide segment is isotope-labeled, and the labeled characteristic peptide segment is used as a heavy peptide, and the position of the isotope labeling is selected from any one of the first lysine to the sixth glutamic acid starting from the C-terminus.

[0090] Preferably, the isotope label is on the C element and / or N element of the corresponding amino acid.

[0091] The detection method described in this application can be applied to the relative or absolute quantification of target proteins / peptides, the qualitative and quantitative study of protein post-translational modifications, and can be applied to a variety of non-model organisms.

[0092] In this application, we selected products with no post-translational modification, except for reductive alkylation, high fragmentation efficiency, strong signal, narrow and symmetrical chromatographic peak, stable retention time, consistent peak area percentage, and the best performance after parent ion fragmentation. + There are 3-6 peptides as internal standard peptides.

[0093] After comparative screening, the present application finally selected the peptide KDVLETFTVK (SEQ ID NO: 2) in the CD9 protein as the internal standard to synthesize light peptides and heavy peptides.

[0094] The numerical range described in this application includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to limited space and for the sake of simplicity, this application no longer exhaustively lists the specific point values ​​included in the range.

[0095] Compared with the prior art, this application has the following beneficial effects:

[0096] (1) The CD9 antibody-coupled solid support material constructed in this application has good targeting and good stability in repeated experiments, and can accurately quantify the protein content of exosomes.

[0097] (2) The LOD of the detection method is 1 pg and the LOQ is 7 pg. The method of the present application can quantify low-abundance proteins.

[0098] (3) The detection method of this application has high sensitivity, good reproducibility, and high accuracy, with a quantitative range of up to 5 orders of magnitude, a quantitative reproducibility CV <10%, and a relative error <15%. Compared with classic flow cytometry and ELISA techniques, this method can obtain more accurate and reproducible quantitative verification results.

[0099] (4) The method of this application enables high-throughput antibody validation on large-scale biological samples, improving experimental efficiency. Quantitative detection of 50 proteins can be achieved simultaneously within one hour.

[0100] (5) The detection method can be applied to the relative or absolute quantification of target proteins / peptides, the qualitative and quantitative study of protein post-translational modifications, and can be applied to a variety of non-model organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] FIG1 shows the retention time results of CD9-KDVLETFTVK (SEQ ID NO: 2).

[0102] FIG2 shows the peak area percentage results of CD9-KDVLETFTVK (SEQ ID NO: 2).

[0103] FIG3 shows the retention time results of CD9-DVLETFTVK (SEQ ID NO: 1).

[0104] FIG4 shows the peak area percentage results of CD9-DVLETFTVK (SEQ ID NO: 1).

[0105] FIG5 shows the retention time results of CD9-EVQEFYK (SEQ ID NO: 3).

[0106] FIG6 shows the peak area percentage results of CD9-EVQEFYK (SEQ ID NO: 3).

[0107] Figure 7 shows the concentration of 2×10 7 PRM_1 detection results of exosomes containing CD9 protein from HEK293 supernatant at 100 particles / mL.

[0108] Figure 8 shows the concentration of 2×10 7 PRM_2 detection results of exosomes containing CD9 protein from HEK293 supernatant at 100 particles / mL.

[0109] Figure 9 shows the concentration of 2×10 7 PRM_2 detection results of exosomes containing CD9 protein from HEK293 supernatant at 100 particles / mL.

[0110] Figure 10 shows the concentration of 2×10 8 PRM_1 detection results of exosomes containing CD9 protein from HEK293 supernatant at 100 particles / mL.

[0111] Figure 11 shows the concentration of 2×10 8 PRM_2 detection results of exosomes containing CD9 protein from HEK293 supernatant at 100 particles / mL.

[0112] Figure 12 shows the concentration of 2×10 8 PRM_3 detection results of exosomes containing CD9 protein from HEK293 supernatant at 100 particles / mL.

[0113] Figure 13 shows the concentration of 2×10 9 PRM_1 detection results of exosomes containing CD9 protein from HEK293 supernatant at 100 particles / mL.

[0114] Figure 14 shows the concentration of 2×10 9PRM_2 detection results of exosomes containing CD9 protein from HEK293 supernatant at 100 particles / mL.

[0115] Figure 15 shows the concentration of 2×10 9 PRM_3 detection results of exosomes containing CD9 protein from HEK293 supernatant at 100 particles / mL.

[0116] Figure 16 shows the concentration of 2×10 10 PRM_1 detection results of exosomes containing CD9 protein from HEK293 supernatant at 100 particles / mL.

[0117] Figure 17 shows the concentration of 2×10 10 PRM_2 detection results of exosomes containing CD9 protein from HEK293 supernatant at 100 particles / mL.

[0118] Figure 18 shows the concentration of 2×10 10 PRM_3 detection results of exosomes containing CD9 protein from HEK293 supernatant at 100 particles / mL.

[0119] FIG19 shows the results of WB detection of the extracted exosomes.

[0120] FIG20 shows the results of WB detection of the supernatant after incubation with a solid support material coupled with an antibody that recognizes a target surface protein in the step of extracting exosomes.

[0121] FIG21 is a graph showing the retention time detection results when the elution program is 20 min.

[0122] FIG22 is a graph showing the retention time detection results when the elution program is 30 min.

[0123] FIG23 is a flowchart of the enrichment and quantification of CD9 protein on the surface of plasma exosomes.

[0124] FIG24 shows the PRM_1 detection results of human plasma exosomes containing CD9 protein (200 μL).

[0125] FIG25 shows the PRM_2 detection results of human plasma exosomes containing CD9 protein (200 μL).

[0126] FIG26 shows the PRM_3 detection results of human plasma exosomes containing CD9 protein (200 μL). DETAILED DESCRIPTION

[0127] The technical solution of the present application is further described below through specific implementation methods. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations of the present application.

[0128] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0129] Example 1

[0130] 1. Target peptide screening

[0131] Using proteomics, we extracted the entire protein of HEK293 exosomes and performed DDA (Data Dependent Acquisition) mass spectrometry. We then used Proteome Discoverer software to identify unique peptides for the signature proteins. We then used Picky software to verify that the peptides we identified were unique peptides. We then extracted data from ProteomeTools, designed a targeted acquisition method for the protein of interest, and filtered the peptides based on the maximum number of co-eluting features to select the optimal peptide set for the protein of interest.

[0132] The proteotypic score (protein cleavage score) and the Organisms in which the peptide was found (organisms in which the peptide was found) of the peptide were viewed in the PeptideAtlas software to analyze the specificity and enzymatic efficiency of the peptide.

[0133] Total protein was extracted from HEK293 cells, HEK293 supernatant, human saliva, human urine, and human plasma samples, and the raw data after three repeated PRM tests were imported into Skyline.

[0134] After preliminary screening, three characteristic peptides were obtained, including (1) DVLETFTVK (SEQ ID NO: 1); (2) KDVLETFTVK (SEQ ID NO: 2); and (3) EVQEFYK (SEQ ID NO: 3). All of the above three characteristic peptides can be used as characteristic peptides of CD9 protein.

[0135] The mass spectrometry characteristics evaluation results of the above peptides are shown in Figures 1-6.

[0136] Figure 1 shows the retention time results of CD9-KDVLETFTVK (SEQ ID NO: 2), and Figure 2 shows the peak area percentage results of CD9-KDVLETFTVK (SEQ ID NO: 2); the abscissas in Figures 1 and 2 are samples of HEK293 cells, HEK293 supernatant, human saliva, human urine, and human plasma processed by three people A, B, and C, and the results of CD9-KDVLETFTVK (SEQ ID NO: 2) peptide PRM detection were obtained by performing two replicates for each sample. A1, A2, B1, B2, C1, and C2 are HEK293 cell samples; A3, A4, B3, B4, C3, and C4 are HEK293 supernatant samples; A5, A6, B5, B6, C5, and C6 are human saliva samples; A7, A8, B7, B8, C7, and C8 are human urine samples; and A9, A10, B9, B10, C9, and C10 are human plasma samples. In Figure 1, taking A1 as an example, the retention time results for A1 are shown, from left to right, for y7, y6, and y5; the results for the remaining samples refer to A1. In Figure 2, taking A1 as an example, the peak area percentage results for A1 are shown, from bottom to top, for y7, y6, and y5; the results for the remaining samples refer to A1.

[0137] Figure 3 shows the retention time results of CD9-DVLETFTVK (SEQ ID NO: 1), and Figure 4 shows the peak area percentage results of CD9-DVLETFTVK (SEQ ID NO: 1); the abscissas in Figures 3 and 4 are samples of HEK293 cells, HEK293 supernatant, human saliva, human urine, and human plasma processed by three people A, B, and C, and the results of CD9-DVLETFTVK (SEQ ID NO: 1) peptide PRM detection were obtained by performing two replicates for each sample. A1, A2, B1, B2, C1, and C2 are HEK293 cell samples; A3, A4, B3, B4, C3, and C4 are HEK293 supernatant samples; A5, A6, B5, B6, C5, and C6 are human saliva samples; A7, A8, B7, B8, C7, and C8 are human urine samples; and A9, A10, B9, B10, C9, and C10 are human plasma samples. In Figure 3, taking A1 as an example, the retention time results for A1 are shown, from left to right, for y7, y6, and y5; the results for the remaining samples refer to A1. In Figure 4, taking A1 as an example, the peak area percentage results for A1 are shown, from bottom to top, for y7, y6, and y5; the results for the remaining samples refer to A1.

[0138] Figure 5 shows the retention time results of CD9-EVQEFYK (SEQ ID NO: 3), and Figure 6 shows the peak area percentage results of CD9-EVQEFYK (SEQ ID NO: 3). The horizontal axes in Figures 5 and 6 are samples of HEK293 cells, HEK293 supernatant, human saliva, human urine, and human plasma processed by three people, A, B, and C. Each sample was subjected to two replicates of CD9-EVQEFYK (SEQ ID NO: 3) peptide PRM detection results. Among them, A1, A2, B1, B2, C1, and C2 are HEK293 cell samples, A3, A4, B3, B4, C3, and C4 are HEK293 supernatant samples, A5, A6, B5, B6, C5, and C6 are human saliva samples, A7, A8, B7, B8, C7, and C8 are human urine samples, and A9, A10, B9, B10, C9, and C10 are human plasma samples. In Figure 5, taking A1 as an example, the retention time results for A1 are shown from left to right for y6, y5, and y4; the results for the remaining samples refer to A1. In Figure 6, taking A1 as an example, the peak area percentage results for A1 are shown from bottom to top for y6, y5, and y4; the results for the remaining samples refer to A1.

[0139] As can be seen from Figures 1 to 6, the retention time and peak area percentage results of the peptide CD9-KDVLETFTVK (SEQ ID NO: 2) are relatively consistent.

[0140] Peptide selection criteria: select peptides without post-translational modifications, excluding reductive alkylation, with high fragmentation efficiency and strong signals, peptides with 2-3 charges, and peptides with narrow and symmetrical chromatographic peaks. It is best to select target peptides corresponding to the same protein with different retention times. Select the product ion y with consistent retention time, consistent peak area percentage, and the best performance after fragmentation of the parent ion. + According to the results of Figures 1 to 6 and the conditions for peptide selection, the peptide KDVLETFTVK (SEQ ID NO: 2) in the CD9 protein was finally selected as the internal standard to synthesize light and heavy peptides.

[0141] 2. PRM detection of light and heavy peptide standards

[0142] The positions on the internal standard peptide (KDVLETFTVK (SEQ ID NO: 2)) that can be labeled with heavy isotopes include: from the C-terminus to the N-terminus (i.e., from right to left), any of the following: glutamic acid at position 6, threonine at position 5, phenylalanine at position 4, threonine at position 3, valine at position 2, or lysine at position 1. Furthermore, the isotopes can be labeled on the carbon and / or nitrogen elements of the corresponding amino acids.

[0143] The heavy isotope-labeled amino acid in the heavy isotope-labeled standard peptide in this example is located at the glutamic acid in the middle of the peptide (i.e., glutamic acid at position 6). The concentration of the isotope-labeled peptide in the standard is 10 pg / μL. Standards were prepared to test the concentration of unknown samples (Unknown) according to Table 1.

[0144] Table 1

[0145] The HPLC chromatographic conditions are as follows: the chromatographic column is a C18 capillary chromatographic column 1.9 μm, 75 μm×20 cm; the injection volume is 2 μL; the mobile phase consists of mobile phase A and mobile phase B, mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is an 80% acetonitrile aqueous solution containing 0.1% formic acid.

[0146] The elution program is shown in Table 2.

[0147] Table 2

[0148] The mass spectrometry conditions of the MS are:

[0149] Primary mass spectrometry, scan range: 400-1200 m / z, resolution: 60000, AGC target: 1e5, Maximum IT: 50 ms;

[0150] Secondary PRM scan, isolation window: 2.0 m / z, resolution: 60000, AGC target: 1e6, maximum IT: 100 ms.

[0151] Light peptides were diluted to different gradient concentrations, heavy peptides were diluted to a fixed concentration, and the standard samples mixed in a 1:1 ratio were subjected to nano-liquid chromatography-mass spectrometry analysis, and data were collected under the parallel reaction monitoring mode. The data collected under the above parallel reaction monitoring mode were analyzed by Skyline software, and the three fragment ions with the highest peak intensity after fragmentation of the peptide parent ion were selected as the corresponding target peptide daughter ions to complete the screening and optimization of ion pairs of light peptides and heavy peptides.

[0152] The PRM detection ion pair settings are shown in Table 3:

[0153] Table 3

[0154] Example 2

[0155] This embodiment provides a method for absolute quantification of exosome surface proteins, which comprises the following steps:

[0156] (1) Exosomes are extracted using a solid support material coupled with CD9 antibodies, and then the exosomes are lysed and desalted in sequence, and the desalted protein is prepared as a test sample.

[0157] The solid phase support material coupled to the CD9 antibody is prepared by a method comprising the following steps:

[0158] a. The solid support material was washed with MES buffer. The solid support material was magnetic beads (Cat. No. MS160, Source: Beijing Borma Biotechnology Co., Ltd.). The MES buffer was mixed with the solid support material and incubated on ice for 0.5 h. After the ice bath, EDC and NHS were added and mixed for 30 min. The final concentrations of EDC and NHS were 1.2 mg / mL and 1.2 mg / mL, respectively, to obtain an activated solid support material.

[0159] b. washing the activated solid support material with MES buffer;

[0160] c. The CD9 antibody (Cat. No. SR70S01, source: Langfang Tianguang Biotechnology Co., Ltd.) was mixed with the activated solid support material at a mass ratio of 250 μg antibody / 1 mg solid support material, allowed to react for 4 h, and the reaction product was washed with MES buffer and blocked;

[0161] d. The blocked solid support material coupled to the CD9 antibody was placed in a preservation solution, wherein the preservation solution comprises, by concentration, PBS + 1‰ p-300 + 1% Tween 20, pH 7.4.

[0162] Collect the HEK293 supernatant and centrifuge at 10,000 g for 30 minutes. After collecting the supernatant, resuspend the precipitate at the bottom of the centrifuge tube with PBS. Filter through a 0.22 μm filter membrane and centrifuge the filtered supernatant at 130,000 g for 70 minutes. Discard the supernatant and resuspend the precipitate with PBS. Centrifuge at 130,000 g for 70 minutes. Discard the supernatant and resuspend the precipitate with 1 mL of PBS to obtain exosomes derived from the HEK293 supernatant. Measure the NTA concentration to determine the concentration, and then dilute with PBS to 2 × 10 10 particles / mL, 2×10 9 particles / mL, 2×10 8 particles / mL, 2×10 71 mL of sample was added to 40 μL of CD9 antibody-coupled solid support material (hereinafter referred to as CD9 magnetic beads) per tube and incubated for 1 hour. After 1 hour of incubation, the supernatant was removed by magnetic aspiration, and the cells were washed twice with 1 mL of Urine Loading Buffer, the supernatant was discarded by magnetic aspiration, and then washed twice with 1 mL of 1× PBS, the supernatant was discarded by magnetic aspiration to obtain exosomes containing CD9 protein.

[0163] The exosome lysis and desalination treatment is performed using a method comprising the following steps:

[0164] Exosomes were lysed using exosome lysis buffer (Yiwei Jianhua EV02-08-01), heated to 95°C, cooled, diluted 5-fold with 50 mM tetraethylammonium bromide solution, and digested for 15 h using recombinant trypsin and lysyl endonuclease, wherein the mass ratio of the recombinant trypsin to the substrate was 1 μg:40 μg, and the mass ratio of the lysyl endonuclease to the substrate was 1 μg:80 μg. 10% TFA was acidified to a final concentration of 1% TFA, ethyl acetate was added and the protein was vigorously vortexed to precipitate the protein, passed through a desalting column, and the eluate was collected and lyophilized and concentrated to obtain the purified protein.

[0165] The sample to be tested is prepared by a method comprising the following steps:

[0166] The protein was dissolved in 0.1% formic acid aqueous solution, vortexed and centrifuged at 20,000 g for 10 min, the supernatant was collected, and the sample and heavy peptide were mixed at a volume ratio of 1:1 and then detected by liquid chromatography-mass spectrometry.

[0167] (2) The samples to be tested were analyzed by HPLC-PRM / MS. The characteristic peptide KDVLETFTVK (SEQ ID NO: 2) in the CD9 protein was selected as the internal standard, and the glutamic acid in the middle of the characteristic peptide was labeled with a heavy isotope to obtain a heavy peptide standard.

[0168] The chromatographic conditions of the HPLC are:

[0169] The chromatographic column was a C18 capillary column 1.9 μm, 75 μm × 20 cm; the injection volume was 2 μL;

[0170] The mobile phase consisted of mobile phase A and mobile phase B, where mobile phase A was a 0.1% formic acid aqueous solution and mobile phase B was an 80% acetonitrile aqueous solution containing 0.1% formic acid;

[0171] The elution program is shown in Table 2.

[0172] The mass spectrometry conditions of the MS are:

[0173] Primary mass spectrometry, scan range: 400-1200 m / z, resolution: 60000, AGC target: 1e5, Maximum IT: 50 ms;

[0174] Secondary PRM scan, isolation window: 2.0 m / z, resolution: 60000, AGC target: 1e6, maximum IT: 100 ms.

[0175] (3) Establish a standard curve for the internal standard and perform absolute quantification of exosome surface proteins based on the standard curve.

[0176] The mass spectrometry results are shown in Table 4.

[0177] Table 4

[0178] As can be seen from Table 4, the method has good targeting, good stability and good repeatability.

[0179] The external calibration curves obtained by performing three repeated PRM detection tests on the above samples are shown in Figures 7-18 respectively.

[0180] Figures 7-9 show the concentration of 2×10 7 PRM_1-3 assay results for CD9 protein-containing exosomes at 400 particles / mL: Figure 7: Slope: 1.0626E-2; Intercept: 1.2810E-2; R-squared = 0.9942; Calculated concentration = 4.7086 pg / μL. Figure 8: Slope: 1.0626E-2; Intercept: 1.2810E-2; R-squared = 0.9942; Calculated concentration = 3.0586 pg / μL. Figure 9: Slope: 1.0626E-2; Intercept: 1.2810E-2; R-squared = 0.9942; Calculated concentration = 3.2212 pg / μL.

[0181] Figure 10-12 shows the concentration of 2×10 8 PRM_1-3 assay results for exosomes containing CD9 protein at 400 μg / mL; Figure 10: Slope: 1.0626E-2; Intercept: 1.2810E-2; R-squared = 0.9942; Calculated concentration = 8.8332 pg / μL. Figure 11: Slope: 1.0626E-2; Intercept: 1.2810E-2; R-squared = 0.9942; Calculated concentration = 8.3442 pg / μL. Figure 12: Slope: 1.0626E-2; Intercept: 1.2810E-2; R-squared = 0.9942; Calculated concentration = 8.0839 pg / μL.

[0182] Figure 13-15 shows the concentration of 2×10 9PRM_1-3 assay results for exosomes containing CD9 protein at 400 μg / mL; Figure 13: Slope: 1.0626E-2; Intercept: 1.2810E-2; R-squared = 0.9942; Calculated concentration = 64.7952 pg / μL. Figure 14: Slope: 1.0626E-2; Intercept: 1.2810E-2; R-squared = 0.9942; Calculated concentration = 63.8832 pg / μL. Figure 15: Slope: 1.0626E-2; Intercept: 1.2810E-2; R-squared = 0.9942; Calculated concentration = 63.8538 pg / μL.

[0183] Figure 16-18 shows the concentration of 2×10 10 PRM_1-3 assay results for exosomes containing CD9 protein at 400 μg / mL; Figure 16: Slope: 1.0626E-2; Intercept: 1.2810E-2; R-squared = 0.9942; Calculated concentration = 174.2369 pg / μL. Figure 17: Slope: 1.0626E-2; Intercept: 1.2810E-2; R-squared = 0.9942; Calculated concentration = 174.6033 pg / μL. Figure 18: Slope: 1.0626E-2; Intercept: 1.2810E-2; R-squared = 0.9942; Calculated concentration = 170.7086 pg / μL.

[0184] From the results in Figures 7-18, it can be seen that the method has good targeting, good stability and good repeatability.

[0185] Example 3

[0186] This example provides a method for absolute quantification of exosome surface proteins. The steps for preparing the exosome surface protein sample are as follows, and the remaining steps refer to Example 2.

[0187] The CD9 antibody-coupled magnetic beads are prepared by a method comprising the following steps:

[0188] a. The magnetic beads were washed with MES buffer, the MES buffer was mixed with the magnetic beads and placed on ice for 1 hour, and EDC and NHS were added after the ice bath and mixed for 40 minutes to obtain activated magnetic beads; the final concentration of the EDC was 0.8 mg / mL, and the final concentration of the NHS was 0.8 mg / mL;

[0189] b. washing the activated magnetic beads with MES buffer;

[0190] c. Mix the CD9 antibody with the activated magnetic beads at a mass ratio of 250 μg antibody / 1.2 mg magnetic beads, allow to react for 6 h, wash the reaction product with MES buffer, and perform blocking treatment;

[0191] d. The blocked CD9 antibody-coupled magnetic beads were placed in a preservation solution comprising, by concentration, PBS + 1.5‰ p-300 + 1.5% Tween 20, pH 7.4.

[0192] Collect the HEK293 supernatant and centrifuge at 10,000 g for 30 minutes. After collecting the supernatant, resuspend the pellet at the bottom of the centrifuge tube in PBS. Filter through a 0.22 μm filter membrane and centrifuge at 130,000 g for 70 minutes. Discard the supernatant, resuspend the pellet in PBS, and centrifuge at 130,000 g for 70 minutes. Discard the supernatant and resuspend the pellet in 1 mL of PBS to obtain the HEK293 supernatant-derived exosomes.

[0193] The solid phase support material (CD9 magnetic beads) coupled with the CD9 antibody is used to capture the exosomes, and the exosomes are lysed and desalted. The lysing and desalting of the exosomes are performed using a method comprising the following steps:

[0194] Exosomes were lysed using an exosome lysis buffer, heated to 100°C, cooled, diluted 5-fold with 60 mM tetraethylammonium bromide solution, and digested for 16 hours using recombinant trypsin and lysyl endonuclease, wherein the mass ratio of the recombinant trypsin to the substrate was 1 μg:30 μg, and the mass ratio of the lysyl endonuclease to the substrate was 1 μg:50 μg. The protein was acidified with 15% TFA to a final concentration of 0.5% TFA, and ethyl acetate was added to vigorously vortex to precipitate the protein. The protein was passed through a desalting column, and the eluate was collected and lyophilized and concentrated to obtain a purified protein.

[0195] The sample to be tested is prepared by a method comprising the following steps:

[0196] The protein was dissolved in 0.2% formic acid aqueous solution, vortexed and centrifuged at 20,000 g for 10 min, the supernatant was collected, and the sample and heavy peptide were mixed at a volume ratio of 1:1 and then detected by liquid chromatography-mass spectrometry.

[0197] The chromatographic conditions of the liquid chromatography-mass spectrometry were also referred to those in Example 2. This example also obtained good detection results with good detection stability and repeatability.

[0198] Example 4

[0199] This example provides a method for absolute quantification of exosome surface proteins. The difference between the method and Example 2 is that the solid phase support material used in this example is a hydrogen bromide activated cross-linked agarose matrix (Cat. No.: A53101-14 Source: Jiaxing Qianchun Biology. The remaining steps refer to

[0200] Example 2.

[0201] The specific method for coupling the cross-linked agarose matrix with the antibody by hydrogen bromide activation includes the following steps:

[0202] a. Dissolve the CD9 antibody (Cat. No. SR70S01, Source: Langfang Tianguang Biotechnology Co., Ltd.) in a coupling buffer containing 0.5 M NaCl, 0.1 M NaHCO3, pH 8.3. The concentration of the CD9 antibody is 5 mg / mL.

[0203] b. Rinse the cross-linked agarose matrix with hydrogen bromide three times with coupling buffer containing 0.5 M NaCl, 0.1 M NaHCO3, pH 8.3, and drain.

[0204] c. Add CD9 antibody to the pretreated hydrogen bromide cross-linked agarose matrix so that the mass ratio of CD9 antibody to activated cross-linked agarose matrix is ​​250 μg antibody / 1.2 mg cross-linked agarose matrix, and mix by inversion overnight at 4°C.

[0205] d. After the cross-linking reaction is completed, the protein concentration is collected and the cross-linking value is calculated.

[0206] e. Wash the free protein with 5 volumes of cross-linking buffer and then with 10 volumes of distilled water.

[0207] f. The washed gel was suspended in water, and ethanolamine (1 mol / L) or Tris-HCl buffer (pH 8.0) was added. The reaction was stirred at room temperature for at least 2 h to eliminate residual active groups.

[0208] g. Wash the column alternately with a buffer solution containing 0.5 mol / L NaCl and 0.1 mol / L acetate (pH 4.0) and a buffer solution containing 0.5 mol / L NaCl and 0.1 mol / L Tris-HCl (pH 8). Repeat the above washing cycle at least three times, and then wash with 10 column volumes of PBS.

[0209] h. Use 20% ethanol to store the coupling product.

[0210] Example 5

[0211] This example provides a method for absolute quantification of exosome surface proteins. This method differs from Example 2 in that the solid support material used in this example is NHS agarose microspheres (Cat. No. A51801-05, source: Jiaxing Qianchun Biotechnology). The remaining steps are similar to those in Example 2.

[0212] The specific method of coupling NHS agarose microspheres with antibodies includes the following steps:

[0213] a. Dissolve an appropriate amount of antibody in 100mM 2-morpholinoethanesulfonic acid (MES) to a concentration of 3.0mg / mL. Antibodies already stored in buffer should be dialyzed or desalted to remove any primary amine groups in the buffer. Then, dissolve the antibody in 100mM 2-morpholinoethanesulfonic acid (MES) to a concentration of 3.0mg / mL. Store the prepared antibody solution at 4°C until ready for use.

[0214] b. Quickly wash the agarose beads with pre-cooled 1 mM hydrochloric acid solution to prevent hydrolysis of the NHS group during the washing process.

[0215] c. Immediately after washing the agarose beads, add the antibody solution at a mass ratio of 250 μg of CD9 antibody to 1.2 mg of agarose beads. Place the solution on a vertical mixer and mix at room temperature for 3 h. If mixing is uneven, remove the EP tube and vortex for 15 s every 15 min.

[0216] d. Add 1 mL of 3 M ethanolamine, vortex for 30 seconds, place the EP tube in a vertical mixer and react at room temperature for 2 hours, centrifuge and discard the supernatant.

[0217] e. Add 1 mL of PBS buffer containing 0.05% sodium azide to the EP tube, mix thoroughly, centrifuge and discard the supernatant, and repeat twice.

[0218] f. Add 400 μL of PBS buffer containing 0.05% sodium azide to the EP tube, mix thoroughly, and store at 4°C until use.

[0219] Example 6

[0220] This example provides a method for absolute quantification of exosome surface proteins. This method differs from Example 2 in that the solid support material used in this example is amino-functionalized agarose beads (Cat. No. C740001, source: Jiaxing Qianchun Biotechnology). The remaining steps are similar to those in Example 2.

[0221] The specific method for coupling amino-functionalized agarose beads with antibodies includes the following steps:

[0222] a. Remove the protective agent, wash the amino agarose beads with MES buffer, and shake on a mixer.

[0223] b. Wash three times and discard the MES buffer with a pipette.

[0224] c. MES buffer was mixed with the amino-functionalized agarose matrix and placed on ice for 0.5 h. After the ice bath, EDC and NHS were added and mixed for 30 min. The final concentration of the EDC was 1.2 mg / mL, and the final concentration of the NHS was 1.2 mg / mL to obtain an activated solid support material.

[0225] d. Add the ligand (the ligand is CD9 antibody, product number: SR70S01, source: Langfang Tianguang Biotechnology Co., Ltd.).

[0226] e. If the ligand is unstable at room temperature, perform the following steps in a refrigerator:

[0227] Add 1 mL of activated amino-agarose beads to the previous CD9 antibody solution. The mass ratio of CD9 antibody to amino-functionalized agarose beads is 250 μg antibody / 1.2 mg agarose beads. Stir gently, remove an aliquot of the suspension, and test for activity and / or absorbance at 280 nm. Continue gentle stirring for several 2.5 hours or until the activity measurement remains constant, indicating complete immobilization. Avoid magnetic stirring. (Stirring time should not exceed 3 hours because CDI will decompose. However, if immobilization must be performed in a cold room, stirring time may be longer due to the low stability of the ligand). Wash the suspension with distilled water to eliminate excess reagent, then wash with 1.0 M NaCl solution, and finally wash with distilled water.

[0228] f. The ligand is conjugated to the amino matrix and can be stored in a 0.03% sodium azide solution at 4°C.

[0229] The same volume of solid support material coupled with antibodies that recognize target surface proteins in Examples 2 and 4-6 was used to extract 200 μL of plasma exosomes for WB and PRM detection.

[0230] The WB test results are shown in Figures 19 and 20. Figure 19 shows the results of WB testing of the extracted exosomes, and Figure 20 shows the results of WB testing of the supernatant after incubation with a solid phase support material coupled to an antibody that recognizes the target surface protein in the exosome extraction step. It can be seen from Figures 19 and 20 that the extraction material prepared by the coupling method of carboxyl magnetic beads has a better extraction effect, and basically all exosomes containing the target surface protein can be extracted, and the supernatant basically does not contain target exosomes.

[0231] The PRM test results are shown in Table 5.

[0232] Table 5

[0233] As shown in Table 5, the extraction material prepared by the coupling method of carboxyl magnetic beads has a better extraction effect on exosomes, is more suitable for subsequent quantitative detection, and has more accurate detection results.

[0234] Example 7

[0235] This example investigates the elution procedure of liquid chromatography in a method for absolute quantification of exosome surface proteins. The remaining chromatographic conditions are similar to those in Example 2.

[0236] Elution program 1 is shown in Table 6:

[0237] Table 6

[0238] Elution procedure 2 is shown in Table 7:

[0239] Table 7

[0240] Std standards were mixed in a 1:1 ratio as shown in Table 8 and loaded in equal volumes.

[0241] Table 8

[0242] When the elution program was 20 minutes (Table 6), the corresponding results are shown in Figure 21, which shows the retention time detection results for an elution program of 20 minutes. In Figure 21, taking the results of Std_1 as an example, the left side of the Std_1 results shows the retention time result of KDVLETFTVK (SEQ ID NO: 2) -590.3346++, and the right side shows the retention time result of KDVLETFTVK (SEQ ID NO: 2) -590.8331++ (heavy). The results of the remaining standards refer to Std_1.

[0243] When the elution program is 30 minutes (the gradient in Example 2), the corresponding results are shown in Figure 22, which shows the retention time detection results for an elution program of 30 minutes. In Figure 22, the results of Std_1 are used as an example. The left side of the results of Std_1 shows the retention time result of KDVLETFTVK (SEQ ID NO: 2)-590.3346++, and the right side shows the retention time result of KDVLETFTVK (SEQ ID NO: 2)-590.8331++ (heavy). The results of the remaining standards refer to Std_1.

[0244] From the above results, we can see that when the elution time is 15 minutes, the retention time is unstable; when the elution time is 20 minutes, the retention time is not as stable as 30 minutes; when the elution time is set to 30 minutes, the retention time does not fluctuate, so the 30-minute gradient is better.

[0245] Example 8

[0246] This example provides a method for absolute quantification of exosome surface proteins. This example quantitatively detects exosomes derived from human plasma. FIG23 is a flowchart of the enrichment and quantification of CD9 protein on the surface of plasma exosomes, including the following steps:

[0247] (1) Exosomes are extracted using a solid support material coupled with CD9 antibodies, and then the exosomes are lysed and desalted in sequence, and the desalted protein is prepared as a test sample.

[0248] The solid phase support material coupled to the CD9 antibody is prepared by a method comprising the following steps:

[0249] a. The solid support material was washed with MES buffer, wherein the solid support material was magnetic beads. The MES buffer was mixed with the magnetic beads and placed in an ice bath. After the ice bath, EDC and NHS were added and mixed for 30 minutes. The final concentration of the EDC was 1.2 mg / mL, and the final concentration of the NHS was 1.2 mg / mL to obtain activated magnetic beads.

[0250] b. washing the activated magnetic beads with MES buffer;

[0251] c. The CD9 antibody was mixed with activated magnetic beads at a mass ratio of 250 μg antibody / 1 mg magnetic beads, and the reaction was allowed to stand for 5 h. The reaction product was washed with MES buffer and blocked;

[0252] d. The blocked CD9 antibody-coupled magnetic beads (abbreviated as CD9 magnetic beads) were placed in a preservation solution, wherein the preservation solution comprises, by concentration, PBS + 1‰ p-300 + 1% Tween 20, pH 7.4.

[0253] Human plasma was centrifuged at 3000 g for 10 minutes at 4°C. 200 μL of the middle layer was collected. 1 mL of 1× PBS was added, and 40 μL of CD9 magnetic beads were added for 1 hour. After 1 hour of incubation, the supernatant was removed by magnetic aspiration. The cell suspension was washed twice with 1 mL of Urine Loading Buffer, the supernatant was discarded by magnetic aspiration, and the cell suspension was washed twice with 1 mL of 1× PBS, the supernatant was discarded by magnetic aspiration. Exosomes were obtained.

[0254] The exosome lysis and desalination treatment is performed using a method comprising the following steps:

[0255] Exosomes were lysed using an exosome lysis buffer, heated to 95°C, cooled, diluted 5-fold with 50 mM tetraethylammonium bromide solution, and digested for 14 h using recombinant trypsin and lysyl endonuclease, wherein the mass ratio of the recombinant trypsin to the substrate was 1 μg:40 μg, and the mass ratio of the lysyl endonuclease to the substrate was 1 μg:50 μg. The protein was acidified with 15% TFA to a final concentration of 0.5% TFA, and ethyl acetate was added to vigorously vortex to precipitate the protein. The protein was passed through a desalting column, and the eluate was collected and lyophilized and concentrated to obtain the purified protein.

[0256] The sample to be tested is prepared by a method comprising the following steps:

[0257] The protein was dissolved in 0.1% formic acid aqueous solution, vortexed and centrifuged at 20,000 g for 10 min, the supernatant was collected, and the sample and heavy peptide were mixed at a volume ratio of 1:1 and then detected by liquid chromatography-mass spectrometry.

[0258] (2) The samples to be tested were analyzed by HPLC-PRM / MS. The characteristic peptide KDVLETFTVK (SEQ ID NO: 2) in the CD9 protein was selected as the internal standard, and the glutamic acid in the middle of the characteristic peptide was labeled with a heavy isotope to obtain a heavy peptide standard.

[0259] The chromatographic conditions of the HPLC are:

[0260] The chromatographic column was a C18 capillary column 1.9 μm, 75 μm × 20 cm; the injection volume was 2 μL;

[0261] The mobile phase consisted of mobile phase A and mobile phase B, where mobile phase A was a 0.1% formic acid aqueous solution and mobile phase B was an 80% acetonitrile aqueous solution containing 0.1% formic acid;

[0262] The elution program is shown in Table 2.

[0263] The mass spectrometry conditions of the MS are:

[0264] Primary mass spectrometry, scan range: 400-1200 m / z, resolution: 60000, AGC target: 1e5, Maximum IT: 50 ms;

[0265] Secondary PRM scan, isolation window: 2.0 m / z, resolution: 60000, AGC target: 1e6, maximum IT: 100 ms.

[0266] (3) Establish a standard curve for the internal standard and perform absolute quantification of exosome surface proteins based on the standard curve.

[0267] The mass spectrometry results are shown in Table 9.

[0268] Table 9

[0269] As can be seen from Table 9, the method has good targeting, good stability, and good repeatability, and can achieve quantitative detection of exosomes derived from human plasma.

[0270] The external calibration curves obtained by performing three repeated PRM detection tests on the above samples are shown in Figures 24-26 respectively.

[0271] FIG24 shows the PRM_1 detection results of human plasma exosomes containing CD9 protein (200 μL); in FIG24 , the slope is 1.0626E-2; the intercept is 1.2810E-2; the R-square is 0.9942; and the calculated concentration is 88.4471 pg / μL.

[0272] Figure 25 shows the PRM_2 detection results of human plasma exosomes containing CD9 protein (200 μL); slope in Figure 25: 1.0626E-2; intercept: 1.2810E-2; R square = 0.9942; calculated concentration = 84.9927 pg / μL.

[0273] Figure 26 shows the PRM_3 detection results of human plasma exosomes containing CD9 protein (200 μL); slope in Figure 26: 1.0626E-2; intercept: 1.2810E-2; R square = 0.9942; calculated concentration = 84.9735 pg / μL.

[0274] The results in Figures 24-26 further demonstrate that the method has good targeting, good stability and good repeatability.

[0275] Example 9

[0276] This example methodologically validates the absolute quantification method of exosome surface proteins, including the limit of quantification and the limit of detection.

[0277] The specific method includes the following steps:

[0278] Collect the HEK293 supernatant and centrifuge at 10,000 g for 30 minutes. After collecting the supernatant, resuspend the precipitate at the bottom of the centrifuge tube with PBS. Filter through a 0.22 μm filter membrane and centrifuge the filtered supernatant at 130,000 g for 70 minutes. Discard the supernatant and resuspend the precipitate with PBS. Centrifuge at 130,000 g for 70 minutes. Discard the supernatant and resuspend the precipitate with 1 mL of PBS to obtain exosomes derived from the HEK293 supernatant. Measure the NTA concentration to determine the concentration, and then dilute with PBS to 2 × 10 10 particles / mL, 2×10 9 particles / mL, 2×10 8 particles / mL, 2×10 7 40 μL CD9 magnetic beads were added to 1 mL of sample per tube and incubated for 1 hour. After 1 hour of incubation, the supernatant was removed by magnetic aspiration, and the cells were washed twice with 1 mL of Urine Loading Buffer, the supernatant was discarded by magnetic aspiration, and then washed twice with 1 mL of 1× PBS, the supernatant was discarded by magnetic aspiration to obtain exosomes containing CD9 protein.

[0279] The exosome lysis and desalination treatment is performed using a method comprising the following steps:

[0280] Exosomes were lysed using exosome lysis buffer (Yiwei Jianhua EV02-08-01), heated to 95°C, cooled, diluted 5-fold with 50 mM tetraethylammonium bromide solution, and digested for 16 h using recombinant trypsin and lysyl endonuclease, wherein the mass ratio of the recombinant trypsin to the substrate was 1 μg:50 μg, and the mass ratio of the lysyl endonuclease to the substrate was 1 μg:100 μg. 10% TFA was acidified to a final concentration of 0.5% TFA, ethyl acetate was added to vigorously vortex and precipitate the protein, passed through a desalting column, and the eluate was collected and lyophilized and concentrated to obtain the purified protein.

[0281] The sample to be tested is prepared by a method comprising the following steps:

[0282] The protein was dissolved in 0.1% formic acid aqueous solution, vortexed and centrifuged at 20,000 g for 10 min, the supernatant was collected, and the sample and heavy peptide were mixed at a volume ratio of 1:1 and then detected by liquid chromatography-mass spectrometry.

[0283] (2) HPLC-PRM / MS was used to analyze the test samples; the characteristic peptide KDVLETFTVK (SEQ ID NO: 2) in the CD9 protein was selected as the internal standard, and the glutamic acid in the middle of the characteristic peptide was labeled with a heavy isotope. The synthesized light peptide was diluted into different gradient concentrations, and the heavy peptide was diluted to a fixed concentration, and then mixed into a standard at a ratio of 1:1.

[0284] The chromatographic conditions of the HPLC are:

[0285] The chromatographic column was a C18 capillary column 1.9 μm, 75 μm × 20 cm; the injection volume was 2 μL;

[0286] The mobile phase consisted of mobile phase A and mobile phase B. Mobile phase A was a 0.1% formic acid aqueous solution, and mobile phase B was an 80% acetonitrile aqueous solution containing 0.1% formic acid.

[0287] The elution program is shown in Table 2. The mass spectrometry conditions refer to Example 2.

[0288] The verification results are shown in Table 10.

[0289] Table 10

[0290] As shown in Table 10, the lower limit of quantification of exosomes after extracting exosomes of different concentrations using CD9 magnetic beads is 7 pg, and the experimental R square is equal to 0.9987.

[0291] In summary, this application provides a method for absolute quantification of exosome surface proteins. The detection method is highly sensitive, reproducible, and accurate, with a quantitative range of up to 5 orders of magnitude, a quantitative reproducibility CV <10%, and a relative error <15%. The detection method has an LOD of 1 pg and an LOQ of 7 pg, allowing the method of this application to quantify low-abundance proteins. In addition, the solid phase support material conjugated to the CD9 antibody constructed in this application has good targeting and good stability in repeated experiments, enabling accurate quantification of exosome protein content and more reproducible quantitative verification results.

[0292] The applicant declares that the above is only a specific implementation method of the present application, but the protection scope of the present application is not limited thereto. Technical personnel in the relevant technical field should understand that any changes or substitutions that can be easily thought of by technical personnel in the relevant technical field within the technical scope disclosed in this application fall within the protection scope and disclosure scope of this application.

Claims

1. A method for absolute quantification of exosome surface proteins, wherein include: (1) extracting exosomes containing the target surface protein using a solid support material coupled to an antibody that recognizes the target surface protein, then sequentially lysing the exosomes and desalting, and preparing the desalted protein into a sample to be tested; (2) Analyzing the sample by HPLC-PRM / MS; selecting a characteristic peptide in the target surface protein as an internal standard, labeling the characteristic peptide with a heavy isotope to obtain a heavy peptide, and using an unlabeled isotope characteristic peptide as a light peptide, diluting the synthesized light peptide into different gradient concentrations, diluting the synthesized heavy peptide into a fixed concentration, and mixing them into a standard at a volume ratio of 1:1; and (3) Establish a standard curve for the internal standard and perform absolute quantification of exosome surface proteins based on the standard curve.

2. The method for absolute quantification of exosome surface proteins according to claim 1, in, The solid support material is selected from magnetic beads or agarose medium; The antibody that recognizes the target surface protein is selected from monoclonal antibodies and / or nanobodies.

3. The method for absolute quantification of exosome surface proteins according to claim 2, in, When the solid phase support material is magnetic beads, the coupling method of the magnetic beads and antibodies is a coupling method of carboxyl magnetic beads.

4. The method for absolute quantification of exosome surface proteins according to claim 1, in, In step (1), the exosome lysis and desalination treatment are performed by a method comprising the following steps: The exosomes were lysed with an exosome lysis buffer, heated, cooled, diluted 4-5 times with tetraethylammonium bromide solution, digested with protease, acidified with 10-15% TFA to a final concentration of 0.5-5% TFA, vortexed with ethyl acetate to precipitate the protein, passed through a desalting column, and the eluate was collected and freeze-dried and concentrated to obtain a purified protein.

5. The method for absolute quantification of exosome surface proteins according to claim 1, in, In step (1), the sample to be tested is prepared by a method comprising the following steps: The protein was dissolved in a 0.1-0.2% formic acid aqueous solution, vortexed and centrifuged at 20000-21000g for 8-12 minutes, the supernatant was collected, and the sample and heavy peptide were mixed at a volume ratio of 1:1 and then subjected to liquid chromatography-mass spectrometry detection.

6. The method for absolute quantification of exosome surface proteins according to claim 1, in, In step (2), the chromatographic conditions of the HPLC are: The chromatographic column was a C18 capillary column 1.9 μm, 75 μm × 20 cm; the injection volume was 2 ± 0.5 μL; The mobile phase consists of mobile phase A and mobile phase B, mobile phase A is 0.1% formic acid in water, and mobile phase B is 80% acetonitrile in water containing 0.1% formic acid; The elution program is as follows:

7. The method for absolute quantification of exosome surface proteins according to claim 1, in, In step (2), the mass spectrometry conditions of the MS are: primary mass spectrometry, scanning range: 400-1200 m / z, resolution: 60000, AGC target: 1e5, Maximum IT: 50 ms; Secondary PRM scan, Isolation window: 2.0m / z, resolution: 60000, AGC target: 1e6, Maximum IT: 100ms.

8. Use of the method for absolute quantification of exosome surface proteins according to any one of claims 1 to 7 in exosome quality detection.

9. The use according to claim 8, in, In the application of exosome quality detection, when absolutely quantifying CD9 protein in exosomes, the characteristic peptide segment used is selected from any one of (1), (2) or (3) or a combination of at least two of them: (1) DVLETFTVK (SEQ ID NO: 1); (2) KDVLETFTVK (SEQ ID NO: 2); (3) EVQEFYK (SEQ ID NO: 3).

10. The use according to claim 9, in, In the application of exosome quality detection, the characteristic peptide used for absolute quantification of CD9 protein in exosomes is KDVLETFTVK (SEQ ID NO: 2).

11. The use according to claim 10, in, The characteristic peptide segment is isotopically labeled, and the labeled characteristic peptide segment is used as a heavy peptide, and the position of the isotope labeling is selected from any one of the first lysine to the sixth glutamic acid starting from the C-terminus; The isotope is labeled on the C element and / or N element of the corresponding amino acid.

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