CN116660549A - Proteomics analysis method of exosomes derived from nervous system - Google Patents
Proteomics analysis method of exosomes derived from nervous system Download PDFInfo
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Abstract
The invention relates to a proteomics analysis method of an exosome from a nervous system, belonging to the technical field of protein analysis. Separating exosomes from a nervous system source, eluting, performing enzymolysis, concentrating to obtain peptide fragment dry powder, and finally performing mass spectrometry detection. By optimizing the separation flow and elution method of the nerve cell exosomes, the separation efficiency of the nerve cell source exosome protein in the blood sample is effectively improved. Meanwhile, the invention carries out proteomic analysis of the exosomes based on the optimized FASP method and mass spectrum detection method, thereby effectively improving the number of proteins identified in the exosomes.
Description
Technical Field
The invention relates to a proteomics analysis method of an exosome from a nervous system, belonging to the technical field of protein analysis.
Background
Exosomes are a class of extracellular vesicles produced by endosomal pathways that are involved in cellular communication, immune response, cell migration, etc. processes in the body and are widely found in a variety of body fluids. Particularly in the central nervous system, exosomes play important physiological roles in intercellular communication, myelination, synaptic plasticity, antigen presentation, and nutritional support. Exosomes have unique advantages in neurological disease research: firstly, the exosomes are phospholipid bilayer nano vesicles with diameters of about 30-150 nm, can bidirectionally cross the blood brain barrier, and have congenital advantages in the aspects of biomarker screening, drug delivery and the like. Second, exosomes contain various active ingredients derived from the source cell, such as proteins, RNAs, lipids, capable of reflecting physiological or pathological changes of the source cell, and are detectable in a variety of body fluids without invasive means. Thirdly, after recognizing exosome specific receptors, exosomes of different cell sources can be effectively separated, especially in the central nervous system, neurons, astrocytes, oligodendrocytes and the like can generate and release exosomes, and can be captured by specific antibodies. The exosomes derived from the nerve cells are used as a new research object, and have great research value and application prospect in the pathology and diagnosis research of nervous system diseases.
2021, federica Anastasi et al developed a workflow for isolation of ndsevs (small extracellular vesicles derived from neurons) from plasma of individual patients and proteomic analysis, and authors were able to identify 349±38 proteins by protein isolation, enzymatic hydrolysis (S-trap method) and mass spectrometry using an immunoplate covalently coated with L1CAM antibodies to isolate ndsevs. While, as early as before, zhang at university of washington and Goetzl et al at university of california used NCAM or L1CAM antibodies for isolation of NDE (neuron-derived exosomes) in blood, but were limited to isolation of exosomes and did not involve proteomic methods of exosomes; later, this method was also used to isolate astrocytes and oligodendrocyte-derived exosomes in human blood in addition to neuron-derived exosomes, except that the antibodies used were changed to GLAST and OMG. Of the above methods and prior studies, only a few have studied the exosome protein component of neuronal cells by proteomics methods, with low amounts of identified proteins; the methods which are widely extended and applied at present only relate to the separation method of exosomes, and the yield of the exosome protein obtained by separation is lower due to the limitation of the elution method.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a proteomic analysis method of an exosome derived from a nervous system. The method can realize the high-efficiency separation and proteomics analysis of exosomes from different nerve cells in blood, and solves the problems of low exosome protein separation efficiency, small protein quantity identified by mass spectrum and the like in the prior art.
In order to achieve the above object, the technical scheme of the present invention is as follows.
A method of proteomic analysis of a nervous system-derived exosome, the method comprising the steps of:
(1) Separation of exosomes derived from the nervous system: separating exosome precipitate in blood plasma or serum, dispersing the exosome precipitate in PBS solution, adding nerve cell exosome marker biotinylation antibody, incubating to make the antibody fully combine with exosome of nerve cell, then adding streptavidin resin microbead, incubating to make streptavidin fully combine with biotin, forming exosome-antibody-microbead compound in the solution, centrifuging, washing the precipitate with PBS solution, centrifuging, collecting precipitate to obtain exosome-antibody-microbead compound; the nerve cells include neurons and glial cells;
preferably, in the step (1), blood is collected by using a blood collection tube containing a coagulant, and the supernatant is centrifugally separated to obtain serum; using a composition containing K 2 EDTA (ethylene diamine tetraacetic acid) or sodium citrate anticoagulant blood collection tube, and centrifugally separating the supernatant to obtain the plasma.
Preferably, in the step (1), thrombin is added into the plasma, the plasma is incubated for 5-20 min at 20-25 ℃, and the supernatant is centrifugally separated; filtering the supernatant by using a filter membrane with the pore diameter of 0.22 mu m to obtain filtered plasma;
adding an exosome precipitation reagent into the filtered blood plasma or serum, incubating, centrifuging to remove the supernatant, and obtaining exosome precipitation;
preferably, the exosome precipitation reagent is ExoQuick TM The ratio of reagent amount to plasma or serum amount was 126 μl:500 μl, incubating at 4deg.C for 1-2 hours; adding PBS solution containing protease inhibitor and phosphatase inhibitor into exosome precipitate to resuspend exosome precipitate, and repeatedly blowing to uniformly distribute exosome particles in the solution; then adding the nerve cell exosome marker biotinylation antibody, wherein the ratio of the plasma or serum dosage to the exosome marker biotinylation antibody dosage is 500 μl: incubating for 1-2 hours at 2-8 ℃ with 3-5 μl to fully combine the antibody with the exosomes of the nerve cells;
then adding streptavidin resin microbeads, wherein the ratio of the plasma or serum dosage to the streptavidin resin microbeads is 500 μl: incubating for 1-2 hours at the temperature of 2-8 ℃ under the condition of 15-25 μl and rotating uniformly so as to fully combine streptavidin and biotin; centrifuging to remove the supernatant, adding PBS solution for washing the precipitate, centrifuging to remove the supernatant, and obtaining the exosome-antibody-microbead compound.
Preferably, in the step (1), the neuronal exosome marker is one or more of neuronal exosome markers L1CAM (neuronal adhesion molecule L1), NCAM (neuronal adhesion molecule), astrocyte exosome markers GLAST (glutamate aspartate transporter) and oligodendrocyte exosome markers OMG (oligodendrocyte myelin glycoprotein).
(2) Exosome elution: adding a Sodium Dodecyl Sulfate (SDS) solution into the exosome-antibody-microbead compound, boiling for 5-10 min at 90-100 ℃, centrifugally separating, collecting supernatant, namely the SDS solution of exosome protein from nerve cells, and detecting the protein content;
preferably, in the step (2), the exosome-antibody-microbead complex is added into an SDS solution with a mass fraction of 0.1% -0.5% by volume of 10-20 times.
Preferably, in step (2), the BCA method is used to detect protein content.
(3) Exocrine deviceAnd (3) performing body enzymolysis: adding SDS solution of nerve cell source exosome protein into a ultrafilter tube, centrifuging to remove SDS, intercepting the nerve cell source exosome protein at the upper layer of the ultrafilter tube, adding mixed solution of Urea (UA) and Dithiothreitol (DTT) into the nerve cell source exosome protein, and incubating for 30-60 minutes at 37 ℃ to reduce disulfide bonds in the protein; then adding sulfhydryl-reactive alkylating reagent to make alkylation modification, then using urea solution and ammonium bicarbonate (NH) 4 HCO 3 ) Washing and centrifuging the solution, replacing a solution system, replacing a collecting pipe of the ultrafiltration pipe, adding trypsin or trypsin/Lys-C protease mixture solution, carrying out enzymolysis and denaturation reaction for 12-16 hours at 37 ℃, adding trypsin or trypsin/Lys-C protease mixture solution, carrying out denaturation reaction for 4-8 hours at 37 ℃, centrifuging, washing with ammonium bicarbonate solution, and collecting the solution in the collecting pipe; detecting the amount of peptide fragments in a sample, and carrying out vacuum centrifugal concentration to obtain peptide fragment dry powder;
preferably, in the step (3), firstly, 8M urea solution is added into a 10kDa ultrafiltration tube for washing, after centrifugation, SDS solution of the nerve cell source exosome protein is added into the 10kDa ultrafiltration tube, and SDS is removed by centrifugation;
adding 100-200 μl of mixed solution of Urea (UA) and Dithiothreitol (DTT) into a 10kDa ultrafiltration tube, wherein the concentration of urea in the mixed solution is 8M, the concentration of dithiothreitol is 10-50 mM, and placing the mixed solution in a 37 ℃ incubator for 30-60 minutes to reduce disulfide bonds in proteins;
adding a sulfhydryl-reactive alkylating reagent into a 10kDa ultrafiltration tube to make the final concentration of the reagent be 50-100 mM, standing in a dark place at 20-25 ℃ for 30 minutes, carrying out alkylation modification on protein molecules, and centrifuging;
adding 100-200 mu l of 8M urea solution into a 10kDa ultrafiltration tube, centrifuging, and repeating twice;
adding 100-200 mu l of 50mM ammonium bicarbonate solution into a 10kDa ultrafiltration tube, centrifuging, and repeating for three times;
the bottom collecting pipe of the 10kDa ultrafiltration pipe is replaced, and the mass ratio of the exosome protein amount to trypsin is 50: 1-20: 1 adding trypsin or a trypsin/Lys-C protease mixture solution; placing the mixture in a 37 ℃ incubator for denaturation reaction for 12-16 hours, and then carrying out denaturation reaction according to the mass ratio of the exosome protein to trypsin of 100: 1-50: 1 supplementing trypsin or a trypsin/Lys-C protease mixture, and continuously placing the mixture in a 37 ℃ incubator for denaturation reaction for 4-8 hours;
and (3) centrifuging, then supplementing 50-100 mu l of 50mM ammonium bicarbonate, centrifuging, and reserving protein (peptide fragment) solution in a collecting pipe after two times of centrifuging.
Preferably, the centrifugation condition is 14000g, the centrifugation time is 20-30 minutes, and the temperature is 20-25 ℃.
Preferably, in the step (3), the thiol-reactive alkylating agent is Chloroacetamide (CAA) or Iodoacetamide (IAA), and the final concentration is 50-100 mm.
Preferably, in the step (3), the addition amount of the trypsin is that the ratio of the mass of the exosome protein derived from the nerve cells is 1/50-1/20; the volume of the ammonium bicarbonate solution is 100-200 mu l, and the concentration is 25-50 mM; the mass of the supplemented trypsin is 1/100-1/50 of the mass of the protein of the exosome from the nerve cell.
Preferably, in step (3), the amount of peptide fragments in the sample is detected using a quantitative peptide detection reagent.
(4) Mass spectrometry detection: and redissolving the peptide fragment dry powder in a formic acid aqueous solution, and performing high performance liquid chromatography-tandem mass spectrometry analysis.
Preferably, in the step (4), the mobile phase A is an aqueous solution of formic acid with the volume fraction of 0.1%, the mobile phase B is an acetonitrile/aqueous solution of formic acid with the volume fraction of 0.1%, and the volume ratio of acetonitrile to water is 20:80, the chromatographic gradient was varied as follows: 0-5 min, and the linear gradient of the mobile phase B is 2-8%; 5min-90min, and the linear gradient of the mobile phase B is 8% -24%;90min-110min, and the linear gradient of the mobile phase B is 24% -32%; 110-115 min, and the linear gradient of the mobile phase B is 32-90%; 115-125 min, wherein the linear gradient of the mobile phase B is 90-5%; 125-130 min, and maintaining the mobile phase B at 5%; the flow rate was 300nL/min.
Preferably, in the step (4), the mass spectrometry adopts a data independent acquisition mode, the primary mass spectrometry scanning range is 350-1250m/z, the resolution is 120000, the maximum gain is controlled to be 3e6, and the maximum injection time is 60ms; the resolution of the secondary mass spectrum is 30000, the maximum gain is controlled to be 1e6, the maximum injection time is 55ms, and the collision energy is 25.5eV, 27.0eV and 30.0eV.
Advantageous effects
The invention provides a proteomics analysis method of exosomes from different nervous systems in blood, which effectively improves the separation efficiency of exosome proteins from nerve cells in blood samples by optimizing the separation flow and elution method of the exosomes from the nerve cells. Meanwhile, the invention carries out proteomic analysis of the exosomes based on the optimized FASP method and mass spectrum detection method, thereby effectively improving the number of proteins identified in the exosomes.
According to the invention, the separation process of exosomes is optimized, so that the traditional elution method is changed, and the separation efficiency of the exosomes from nerve cells in blood is greatly improved; SDS solution is used as an elution solution, so that the membrane structure of exosomes combined with the microbeads can be destroyed, exosome proteins are released in a solution system, the supernatant is collected through centrifugation, the separation efficiency of the exosome proteins is improved, SDS in the exosome protein solution can be effectively removed through a FASP enzymolysis method, a sample meets the requirement of mass spectrometry detection, and the method is suitable for proteomics analysis.
Compared with the existing proteomics method, the method provided by the invention has the advantages that the number of the identified proteins is obviously increased, and the reliability and accuracy of the identification of the proteins in the exosome samples from nerve cells are greatly improved.
Drawings
FIG. 1 shows the results of protein quantification of neuronal-derived exosomes and astrocyte-derived exosomes in example 1 and comparative example 1 of the present invention.
FIG. 2 is a graph showing the results of Western blotting experiments on neuronal-derived exosomes and astrocyte-derived exosomes in example 1 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
Example 1: separation of nerve cell-derived exosomes in blood
(1) 12 healthy plasma samples, 0.5ml each, were taken and labeled as samples 1-12. After 5. Mu.l of thrombin was added to the sample and incubated at 25℃for 5 minutes, the supernatant was centrifuged at 10,000g at 25℃for 20 minutes.
(2) The supernatant was filtered using a filter membrane with a pore size of 0.22. Mu.m, and an appropriate amount of Exoquick was added to the sample TM Reagent (126. Mu.l), incubated at 4℃for 1 hour, followed by centrifugation at 1500g at 4℃for 30min, and the supernatant removed.
(3) The exosome pellet was resuspended by adding 0.5ml of PBS solution containing protease inhibitor and phosphatase inhibitor, and the exosome pellet was uniformly distributed in the solution system by repeated blowing. To samples 1-6, 4. Mu. l L1CAM biotinylated antibody was added and to samples 7-12, 4. Mu.l GLAST biotinylated antibody was added and incubated at 4℃for 1 hour. Add 25. Mu.l streptavidin resin beads and incubate for 1.5 hours at 4℃with a spin mixer. Centrifugation was performed at 4000g at 4℃for 15 minutes, the supernatant was removed, the microbeads were rinsed with 0.3ml PBS solution, and centrifugation was performed at 4000g at 4℃for 15 minutes, the supernatant was removed.
(4) Mu.l of 0.1% SDS was added and boiled for 5 minutes. The supernatant was collected by centrifugation at 4000 Xg at 4℃for 20 minutes.
Samples 1 to 6 are obtained by separating the obtained neural cell-derived exosome protein, and samples 7 to 12 are obtained by separating the obtained astrocyte-derived exosome protein, and the protein content is detected by adopting a BCA method. As shown in FIG. 1, the quantitative results of the neuron-derived exosome and astrocyte-derived exosome proteins show that the amount of the neuron-derived exosome proteins isolated from 0.5ml of plasma by the method of the present invention reaches 134.69.+ -. 13.37. Mu.g, and the amount of the astrocyte-derived exosome proteins isolated from 0.5ml of plasma reaches 132.86.+ -. 13.89. Mu.g.
The exosome samples obtained in this example were tested using antibodies to exosome marker proteins TSG101, CD63 and the cytoplasmic omentum protein Calnexin, respectively, and the experimental results are shown in fig. 2, where it is seen that the exosome marker proteins TSG101, CD63 are highly expressed and the exosome negative marker protein Calnexin is not expressed.
Example 2: proteomic analysis of nerve cell-derived exosomes in blood
(1) 1 sample of healthy plasma was taken as 0.5. 0.5ml, 5. Mu.l of thrombin was added to the sample, incubated at 25℃for 5 minutes, and centrifuged at 25℃for 20 minutes at a rate of 10,000g, and the supernatant was taken.
(2) The supernatant was filtered using a filter membrane with a pore size of 0.22. Mu.m, and an appropriate amount of Exoquick was added to the sample TM Reagent (126. Mu.l), incubated at 4℃for 1 hour, followed by centrifugation at 1500g at 4℃for 30min, and the supernatant removed.
(3) The exosome pellet was resuspended by adding 0.5ml of PBS solution containing protease inhibitor and phosphatase inhibitor, and the exosome pellet was uniformly distributed in the solution system by repeated blowing. To the sample, 4 μ l L1CAM biotinylated antibody was added and incubated at 4 ℃ for 1 hour. Add 25. Mu.l streptavidin resin beads and incubate for 1.5 hours at 4℃with a spin mixer. Centrifugation was performed at 4000g at 4℃for 15 minutes, the supernatant was removed, the microbeads were rinsed with 0.3ml PBS solution, and centrifugation was performed at 4000g at 4℃for 15 minutes, the supernatant was removed. Mu.l of 0.1% SDS was added and boiled for 5 minutes. Centrifuge at 4000g at 4℃for 20min and collect the supernatant.
(4) To a 10kDa ultrafiltration tube was added 100. Mu.l of 8M urea for rinsing and centrifuged at 25℃for 20 minutes at a rate of 14,000 g. The above exosome protein samples were then transferred to a 10kDa ultrafiltration tube and centrifuged at 14,000g for 20min at 25 ℃.
(5) 200 μl of a mixed solution of 8M Urea (UA) and 10mM Dithiothreitol (DTT) was added to a 10kDa ultrafiltration tube, and the tube was placed in an incubator at 37℃for 60 minutes to reduce disulfide bonds in the protein.
(6) 500mM Chloroacetamide (CAA) was added to a 10kDa ultrafiltration tube to a final concentration of 50mM and allowed to stand in the dark at 25℃for 30 minutes to effect alkylation modification of the protein molecules. Followed by centrifugation at 14,000g for 20 minutes at 25 ℃.
(7) 200 μl of 8M urea was added to the 10kDa ultrafiltration tube and centrifuged at 14,000g for 20min at 25℃and repeated twice.
(8) 200 μl of 50mM ammonium bicarbonate (NH) was added to a 10kDa ultrafiltration tube 4 HCO 3 ) Centrifugation was performed at 14,000g for 20min at 25℃and repeated three times.
(9) The bottom collecting tube of the 10kDa ultrafiltration tube was replaced, and 100. Mu.l of 50mM ammonium bicarbonate was added to the 10kDa ultrafiltration tube in a mass ratio of exosome protein to trypsin of 50:1 trypsin was added. The reaction was denatured at 37℃for 14 hours, followed by a mass ratio of exosome protein to trypsin of 100:1, trypsin was supplemented and the reaction was continued in a 37℃incubator for 4 hours.
(10) Centrifugation was performed at 14,000g at 25℃for 20min, followed by addition of 50. Mu.l of 50mM ammonium bicarbonate, centrifugation at 14,000g at 25℃for 20min, and the peptide solution was retained in the collection tube after two centrifugation.
(11) The amount of peptide fragments in the sample is detected by adopting a quantitative peptide detection reagent, and the sample is subjected to vacuum centrifugal concentration at 60 ℃. The sample was reconstituted to 0.2. Mu.g/. Mu.l with 0.1% formic acid, followed by HPLC-MS analysis, and the loading was 1. Mu.g. Mobile phase a of high performance liquid chromatography was 0.1% formic acid in water and mobile phase B was acetonitrile/water (20/80, v/v) solution containing 0.1% formic acid. The liquid phase gradient is as follows: 0min-4min, mobile phase B is maintained at 2%;4min-5min, the linear gradient of the mobile phase B is 2% -8%;5min-90min, the linear gradient of the mobile phase B is 8% -24%;90min-110min, the linear gradient of the mobile phase B is from 24% to 32%;110min-115min, the linear gradient of the mobile phase B is from 32% to 90%;115min-120min, mobile phase B is maintained at 90%;120min-125min, the linear gradient of the mobile phase B is 90% -5%;125min-130min, mobile phase B is maintained at 5%; the flow rate was 300nL/min.
(12) The mass spectrum analysis adopts a data independent acquisition mode, the primary mass spectrum scanning range is 350-1250m/z, the resolution is 120000, the maximum gain is controlled to be 3e6, and the maximum injection time is 60ms. The resolution of the secondary mass spectrum is 30000, the maximum gain is controlled to be 1e6, the maximum injection time is 55ms, and the collision energy is 25.5eV, 27.0eV and 30.0eV.
And (3) carrying out database retrieval on mass spectrum data through DIA-NN software, wherein the database is a Swiss-Prot human database in Uniprot, the mass window of parent ions is 350-1250, the mass window of fragment ions is 200-2000, and the FDR is set to be 1%. A total of 685 proteins were identified by database search.
Comparative example 1:
(1) 12 healthy plasma samples, 0.5ml each, were taken and labeled as samples 1-12. After 5. Mu.l of thrombin was added to the sample and incubated at 25℃for 5 minutes, the supernatant was centrifuged at 10,000g at 25℃for 20 minutes.
(2) The supernatant was filtered using a filter membrane with a pore size of 0.22. Mu.m, and an appropriate amount of Exoquick was added to the sample TM Reagent (126. Mu.l), incubated at 4℃for 1 hour, followed by centrifugation at 1500g at 4℃for 30min, and the supernatant removed.
(3) The exosome pellet was resuspended by adding 0.5ml of PBS solution containing protease inhibitor and phosphatase inhibitor, and the exosome pellet was uniformly distributed in the solution system by repeated blowing. To samples 1-6, 4. Mu. l L1CAM biotinylated antibody was added and to samples 7-12, 4. Mu.l GLAST biotinylated antibody was added and incubated at 4℃for 1 hour. Add 25. Mu.l streptavidin resin beads and incubate for 1.5 hours at 4℃with a spin mixer. Centrifugation was performed at 4000g at 4℃for 15 minutes, the supernatant was removed, the microbeads were rinsed with 0.3ml PBS solution, and centrifugation was performed at 4000g at 4℃for 15 minutes, the supernatant was removed.
(4) Mu.l of 0.05M Glycine-HCl (pH 3.0) was added thereto, and 30. 30 s was vortexed. Centrifugation was performed at 4000 Xg at 4℃for 20 minutes, the supernatant was collected, and 1M Tris-HCl (pH 8.6) was added to adjust the pH of the supernatant to 7.0.
Samples 1 to 6 are obtained by separating the obtained neural cell-derived exosome protein, and samples 7 to 12 are obtained by separating the obtained astrocyte-derived exosome protein, and the protein content is detected by adopting a BCA method. As shown in FIG. 1, the results of the quantification of the neuron-derived exosome and the astrocyte-derived exosome proteins show that the amount of the neuron-derived exosome proteins isolated from 0.5ml of plasma by the comparative method of the present invention is 22.98.+ -. 13.56. Mu.g, and the amount of the astrocyte-derived exosome proteins isolated from 0.5ml of plasma is 17.74.+ -. 8.58. Mu.g, which are significantly lower than those of example 1 (P < 0.0001).
In view of the foregoing, it will be appreciated that the invention includes but is not limited to the foregoing embodiments, any equivalent or partial modification made within the spirit and principles of the invention.
Claims (10)
1. A method for proteomic analysis of an exosome of neurological origin, characterized by: the method comprises the following steps:
(1) Separating exosome precipitate in blood plasma or serum, dispersing the exosome precipitate in PBS solution, adding nerve cell exosome marker biotinylation antibody, incubating to make the antibody fully combine with exosome of nerve cell, then adding streptavidin resin microbead, incubating to make streptavidin fully combine with biotin, forming exosome-antibody-microbead compound in the solution, centrifuging, washing the precipitate with PBS solution, centrifuging, collecting precipitate to obtain exosome-antibody-microbead compound; the nerve cells include neurons and glial cells;
(2) Adding the exosome-antibody-microbead compound into an SDS solution, boiling for 5-10 min at 90-100 ℃, centrifugally separating, collecting supernatant, namely the SDS solution of the exosome protein from nerve cells, and detecting the protein content;
(3) Adding SDS solution of nerve cell source exosome protein into a ultrafilter tube, centrifuging to remove SDS, intercepting the nerve cell source exosome protein at the upper layer of the ultrafilter tube, adding mixed solution of urea and dithiothreitol into the nerve cell source exosome protein, incubating for 30-60 minutes at 37 ℃, and reducing disulfide bonds in the protein; then adding sulfhydryl reactive alkylating reagent for alkylation modification, then washing and centrifuging sequentially by using urea solution and ammonium bicarbonate solution, replacing a solution system, replacing a collecting pipe of a ultrafilter pipe, adding trypsin or trypsin/Lys-C protease mixture solution, carrying out enzymolysis and denaturation reaction for 12-16 hours at 37 ℃, adding trypsin or trypsin/Lys-C protease mixture solution, carrying out denaturation reaction for 4-8 hours at 37 ℃, centrifuging, and collecting the solution in the collecting pipe after washing by using ammonium bicarbonate solution; detecting the amount of peptide fragments in a sample, and carrying out vacuum centrifugal concentration to obtain peptide fragment dry powder;
(4) And redissolving the peptide fragment dry powder in a formic acid aqueous solution, and performing high performance liquid chromatography-tandem mass spectrometry analysis.
2. A god according to claim 1A method of proteomic analysis of a system-derived exosome, characterized by: in the step (1), blood is collected by using a blood collection tube containing a coagulant, and the supernatant is centrifugally separated to obtain serum; using a composition containing K 2 Blood is collected by a blood collecting tube of EDTA or sodium citrate anticoagulant, and the supernatant obtained by centrifugal separation is the plasma.
3. A method of proteomic analysis of a nervous system derived exosome according to claim 1, wherein: in the step (1), thrombin is added into plasma, the plasma is incubated for 5 to 20 minutes at the temperature of 20 to 25 ℃, and the supernatant is centrifugally separated; filtering the supernatant by using a filter membrane with the pore diameter of 0.22 mu m to obtain filtered plasma;
adding an exosome precipitation reagent into the filtered blood plasma or serum, incubating, centrifuging to remove the supernatant, and obtaining exosome precipitation;
the exosome precipitation reagent is Exoquick TM The ratio of reagent amount to plasma or serum amount was 126 μl:500 μl, incubating at 4deg.C for 1-2 hours; adding PBS solution containing protease inhibitor and phosphatase inhibitor into exosome precipitate to resuspend exosome precipitate, and repeatedly blowing to uniformly distribute exosome particles in the solution; then adding the nerve cell exosome marker biotinylation antibody, wherein the ratio of the plasma or serum dosage to the exosome marker biotinylation antibody dosage is 500 μl: incubating for 1-2 hours at 2-8 ℃ with 3-5 μl to fully combine the antibody with the exosomes of the nerve cells;
then adding streptavidin resin microbeads, wherein the ratio of the plasma or serum dosage to the streptavidin resin microbeads is 500 μl: incubating for 1-2 hours at the temperature of 2-8 ℃ under the condition of 15-25 μl and rotating uniformly so as to fully combine streptavidin and biotin; centrifuging to remove the supernatant, adding PBS solution for washing the precipitate, centrifuging to remove the supernatant, and obtaining the exosome-antibody-microbead compound.
4. A method of proteomic analysis of a neurological derived exosome according to claim 1 or 3, wherein: in the step (1), the neuroextracellular body marker is one or more of a neuroextracellular body marker L1CAM, NCAM, an astrocyte extracellular body marker GLAST and an oligodendrocyte extracellular body marker OMG.
5. A method of proteomic analysis of a nervous system derived exosome according to claim 1, wherein: in the step (2), adding the exosome-antibody-microbead compound into an SDS solution with the mass fraction of 0.1% -0.5% and the volume of 10-20 times;
protein content was measured using BCA method.
6. A method of proteomic analysis of a nervous system derived exosome according to claim 1, wherein: in the step (3), firstly, 8M urea solution is added into a 10kDa ultrafiltration tube for flushing, after centrifugation, SDS solution of the nerve cell source exosome protein is added into the 10kDa ultrafiltration tube, and SDS is removed by centrifugation;
adding 100-200 μl of mixed solution of urea and dithiothreitol into a 10kDa ultrafiltration tube, wherein the concentration of urea in the mixed solution is 8M, the concentration of dithiothreitol is 10-50 mM, and placing the mixed solution in a 37 ℃ incubator for 30-60 minutes to reduce disulfide bonds in proteins;
adding a sulfhydryl-reactive alkylating reagent into a 10kDa ultrafiltration tube to make the final concentration of the reagent be 50-100 mM, standing in a dark place at 25 ℃ for 30 minutes, and carrying out alkylation modification on protein molecules; followed by centrifugation;
adding 100-200 mu l of 8M urea solution into a 10kDa ultrafiltration tube, centrifuging, and repeating twice;
adding 100-200 mu l of 50mM ammonium bicarbonate solution into a 10kDa ultrafiltration tube, centrifuging, and repeating for three times;
the bottom collecting pipe of the 10kDa ultrafiltration pipe is replaced, and the mass ratio of the exosome protein amount to trypsin is 50: 1-20: 1 adding trypsin or a trypsin/Lys-C protease mixture solution; placing the mixture in a 37 ℃ incubator for denaturation reaction for 12-16 hours, and then carrying out denaturation reaction according to the mass ratio of the exosome protein to trypsin of 100: 1-50: 1 supplementing trypsin or a trypsin/Lys-C protease mixture, and continuously placing the mixture in a 37 ℃ incubator for denaturation reaction for 4-8 hours;
centrifuging, then supplementing 50-100 mu l of 50mM ammonium bicarbonate, centrifuging, and reserving the solution in the collecting pipe after two times of centrifuging.
7. A method of proteomic analysis of a neurological derived exosome according to claim 6 wherein: the centrifugation condition is 14000g, the centrifugation time is 20-30 minutes, and the temperature is 20-25 ℃.
8. A method of proteomic analysis of a neurological derived exosome according to claim 1 or 6, wherein: in the step (3), the sulfhydryl-reactive alkylating reagent is chloroacetamide or iodoacetamide, and the final concentration is 50-100 mM;
the addition amount of the trypsin is 1/50-1/20 of the mass ratio of the protein of the exosome of the nerve cell source; the volume of the ammonium bicarbonate solution is 100-200 mu l, and the concentration is 25-50 mM; the mass of the supplemented trypsin is 1/100-1/50 of the mass of the protein of the exosome from the nerve cell;
the amount of peptide fragments in the sample is detected using a quantitative peptide detection reagent.
9. A method of proteomic analysis of a nervous system derived exosome according to claim 1, wherein: in the step (4), the mobile phase A is 0.1% formic acid aqueous solution by volume fraction, the mobile phase B is 0.1% formic acid acetonitrile/aqueous solution by volume fraction, and the volume ratio of acetonitrile to water is 20:80, the chromatographic gradient was varied as follows: 0-5 min, and the linear gradient of the mobile phase B is 2-8%; 5min-90min, and the linear gradient of the mobile phase B is 8% -24%;90 The linear gradient of the mobile phase B is from 24% to 32% after min-110 min; 110-115 min, and the linear gradient of the mobile phase B is 32-90%; 115-125 min, wherein the linear gradient of the mobile phase B is 90-5%; 125-130 min, and maintaining the mobile phase B at 5%; the flow rate was 300nL/min.
10. A method of proteomic analysis of a nervous system derived exosome according to claim 1, wherein: in the step (4), a data independent acquisition mode is adopted in mass spectrometry, the primary mass spectrum scanning range is 350-1250m/z, the resolution is 120000, the maximum gain is controlled to be 3e6, and the maximum injection time is 60ms; the resolution of the secondary mass spectrum is 30000, the maximum gain is controlled to be 1e6, the maximum injection time is 55ms, and the collision energy is 25.5eV, 27.0eV and 30.0eV.
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