Exosome protein composition, application thereof and atrial fibrillation screening kit

Abstract

The invention provides an exosome protein composition and application thereof and an atrial fibrillation screening kit.The protein composition is composed of fibronectin and a blood coagulation factor XIII A1 chain, and preliminary research is conducted on detection of the fibronectin and the blood coagulation factor XIII A1 chain and functions in the cardiovascular aspect by means of modern biological technology and bioinformatics analysis; the exosome proteins can be applied to preparation of atrial fibrillation detection preparations or medical instruments for early intervention, and have good diagnosis and treatment efficiency and potential huge application value for atrial fibrillation.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to an exosome protein composition and application thereof and an atrial fibrillation screening kit. Background Art

[0002] Atrial fibrillation (AF) is the most common cardiac arrhythmia worldwide. The prevalence of AF has increased over the past three decades, with estimates of the number of patients reaching 60 million. AF leads to a poor prognosis, adding to the overall burden, morbidity, prevalence, and mortality associated with AF. Previous studies have shown that structural remodeling plays an important role in the development and maintenance of AF, but the exact molecular mechanisms associated with AF are unclear.

[0003] Not only is the electrophysiological mechanism of AF controversial, its underlying biochemical mechanism is also unclear. In the past few years, it has been determined that miRNAs associated with cardiac fibrosis in pericardial fluid exosomes play an important role in atrial remodeling in AF. Recent studies have found that non-coding RNAs carried by exosomes in the body fluids of patients with AF are of great significance for auxiliary diagnosis and prognostic detection of AF. It has been reported that 26 differentially expressed IncRNAs were identified in serum exosomes of patients with PEAF. However, few studies have focused on how exosomal transport of biomarkers affects the pathophysiology of AF. With the development of proteomics technology, qualitative and quantitative studies of exosomal proteins and serum exosomal protein fragments are expected to provide information on AF-induced physiological states and disease conditions, and provide valuable insights into the diagnosis and pathogenesis of arrhythmias and cancer. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an exosome protein composition.

[0005] Another technical problem to be solved by the present invention is to provide an application of the above-mentioned exosome protein composition.

[0006] Another technical problem to be solved by the present invention is to provide an atrial fibrillation screening kit.

[0007] The technical solution adopted by the present invention is:

[0008] An exosomal protein composition consisting of fibronectin and coagulation factor XIII A1 chain.

[0009] The use of the exosome protein composition as a biomarker for predicting atrial fibrillation in the preparation of atrial fibrillation detection preparations or medical devices.

[0010] Preferably, in the application of the above-mentioned exosome protein composition, the medical device is an atrial fibrillation screening kit.

[0011] An atrial fibrillation screening kit using the above exosome protein composition comprises two protein detection groups:

[0012] The first test group Including: fibronectin standard, color developer A solution, color developer B solution, stop solution, sample diluent, concentrated washing solution, enzyme labeling reagent, among which,

[0013] The concentrations of fibronectin standards were: 600, 300, 150, 75, and 37.5 μg / L;

[0014] Color developer solution A is: sodium acetate, citric acid, 30% H2O2;

[0015] Color developer solution B is: EDTA-Na, citric acid;

[0016] The stop solution is: 1 mol / L sulfuric acid;

[0017] The sample diluent was: PBS containing 0.5% BSA, concentration 0.01 M, pH = 7.2-7.4;

[0018] The concentrated washing solution was: PBS containing 0.05% Tween 20;

[0019] The enzyme labeling reagents are: HRP-labeled secondary antibody;

[0020] The second test group It includes: coagulation factor XIII A1 chain standard, color developer A solution, color developer B solution, stop solution, sample diluent, concentrated washing solution, enzyme labeling reagent, among which,

[0021] The concentrations of factor XIII A1 chain standards were: 24, 12, 6, 3, and 1.5 ng / ml;

[0022] Color developer solution A is: sodium acetate, citric acid, 30% H2O2;

[0023] Color developer solution B is: EDTA-Na, citric acid;

[0024] The stop solution is: 1 mol / L sulfuric acid;

[0025] The sample diluent was: PBS containing 0.5% BSA, concentration 0.01 M, pH = 7.2-7.4;

[0026] The concentrated washing solution was: PBS containing 0.05% Tween 20;

[0027] The enzyme-labeled reagent is: HRP-labeled secondary antibody.

[0028] The above detection group can be prepared by conventional methods.

[0029] The beneficial effects of the present invention are:

[0030] The present invention utilizes modern biological technology and bioinformatics analysis to study the proteomic changes of plasma exosomes in PEAF, understand the molecular mechanism of exosomes in AF, and conduct preliminary research on the detection of fibronectin and coagulation factor XIII A1 chain and their cardiovascular functions. Fibronectin and coagulation factor XIII A1 chain have the use of predicting persistent atrial fibrillation. These exosome proteins can be used to prepare atrial fibrillation detection preparations or medical devices for early intervention, and have good diagnostic and therapeutic efficacy for atrial fibrillation and potential huge application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a non-standard proteomics differential protein heat map that presents the clustering relationship of the relative expression amounts of differential proteins by comparing the relative expression levels of multiple differential proteins detected in different samples.

[0032] Figure 2 This is a comparison chart of the contents of fibronectin and coagulation factor XIII A1 chain detected by quantitative proteomics in patients with persistent atrial fibrillation and those without atrial fibrillation (negative coronary angiography). Figure 3 Diagram showing the roles of differentially expressed proteins in cellular pathways. Figure 4 Detection plots of differentially expressed proteins were validated in a new cohort for quantitative proteomics. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0034] Example 1

[0035] 1. Collection of Human Plasma Samples

[0036] Patients with persistent atrial fibrillation were selected as the experimental group, and patients with sinus rhythm were selected as the control group. All patients (experimental group and control group) had their blood samples collected on the morning before surgery, after fasting for more than 10 hours. 2 ml of whole blood was collected from the radial artery of each patient, and immediately centrifuged at 2500 rpm for 10 minutes to separate the upper plasma. The separated plasma was then divided into several equal parts, placed in plasma collection tubes, and frozen in a -80°C refrigerator for testing.

[0037] 2. Non-label proteomics detection

[0038] 1) Exosome extraction

[0039] 1. Will be with EV Particle Binding The EV pellet was resuspended in 20 μL of 8 M urea and 1 μL of 1 M DTT was added.

[0040] 2. Heat the sample at 50°C for 6 minutes.

[0041] 3. Alkylate samples with 6 μL of 500 mM iodoacetamide in 500 mM ammonium bicarbonate and incubate at room temperature in the dark for 10 minutes.

[0042] 4. Dilute the sample with equal amounts of water (27 μL) and a solution of 500 mM ammonium bicarbonate (27 μL).

[0043] 5. Digest the samples with trypsin (1 μL) at 37°C for 4 hours.

[0044] 6. Centrifuge the sample at 16800xg for 10 minutes at room temperature to pellet only EV pellet, while EV peptides remain in the supernatant. The supernatant was collected and frozen at -20 °C overnight to inactivate trypsin.

[0045] 7. Concentrate and purify peptides in supernatant using ZipTip: a. Wash 3 times with 20 μL of Buffer B (0.1% trifluoroacetic acid + 80% acetonitrile). b. Wash ZipTip 3 times with 20 μL of Buffer A (0.1% trifluoroacetic acid). c. Pipette sample through ZipTip and discard flow-through sample. d. Wash ZipTip 3 times with 20 μL of Buffer A (0.1% trifluoroacetic acid). e. Wash 3 times with 20 μL of Buffer B (0.1% trifluoroacetic acid + 80% acetonitrile) and collect in a clean tube.

[0046] 8. Dry the samples using a SpeedVac for 15 minutes.

[0047] 9. Resuspend the sample in 10 μL 0.1% trifluoroacetic acid and load onto the mass spectrometer.

[0048] 2) Plasma protein detection

[0049] Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS / MS) was used to detect the proteins in the two groups of mixed samples.

[0050] 3) Protein extraction

[0051] The sample was taken out from -80℃, centrifuged at 4℃, 12000g for 15min, the supernatant was transferred to a new centrifuge tube, filtered with a 0.22μM microporous membrane, and exosomes were isolated using the qEV kit produced by IZNO according to the instructions. Urea with a final concentration of 8M and protease inhibitors were added for ultrasonic lysis.

[0052] 4) UPLC-MS analysis

[0053] The peptides were dissolved in liquid chromatography mobile phase A and separated using the NanoElute ultra-high performance liquid phase system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was a solution containing 0.1% formic acid and 100% acetonitrile. The liquid phase gradient was set as follows: 0-70min, 5%-20% B; 70-84min, 20%-30% B; 84-87min, 30%-80% B; 87-90min, 80% B, and the flow rate was maintained at 450nL / min. After separation by the ultra-high performance liquid phase system, the peptides were injected into the Capillary ion source for ionization and then analyzed by the timsTOF Pro 2 (Bruker) mass spectrometer. The ion source voltage was set to 1.7kV, and the peptide parent ions and their secondary fragments were detected and analyzed using high-resolution TOF. The secondary mass spectrometer scan range was set to 100-1700. The data acquisition mode used the parallel accumulation serial fragmentation (PASEF) mode. After one primary mass spectrum was acquired, 10 secondary spectra with parent ion charge numbers in the range of 0–5 were acquired in PASEF mode, and the dynamic exclusion time of the tandem mass spectrometry scan was set to 30 s to avoid repeated scanning of parent ions.

[0054] 5) Database search

[0055] Database retrieval is a complex calculation process, which requires the use of professional mass spectrometry data analysis software for data analysis. The raw data of the mass spectrometer was imported into the search software, and the corresponding analysis parameters were set according to the experimental plan. The secondary mass spectrometry data of this experiment was retrieved using Maxquant (v1.6.15.0). Retrieval parameter settings: the database is Homo_sapiens_9606_SP_20220107.fasta (20376 sequences), and the anti-library is added to calculate the false positive rate (FDR) caused by random matching; the enzyme cutting method is set to Trypsin / P; the number of missed cutting sites is set to 2; the minimum length of the peptide is set to 7 amino acid residues; the maximum number of peptide modifications is set to 5; the mass error tolerance of the primary parent ion of First search and Mainsearch is set to 20ppm and 20ppm respectively, and the mass error tolerance of the secondary fragment ion is 20ppm. Cysteine ​​alkylation Carbamidomethyl (C) was set as a fixed modification, and the variable modification was oxidation of methionine and acetylation of the protein N-terminus. The FDR of protein identification and PSM identification was set to 1%.

[0056] 6) Data quality control analysis

[0057] After the database search is completed, the data downloaded from the mass spectrometer needs to undergo a series of quality control evaluations to ensure that the quality of the results meets the standards: including peptide length distribution, peptide number distribution, protein coverage distribution, and protein molecular weight distribution.

[0058] 7) Protein function annotation

[0059] In order to thoroughly understand the functional characteristics of different proteins, we have performed comprehensive functional annotations on the identified proteins. This includes detailed annotations in Gene Ontology (GO), protein domain, KEGG pathway, COG / KOG functional classification, subcellular localization, Reactome, WikiPathways, HallMark, and transcription factor (TF). Note: Reactome, WikiPathways, HallMark, and transcription factors are only for some species. If no relevant analysis results are displayed, it means that the data of this project cannot obtain relevant annotation information.

[0060] 8) Protein Quantification Analysis

[0061] The search results give the LFQ intensity of each protein in different samples (the result of the original intensity value of the protein after correction between samples). The LFQ intensity (1) of the protein in different samples is transformed by centering to obtain the relative quantitative value (R) of the protein in different samples. The calculation formula is as follows: where i represents the sample and j represents the protein. Rij = lij / Mean (Ij).

[0062] 9) Differential protein screening

[0063] Three or more replicates: First, select the samples to be compared, and take the ratio of the mean relative quantitative values ​​of each protein in the multiple replicate samples as the fold change (FC). For example, calculate the fold change of the protein between sample group A and sample group B. The calculation formula is as follows: Where R represents the relative quantitative value of the protein, i represents the sample, and k represents the protein. FCA / B, k = Mean(Rik, i∈A) / Mean(Rik, i∈B).

[0064] In order to determine the significance of the difference, the relative quantitative value of each protein in the comparison group samples was subjected to a T test, and the corresponding P value was calculated as a significance indicator, with a default P value of <0.05. In order to make the test data conform to the normal distribution required by the T test. Before the test, the relative quantitative value of the protein needs to be transformed by Log2. The calculation formula is as follows: Pk = T.test(Log2(Rik, i∈A), Log2(Rik, i∈B).

[0065] Repeat twice: First, select the samples to be compared, and take the mean of the relative quantitative values ​​of each protein in the two repeated comparison groups as the fold change (FC). For example, calculate the fold change of the protein between sample group A and sample group B. The calculation formula is as follows: Where R represents the relative quantitative value of the protein, i represents the sample, and k represents the protein. FCA / B, k = Mean((Rik, i∈A) / (Rik, i∈B).

[0066] In order to determine the significance of the difference, the standard coefficient of variation (CV) of each protein in the two comparison groups was calculated as a significance indicator, with the default CV < 0.1. The calculation formula is as follows:

[0067] CVk=SD(A1k / B1k, A2k / B2k) / Mean(A1k / B1k, A2k / B2k).

[0068] No duplication: First, select the samples to be compared, and take the ratio of the relative quantitative value of each protein in the sample as the fold change (FC). For example, calculate the fold change of the protein between sample A and sample B. The calculation formula is as follows: Where R represents the relative quantitative value of the protein, and k represents the protein. FCA / B, k = RAk / RBk

[0069] Through the above difference analysis, when P value is less than 0.05, the difference expression amount change exceeding 1.5 is regarded as the change threshold of significant upregulation, and less than 1 / 1.5 is regarded as the change threshold of significant downregulation.

[0070] 10) Functional classification of differentially expressed proteins

[0071] 1. Gene Ontology (GO) is an important bioinformatics analysis method and tool used to describe the various properties of genes and gene products. GO annotations are divided into three categories: biological process, cellular component and molecular function, which explain the biological role of proteins from different perspectives. We conducted differential protein classification analysis on the three major categories in GO classification.

[0072] 2. The protein in the eukaryotic tissue cells is located on various elements in the cell according to the difference of the membrane structure combined with it. Based on this, we use WolfPsort software to annotate the subcellular structure of the protein. Relative to eukaryotic cells, prokaryotic cells generally do not have intracellular membranes, there is no formed nucleus wrapped by nuclear membranes, there is no chromosome in the cell, the DNA chain is not spiralized, and is present in the cytoplasm in a free form, and there is no organelle (such as mitochondria or chloroplasts) with membranes in the cytoplasm. Based on this, we use PSORTb (v3.0) software to annotate the subcellular structure of differential proteins. The classification results are displayed in the form of pie charts and rose diagrams.

[0073] 3.COG, or Clusters of Orthologous Groups of proteins. The proteins that make up each COG are assumed to come from an ancestral protein. Orthologs refer to proteins from different species that have evolved through vertical lineages (speciation), and typically retain the same functions as the original protein. The Chinese meaning of COG is "cluster of homologous proteins." COG is divided into two categories, one is prokaryotes and the other is eukaryotes. The prokaryotic database is generally called the COG database; the eukaryotic database is generally called the KOG database. Through database comparison, we performed COG / KOG functional classification statistics on the differential proteins.

[0074] 4. The KEGG database can integrate currently known protein interaction network information, such as pathways and related complexes (Pathway database), genes and gene products (Gene database), biological complexes and related reactions (Compound and Reaction database), etc. The KEGG database divides the collected pathway information into 7 categories and performs KEGG classification on differentially expressed proteins with KEGG annotation information.

[0075] 11) Differential protein functional enrichment analysis

[0076] We conducted enrichment analysis on the differentially expressed proteins in each comparison group at five levels: GO classification, KEGG pathway, protein domain, Reactome, and WikiPathways. Reactome and WikiPathways analysis are only available for samples of some species. Fisher's exact test is used here to calculate the significant Pvalue in order to find out whether the differentially expressed proteins have a significant enrichment trend in certain functional types.

[0077] 12) Differential protein enrichment cluster analysis

[0078] After enriching the differentially expressed proteins in the comparison groups by GO classification, KEGG pathway, protein domain, Reactome, and WikiPathways, we further divided them into four parts according to the differential expression fold, called Q1 to Q4. Then, for each Q group, GO classification, KEGG pathway, protein domain, Reactome, and WikiPathways enrichment were performed, and cluster analysis was performed to find the correlation between the functions of proteins with different differential expression folds in the comparison groups.

[0079] 13) Protein interaction network analysis

[0080] The differential protein database numbers or protein sequences obtained by screening according to the difference factor of 1.5 in the comparison group were compared with the STRING (v.11.5) protein interaction network database, and the differential protein interaction relationships were extracted according to the confidence score>0.7 (high confidence). The differential protein interaction network was then visualized using the R package "networkD3" tool.

[0081] 14) Anova difference analysis

[0082] 1. KEGG pathway enrichment analysis: Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for pathway enrichment analysis of differentially expressed proteins. Fisher's exact test was used to perform pathway enrichment significance analysis on differentially expressed proteins (with the identified proteins as the background), and P value < 0.05 was considered significant.

[0083] 2. Cluster analysis: Based on the Fisher's exact test P value obtained from the enrichment analysis, the hierarchical clustering method was used to cluster the related functions in different Q groups together and draw a heatmap. The horizontal direction of the heatmap is the different Q groups, and the vertical direction is the related functions enriched by the differentially expressed proteins, such as the descriptions of GO, KEGG pathway, Protein domain, Reactome, and WikiPathways, of which Reactome and WikiPathways are only provided for samples of some species. The color blocks corresponding to the functional descriptions of different Q groups and differentially expressed proteins enrichment indicate the level of enrichment significance.

[0084] It was found through Example 1 that two exosomal proteins (FN1 and F13A1) were downregulated in patients with atrial fibrillation. FN1 plays a key role in regulating development (including growth, proliferation, migration and survival) and disease processes. Studies have shown that upregulation of FN1 in exosomes induces overexpression of COL1A1, which leads to excessive precipitation of extracellular matrix (ECM) and myocardial fibrosis, in which the P13K-Akt signaling pathway is involved, and cellular electrophysiological abnormalities caused by fibrous myocardium are related to the pathogenesis of atrial fibrillation. FXIII-A is a glutamine aminotransferase that can be externalized from cells by unknown mechanisms, and FXIII-A plays a vital role in regulating wound healing and tissue repair. Together with coagulation factor 7 and tissue factor, FXIII-A provides a hemostatic barrier to reduce extravasation of cardiac vessels under the mechanical stress of beating and the subsequent development of myocardial fibrosis, thereby reducing the possibility of AF. In the present application, two metabolic pathways, the HIF signaling pathway and glycerolipid metabolism, are associated with the occurrence of AF.

[0085] Example 2

[0086] Quantitative proteomics validation based on fibronectin and coagulation factor XIII A1 chain kit

[0087] Quantitative proteomics was used to validate fibronectin and factor XIII A1 chain in freshly collected plasma samples.

[0088] A protein composition atrial fibrillation screening kit is a detection group comprising the above two proteins:

[0089] The first test group Including: fibronectin standard, color developer A solution: sodium acetate, citric acid, 30% H2O2. Color developer B solution: EDTA-Na, citric acid. Stop solution (1mol / L sulfuric acid), sample diluent (PBS containing 0.5% BSA, concentration 0.01M, PH=7.2-7.4), concentrated washing solution (PBS containing 0.05% Tween 20), enzyme labeling reagent (HRP-labeled secondary antibody) Among them,

[0090] The standard concentrations are: 600, 300, 150, 75, 37.5 μg / L;

[0091] The second test group Including: coagulation factor XIII A1 chain standard, color developer A solution: sodium acetate, citric acid, 30% H202. Color developer B solution: EDTA-Na, citric acid. Stop solution (1mol / L sulfuric acid), sample diluent (PBS containing 0.5% BSA, concentration 0.01M, PH=7.2-7.4), concentrated washing solution (PBS containing 0.05% Tween 20), enzyme labeling reagent (HRP-labeled secondary antibody) Among them,

[0092] The concentrations of the standard substances are: 24, 12, 6, 3, and 1.5 ng / ml.

[0093] The specific operation method of the above-mentioned atrial fibrillation screening kit is as follows:

[0094] 1) Preparation of fibronectin standard

[0095] The standard is set to 10 wells with 5 concentration points, and each concentration is set to parallel wells. 50 microliters of different concentrations of standard are added. The blank well is set to 1 well and 50 microliters of distilled water are added (the blank control well does not add samples and enzyme-labeled reagents, and the rest of the steps are the same). For the sample wells to be tested, 40 μl of sample diluent is first added to the sample wells to be tested on the enzyme-labeled coated plate, and then 10 μl of the sample to be tested is added (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix; S0 is the sample diluent, and the mixture of the 6 concentration gradients of the standard is as shown in Table 1:

[0096] Table 1

[0097] serial number S5 S4 S3 S2 S1 S0 μg / L 600 300 150 75 37.5 0

[0098] 2) Preparation of Factor XIII A1 Chain Standard

[0099] The standard is set with 10 wells at 5 concentration points, and each concentration is set with parallel wells. 50 μl of standard of different concentrations is added. The blank well is set with 1 well and 50 μl of distilled water is added (the blank control well does not add sample and enzyme labeling reagent, and the rest of the steps are the same). For the wells of the samples to be tested, 40 μl of sample diluent is first added to the wells of the samples to be tested on the enzyme-labeled plate, and then 10 μl of the sample to be tested is added (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix. S0 is the sample diluent, and the mixture of the 6 concentration gradients of the standard is as shown in Table 2:

[0100] Table 2

[0101]

[0102]

[0103] 3) Exosome extraction

[0104] 1. Will be with EV Particle Binding EV pellets were resuspended in 20 μL of 8 M urea, and 1 μL of 1 M DTT was added;

[0105] 2. Heat the sample at 50°C for 6 minutes;

[0106] 3. Alkylate samples with 6 μL of 500 mM iodoacetamide in 500 mM ammonium bicarbonate and incubate at room temperature in the dark for 10 minutes;

[0107] 4. Dilute the sample with equal amounts of water (27 μL) and 500 mM ammonium bicarbonate (27 μL).

[0108] 5. Digest the sample with trypsin (1 μL) at 37°C for 4 h;

[0109] 6. Centrifuge the sample at 16800xg for 10 minutes at room temperature to pellet only EV pellet, while EV peptides remain in the supernatant; the supernatant was collected and frozen at -20 °C overnight to inactivate trypsin;

[0110] 7. Concentrate and purify peptides in the supernatant using ZipTip: a. Use 20 μL buffer B (0.1% trifluoroacetic acid + 80% acetonitrile) 3 times; b. Wash ZipTip 3 times with 20 μL buffer A (0.1% trifluoroacetic acid); c. Pipette the sample through ZipTip and discard the flow-through sample; d. Wash ZipTip 3 times with 20 μL buffer A (0.1% trifluoroacetic acid); e. Use 20 μL buffer B (0.1% trifluoroacetic acid + 80% acetonitrile) 3 times and collect in a clean tube;

[0111] 8. Dry the sample using SpeedVac for 15 minutes;

[0112] 9. Resuspend the sample in 10 μL 0.1% trifluoroacetic acid and load it onto the mass spectrometer;

[0113] 4) Protein extraction

[0114] 1. Remove the sample from -80℃ and centrifuge at 12000g for 15min at 4℃;

[0115] 2. The supernatant was transferred to a new centrifuge tube, filtered through a 0.22 μM microporous membrane, and exosomes were isolated using the qEV kit produced by IZNO according to the instructions;

[0116] 3. Add Urea with a final concentration of 8 M and protease inhibitors for ultrasonic lysis;

[0117] 5) Enzymatic hydrolysis with pancreatic enzymes

[0118] Take an equal amount of each sample protein for enzymatic hydrolysis, adjust the volume to the same with lysis buffer, add dithiothreitol (DTT) to a final concentration of 5mM, and reduce at 56°C for 30min; then add iodoacetamide (IAM) to a final concentration of 11mM, and incubate at room temperature in the dark for 15min; transfer the alkylated sample to an ultrafiltration tube, centrifuge at 12000g for 20min at room temperature, replace with 8M urea three times, and then replace urea with replacement buffer three times, add trypsin at a ratio of 1:50 (protease: protein, m / m), and enzymatic hydrolysis overnight; centrifuge at 12000g for 10min at room temperature to recover peptides, and then use ultrapure water to recover peptides once, and combine the two peptide solutions;

[0119] 6) Liquid chromatography-mass spectrometry analysis

[0120] The peptides were dissolved in liquid chromatography mobile phase A and separated using the NanoElute ultra-high performance liquid phase system; mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was a solution containing 0.1% formic acid and 100% acetonitrile; the liquid phase gradient was set as follows: 0-70min, 5%-20% B; 70-84min, 20%-30% B; 84-87min, 30%-80% B; 87-90min, 80% B, and the flow rate was maintained at 450nL / min; the peptides were separated by the ultra-high performance liquid phase system and injected into the Capillary ion source for ionization and then into the timsTOF Pro 2 (Bruker) mass spectrometer for analysis; the ion source voltage was set to 1.7 kV, and the peptide parent ion and its secondary fragments were detected and analyzed using high-resolution TOF; the secondary mass spectrometer scanning range was set to 100-1700; the data acquisition mode used the parallel accumulation serial fragmentation (PASEF) mode; after one primary mass spectrum acquisition, 10 PASEF modes were used to acquire the secondary spectra with parent ion charge numbers in the range of 0-5, and the dynamic exclusion time of the tandem mass spectrometer scan was set to 30 s to avoid repeated scanning of the parent ion;

[0121] 7) Database Search

[0122] The secondary mass spectrometry data were searched using Maxquant (v1.6.15.0); the search parameters were set as follows: the database was Homo_sapiens_9606_SP_20220107.fasta (20376 sequences), a reverse library was added to calculate the false positive rate (FDR) caused by random matching, and a common contamination library was added to the database to eliminate the influence of contaminating proteins in the identification results; the enzyme cleavage method was set to Trypsin / P; the number of missed cleavage sites was set to 2; the minimum length of the peptide was set to 7 amino acid residues; the maximum number of peptide modifications was set to 5; the mass error tolerance of the primary precursor ion of the First search and Main search was set to 20ppm and 4.5ppm, respectively, and the mass error tolerance of the secondary fragment ion was 20ppm; cysteine ​​alkylation Carbamidomethyl (C) was set as a fixed modification, and the variable modifications were oxidation of methionine and acetylation of the protein N-terminus; the FDR of protein identification and PSM identification was set to 1%;

[0123] 8) Data processing

[0124] The data were processed using Skyline 21.2. The peptide parameters were as follows: the protease was set to Trypsin [KR / P], the maximum number of missed cleavage sites was set to 0, the peptide length was set to 7-25 amino acid residues, and cysteine ​​alkylation was set to a fixed modification; Transition parameters: the parent ion charge was set to 2, 3, the daughter ion charge was set to 1, and the ion type was set to b, y; the fragment ions were selected from the third to the last, and the mass error tolerance of the ion match was set to 0.02 Da.

[0125] According to the screening of differentially expressed proteins between patients with atrial fibrillation and patients with normal heart rhythm by non-standard proteomics in Example 1, in order to describe all detected differentially expressed proteins, we classified the detected different proteins by subcellular localization and GO function, see Figure 1 GO enrichment analysis (including biological process, cellular component and molecular function categories) and KEGG pathway analysis, see Figure 2 Then, the differentially expressed proteins were finally screened based on their clinical importance, relevance to atrial fibrillation, and role in cellular pathways related to atrial fibrillation. Figure 3 Quantitative proteomics using ultra-high performance liquid chromatography-mass spectrometry was used to detect differentially expressed proteins (DPs) validated in the new cohort. The ROC method showed that the AUC (area under the curve) was greater than 0.8 and the significance was less than 0.05, indicating that the two proteins had very high sensitivity and specificity, see Figure 4 FN1 and F13A1 have the potential to be further developed as specific potential biomarkers of PEAF. The results are consistent with those in Example 1.

[0126] The above-described embodiments are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineers and technicians in the field should all fall within the protection scope of the present invention.

Claims

1. An exosome protein composition, characterized in that: It is composed of fibronectin and coagulation factor XIIIA1 chains.

2. Use of the exosome protein composition according to claim 1 as a biomarker for predicting atrial fibrillation in the preparation of atrial fibrillation detection preparations or medical devices.

3. The use of the exosome protein composition according to claim 2, characterized in that: The medical device is an atrial fibrillation screening kit.

4. A kit for screening atrial fibrillation using the exosome protein composition of claim 1, characterized in that: Contains 2 protein detection panels: The first test group Including: fibronectin standard, color developer A solution, color developer B solution, stop solution, sample diluent, concentrated washing solution, enzyme labeling reagent, among which, The concentrations of fibronectin standards were: 600, 300, 150, 75, and 37.5 μg / L; Color developer solution A is: sodium acetate, citric acid, 30% H2O2; Color developer solution B is: EDTA-Na, citric acid; The stop solution is: 1 mol / L sulfuric acid; The sample diluent was: PBS containing 0.5% BSA, concentration 0.01 M, pH = 7.2-7.4; The concentrated washing solution was: PBS containing 0.05% Tween 20; The enzyme labeling reagents are: HRP-labeled secondary antibody; The second test group It includes: coagulation factor XIII A1 chain standard, color developer A solution, color developer B solution, stop solution, sample diluent, concentrated washing solution, enzyme labeling reagent, among which, The concentrations of factor XIII A1 chain standards were: 24, 12, 6, 3, and 1.5 ng / ml; Color developer solution A is: sodium acetate, citric acid, 30% H2O2; Color developer solution B is: EDTA-Na, citric acid; The stop solution is: 1 mol / L sulfuric acid; The sample diluent was: PBS containing 0.5% BSA, concentration 0.01 M, pH = 7.2-7.4; The concentrated washing solution was: PBS containing 0.05% Tween 20; The enzyme-labeled reagent is: HRP-labeled secondary antibody.

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