CN114137100A - Exosome analysis method based on SF - Google Patents
Exosome analysis method based on SF Download PDFInfo
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- CN114137100A CN114137100A CN202111263995.7A CN202111263995A CN114137100A CN 114137100 A CN114137100 A CN 114137100A CN 202111263995 A CN202111263995 A CN 202111263995A CN 114137100 A CN114137100 A CN 114137100A
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Abstract
The invention provides an SF-based exosome analysis method, which belongs to the field of biotechnology detection, uses a surfactant capable of decomposing under an acidic condition in a sample protein extraction process to extract protein, and then performs liquid chromatography-tandem mass spectrometry on the extracted protein.
Description
Technical Field
The invention belongs to the field of biotechnology detection, and particularly relates to an exosome modification proteomics analysis method based on SF proteolysis surfactant sample preparation enzymolysis.
Background
Three scientists who discover the transport regulation and control mechanism of vesicles (exosomes) in cells are highlighted in the physiology or medical reward of nobel in 2013, the recognization of exosome research is also triggered, and the method is widely applied to the fields of disease biomarker screening, metabolic disease research, tumor metastasis mechanism, immune regulation and the like. In recent years, with the continuous and deep research of exosome, the application of the exosome relates to the fields of tumor treatment, medical foundation and immunity, and parasite; clinical studies have been directed to the cardiovascular system, endocrine-metabolic system, and the like.
The mainstream view at present holds that the production process of exosomes is that cell membranes are invaginated to form endosomes (endosomes), then multivesicular bodies (MVBs) are formed, and finally the exosomes are secreted to the extracellular space to form exosomes. Exosomes carry important information of the blast's various proteins, lipids, DNA and RNA. .
The exosome is extracted by differential centrifugation, density gradient centrifugation, ultrafiltration, polymer precipitation, immune separation, isolation screening, size exclusion chromatography, etc. Due to the limited extraction method and sample size, the amount of exosomes is in most cases small. Resulting in limited enzymatic methods for proteomic detection of the patch.
Currently, the commonly used methods of proteolysis are SP3, Filter-air Sample Preparation and in-solution proteolysis. The SP3 enzymolysis method has large loss for a small amount of samples, and the level of peptide fragments is inaccurate, so that the method is not beneficial to subsequent omics analysis. FASP enzymolysis steps are tedious, long in time consumption, large in sample loss and not beneficial to small samples. The enzyme digestion in the solution limits the components of the protein solution, and common denaturants (such as SDS, urea and guanidine hydrochloride) can affect the activities of trypsin and Lys C so as to affect the enzymolysis effect, so that the novel patent technology of the SF-based proteolytic surfactant sample preparation enzymolysis method has great significance for the analysis of the exosome protein.
Disclosure of Invention
Based on the problems in the prior art, the invention provides an SF-based exosome analysis method, which uses a surfactant capable of decomposing under an acidic condition in a sample protein extraction process to extract protein, and then performs liquid chromatography-tandem mass spectrometry on the extracted protein.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
an SF-based exosome analysis method is characterized in that an SF proteolytic enzyme surfactant capable of being decomposed under an acidic condition is added in an exosome protein extraction process, the SF proteolytic enzyme surfactant is decomposed after exosome protein extraction is finished, and finally, liquid chromatography-tandem mass spectrometry is used for analyzing exosome protein.
According to the above scheme, the SF-based exosome analysis method comprises the following detailed steps:
step S1, adding SF proteolytic surfactant into the blood exosome sample, wherein the final concentration of the SF proteolytic surfactant is 20%;
step S2, crushing the exosome sample in the step S1, centrifuging after crushing, and taking supernate, namely exosome protein;
step S3, taking the exosome protein to carry out reduction treatment and alkylation treatment in sequence;
step S4, adding NH after alkylation treatment4HCO3A solution that reduces the concentration of SF proteolytic surfactant in the mixture to 2%;
step S5, carrying out enzymolysis on the exosome protein, adding TFA after the enzymolysis is finished, and acidifying until the pH value is less than 3;
and step S6, performing freeze drying on the mixture finally obtained in the step S5, redissolving the mixture by using FA, measuring an OD value, and then loading the mixture for liquid chromatography-tandem mass spectrometry detection analysis.
According to the scheme, in the step S2, ultrasonic crushing is carried out on exosomes, an ultrasonic crusher is used, the power is set to be 80W, the ultrasonic time is 10S, the intermittent time is 15S, and the working times are 10 times; after the exosomes are crushed, centrifuging for 20min at 25 ℃ and 20000g, and taking the supernatant.
According to the above scheme, the step S3 further comprises a step S31, and the BCA quantification method is used to perform quantitative analysis on the exosome protein obtained in the step S2.
According to the above scheme, the reduction treatment in step S3 is to add Dithiothreitol (DTT) to the exosome protein, shake at 800rpm, and incubate at room temperature for 1 h; the alkylation treatment is performed by adding Iodoacetamide (IAA), shaking at 800rpm, and incubating for 15min at room temperature in the dark.
According to the scheme, the enzymolysis in the step S5 is to add intracellular protease (Lys-C) firstly, shake for 1min at 800rpm, incubate for 2h at 37 ℃; then adding trypsin, and reacting for 8h at 37 ℃.
According to the above scheme, in the step S6, the conditions of the liquid chromatography are:
chromatography column 25cm x 75 μm, packing c18 1.9 um; mobile phase A: 0.1% aqueous formic acid; mobile phase B: 0.1% acetonitrile formic acid water solution, the concentration of acetonitrile is 84%; mobile phase A + mobile phase B is 100%; flow rate: 300 nl/min; the liquid phase separation gradient was as follows:
0 min-70 min, and the linear gradient of the b phase is from 2% to 22%;
70 min-78 min, the linear gradient of the b phase is from 22% to 37%;
78 min-83 min, linear gradient of b phase from 37% to 95%;
the linear gradient of the phase b is maintained from 95% to 90min from 83min to 90 min.
According to the above scheme, in step S6, the mass spectrum conditions are: adopting a positive ion scanning mode, analyzing the time length: 90min, parent ion scanning range: 100-1700m/z, integration time 100ms, ion mobility from 0.6 to 1.6Vs/cm2, single cycle time 1.16s, DIA mode.
The invention has the beneficial effects that:
1. and (3) realizing proteomic analysis of a small amount of clinical sample exosomes by adopting an in-solution enzymolysis method without desalting.
2. The SF proteolysis surfactant is applied to the preparation of a sample of exosome proteomics, a new preparation method is provided for the preparation of a protein sample, the peptide recovery rate of the protein extracted by the method in the enzymolysis process in solution, the sample repeatability and other evaluation indexes are not inferior to those of the traditional proteolysis method, and meanwhile, the enzymolysis operation can be automated, and the flux is improved.
Drawings
FIG. 1 is a graph of the results of the number of peptide fragments of proteins repeatedly identified in three experiments in the examples.
FIG. 2 is a venn diagram of the protein repeatedly identified in three sets of experiments in the example.
FIG. 3: in the embodiment, the three sets of experimental repeated positive ion mode TIC graphs are results of the embodiment, and can change according to the result of each detection and analysis, that is, characters in the graphs are irrelevant to whether the detection method provided by the invention can be repeatedly implemented, and the characters in the graphs are unclear, so that the detection method provided by the invention can be repeatedly implemented by a person skilled in the art.
Detailed Description
The technical solution of the present invention will be described below with reference to the specific embodiments and the accompanying drawings.
An SF-based exosome analysis method is characterized in that an SF proteolytic surfactant capable of being decomposed under acidic conditions is added in an exosome protein extraction process, the SF proteolytic surfactant is decomposed after exosome protein extraction is completed, and finally, the exosome protein is analyzed by using liquid chromatography-tandem mass spectrometry, and the method comprises the following detailed steps:
step S1, adding SF proteolytic surfactant into the blood exosome sample, wherein the final concentration of the SF proteolytic surfactant is 20%;
step S2, carrying out ultrasonic crushing on the exosome sample in the step S1, firstly, starting a power supply of an ultrasonic crusher, setting the power to be 80W, the ultrasonic time to be 10S, the intermittent time to be 15S and the working times to be 10 times, rinsing an ultrasonic probe by alcohol, wiping the probe by dust-free paper, inserting the probe into the sample to a position which is about 0.5cm away from the bottom of the tube, fixing the sample tube, and pressing a working reset button to start ultrasonic crushing; crushing exosome, centrifuging for 20min at 25 ℃ of 20000g, and taking supernatant, namely exosome protein;
step S3:
step S31, performing quantitative analysis on the exosome protein obtained in step S2 by BCA quantification.
Step S32, taking 30ug of exosome protein, fixing the volume of a sample to 35ul, adding a proper amount of 1M DTT to a final concentration of 20mM, shaking at 800rpm, and incubating for 1h at room temperature; then adding a proper amount of 1M IAA to a final concentration of 50mM, shaking at 800rpm, and incubating for 15min at room temperature in a dark place.
Step S4, adding 450ul 100mM NH after alkylation treatment4HCO3A solution that reduces the concentration of SF proteolytic surfactant in the mixture to 2%;
step S5, carrying out enzymolysis on the exosome protein, namely adding 0.3ug Lys-C (the adding ratio is 1: 100) firstly, shaking at 800rpm for 1min, and incubating for 2h at 37 ℃; adding 0.3-0.6 ug of trypsin (the adding ratio is 1: 100-1: 50), reacting for 8h at 37 ℃, adding 10% TFA after enzymolysis is finished, acidifying until the pH is less than 3, stopping enzyme digestion reaction, and decomposing SF;
and step S6, freeze-drying the mixture finally obtained in the step S5, taking 200ng of peptide fragments, re-dissolving the peptide fragments to 32ul by using 0.1% FA, measuring OD values, and then loading the sample for liquid chromatography-tandem mass spectrometry detection and analysis.
The conditions of the liquid chromatography were:
chromatography column 25cm x 75 μm, packing c18 1.9 um; mobile phase A: 0.1% aqueous formic acid; mobile phase B: 0.1% acetonitrile formic acid water solution, the concentration of acetonitrile is 84%; mobile phase A + mobile phase B is 100%; flow rate: 300 nl/min; the liquid phase separation gradient was as follows:
0 min-70 min, and the linear gradient of the b phase is from 2% to 22%;
70 min-78 min, the linear gradient of the b phase is from 22% to 37%;
78 min-83 min, linear gradient of b phase from 37% to 95%;
the linear gradient of the phase b is maintained from 95% to 90min from 83min to 90 min.
The conditions of the mass spectrum were: adopting a positive ion scanning mode, analyzing the time length: 90min, parent ion scanning range: 100-1700m/z, integration time 100ms, ion mobility from 0.6 to 1.6Vs/cm2, single cycle time 1.16s, DIA mode.
Library check and qualitative and quantitative analysis were performed using thermoproteomediscover software (version number 2.4) and spectronaut software (spectronaut _ 15.1.210713.50606).
The experiment is carried out in three times according to the embodiment, after the original mass spectrum data of the experiment is analyzed and identified by software, the result is shown in fig. 1, fig. 2 and fig. 3, as can be seen from fig. 1, the proteins identified by the three repeated experiments are all kept at about 1300, which shows that the enzymolysis effect of the method is good, a wien diagram (venndiagram) is made by the proteins repeatedly identified by the three repeated experiments, as can be seen from fig. 2, the protein identified by the three repeated experiments is up to 99%, which shows that the repeatability of the method can meet the requirement of proteomics analysis.
The present invention is provided by the above embodiments only for illustrating and not limiting the technical solutions of the present invention, and although the above embodiments describe the present invention in detail, those skilled in the art should understand that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and any modifications and equivalents may fall within the scope of the claims.
Claims (8)
1. An SF-based exosome analysis method is characterized in that an SF proteolysis surfactant capable of being decomposed under an acidic condition is added in an exosome protein extraction process, the SF proteolysis surfactant is decomposed after exosome protein extraction is completed, and finally, liquid chromatography-tandem mass spectrometry is used for analyzing exosome protein.
2. An SF-based exosome analysis method according to claim 1, characterized in that it comprises the following detailed steps:
step S1, adding SF proteolytic surfactant into the blood exosome sample, wherein the final concentration of the SF proteolytic surfactant is 20%;
step S2, crushing the exosome sample in the step S1, centrifuging after crushing, and taking supernate, namely exosome protein;
step S3, taking the exosome protein to carry out reduction treatment and alkylation treatment in sequence;
step S4, adding NH after alkylation treatment4HCO3A solution that reduces the concentration of SF proteolytic surfactant in the mixture to 2%;
step S5, carrying out enzymolysis on the exosome protein, adding TFA after the enzymolysis is finished, and acidifying until the pH value is less than 3;
and step S6, performing freeze drying on the mixture finally obtained in the step S5, redissolving the mixture by using FA, measuring an OD value, and then loading the mixture for liquid chromatography-tandem mass spectrometry detection analysis.
3. The SF-based exosome analysis method according to claim 2, wherein in step S2, ultrasonication is applied to exosomes, ultrasonication is applied, power is set to 80W, ultrasonication time is set to 10S, pause time is set to 15S, and number of operations is set to 10; after the exosomes are crushed, centrifuging for 20min at 25 ℃ and 20000g, and taking the supernatant.
4. The SF-based exosome analysis method according to claim 3, wherein said step S3 further comprises step S31, using BCA quantification method to perform quantitative analysis on exosome protein obtained in step S2.
5. The SF-based exosome analysis method according to claim 4, wherein said reducing treatment in step S3 is adding dithiothreitol to exosome protein, shaking at 800rpm, incubating for 1h at room temperature; the alkylation treatment is to add iodoacetamide, shake at 800rpm, and incubate for 15min at room temperature in the dark.
6. The SF-based exosome analysis method according to claim 5, wherein in step S5, the enzymatic hydrolysis is adding intracellular protease, shaking at 800rpm for 1min, and incubating at 37 ℃ for 2 h; then adding trypsin, and reacting for 8h at 37 ℃.
7. An SF-based exosome analysis method according to claim 6, characterized in that in step S6, liquid chromatography conditions are:
chromatography column 25cm x 75 μm, packing c18 1.9 um; mobile phase A: 0.1% aqueous formic acid; mobile phase B: 0.1% acetonitrile formic acid water solution, the concentration of acetonitrile is 84%; mobile phase A + mobile phase B is 100%; flow rate: 300 nl/min; the liquid phase separation gradient was as follows:
0 min-70 min, and the linear gradient of the b phase is from 2% to 22%;
70 min-78 min, the linear gradient of the b phase is from 22% to 37%;
78 min-83 min, linear gradient of b phase from 37% to 95%;
the linear gradient of the phase b is maintained from 95% to 90min from 83min to 90 min.
8. An SF-based exosome analysis method according to claim 7, wherein in step S6, the mass spectrometry conditions are: adopting a positive ion scanning mode, analyzing the time length: 90min, parent ion scanning range: 100-1700m/z, integration time 100ms, ion mobility from 0.6 to 1.6Vs/cm2, single cycle time 1.16s, DIA mode.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115197895A (en) * | 2022-07-28 | 2022-10-18 | 中国人民解放军军事科学院军事医学研究院 | Based on TiO 2 Exosomes omics series extraction technology and application thereof |
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