CN112326970A - Protein composition, application thereof and new atrial fibrillation screening kit - Google Patents

Protein composition, application thereof and new atrial fibrillation screening kit Download PDF

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CN112326970A
CN112326970A CN202010959218.5A CN202010959218A CN112326970A CN 112326970 A CN112326970 A CN 112326970A CN 202010959218 A CN202010959218 A CN 202010959218A CN 112326970 A CN112326970 A CN 112326970A
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antibody
diluent
biotin
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何国伟
李新亚
侯海涛
陈焕新
杨沁
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TEDA INTERNATIONAL CARDIOVASCULAR HOSPITAL
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Abstract

The invention provides a protein composition and application thereof and a new atrial fibrillation screening kit, the composition consists of a cholesteryl ester transfer protein antibody, a translipoprotein-C3 antibody, a phospholipid transfer protein antibody, a glutathione peroxidase antibody and a adiponectin, the detection and the function of the cholesteryl ester transfer protein, the translipoprotein-C3, the phospholipid transfer protein, the glutathione peroxidase and the adiponectin are preliminarily researched by utilizing the modern biological technology and the bioinformatics analysis, the cholesteryl ester transfer protein, the glutathione peroxidase and the adiponectin with high concentration, the transesterified lipoprotein-C3 and the phospholipid transfer protein with low concentration have the application of predicting new atrial fibrillation, the method can be applied to the preparation of detection preparations or medical devices for early intervention, and has better evaluation efficiency and potential huge application value on new atrial fibrillation.

Description

Protein composition, application thereof and new atrial fibrillation screening kit
Technical Field
The invention relates to the field of molecular biology, in particular to a protein composition, application thereof and a new atrial fibrillation screening kit.
Background
Atrial Fibrillation (AF) is the most common sustained arrhythmia in the clinic, with 30-50% of post-CABG patients having new atrial fibrillation (POAF). At atrial fibrillation, the atria switch from a regular sinus rhythm to an unordered conduction pattern that has three major effects on the patient: 1. palpitation, chest distress, anxiety, and other symptoms; 2. end-diastolic volume of the ventricles decreases, impairing cardiac function; 3. atrial thrombosis, embolism. Post-operative atrial fibrillation increases mortality following cardiac surgery.
Although there are many treatments including drug prophylaxis, the incidence of atrial fibrillation after adult cardiac surgery is still high and also increases with the average age of the population. Because no effective treatment means for atrial fibrillation exists at present, the generation and maintenance mechanism of atrial fibrillation is clarified, a new effective target point is provided for the research and development of effective medicaments of atrial fibrillation, and the method has great clinical significance. Although the mechanisms of electrical, structural, contractile remodeling of atrial fibrillation have been advanced, more profound mechanisms remain to be studied.
The protein is a basic unit for performing cell functions, proteomics is large-scale gene expression research at a protein level, and comprises structural component proteins, protein interaction and protein expression, so that the protein posttranscriptional modification is clarified, the change in functional metabolism of cells can be reflected better than that of the proteomics, and the research of proteomics not only can provide a material basis for the life activity rule, but also can provide theoretical basis and solution for the clarification and attack of various disease mechanisms. Through comparative analysis of proteome between normal individuals and pathological individuals, we can find some 'disease-specific protein molecules', which can become molecular targets for new drug design, or can provide molecular markers for early diagnosis of diseases. The current study of proteins in post-CABG atrial fibrillation is relatively rare and these proteins can be roughly classified into: 1. a heat shock protein; 2. an antioxidant protein; 3. a cytoskeletal protein; 4. a contractile protein; 5. a metabolic-related protein. More accurate and more comprehensive protein is searched, theoretical basis can be provided for mechanism development and maintenance of atrial fibrillation after coronary heart disease operation, and meaningful clinical biological markers and drug treatment targets can be provided for atrial fibrillation.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a composition of a plurality of proteins in blood.
Another technical problem to be solved by the present invention is to provide the use of the above-mentioned polyprotein composition.
Another technical problem to be solved by the present invention is to provide a new atrial fibrillation screening kit.
The technical scheme adopted by the invention is as follows:
a blood polyprotein composition comprises cholesteryl ester transfer protein antibody, lipoprotein-C3 antibody, phospholipid transfer protein antibody, glutathione peroxidase antibody, and titin.
The application of the multi-protein composition in preparing a novel atrial fibrillation detection preparation or a medical appliance.
The multi-protein composition can be used as a biomarker for predicting new atrial fibrillation.
Preferably, the medical apparatus is a new atrial fibrillation screening kit.
An atrial fibrillation screening kit applying the multi-protein composition comprises 5 protein detection groups:
first detection groupThe method comprises the following steps: multi-well plate coated with cholesteryl ester transfer protein antibody, cholesteryl ester transfer protein pure product as standard substance, biotin-labeled antibody, horseradish peroxidase-labeled avidin, raw materialAn avidin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the cholesteryl ester transfer protein standard substance is
0,0.78,1.56,3.12,6.25,12.5,25,50ng/ml；
The biotin labeled antibody is a biotinylated anti-cholesteryl ester transfer protein antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M phosphate buffer pH 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M phosphate buffer pH7.20.05% thimerosal;
the sample diluent is 86% of sodium chloride, 4.5% of disodium hydrogen phosphate, 3.5% of sodium dihydrogen phosphate, 5% of goat serum, 1% of Proclin-300 and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid;
the above-mentioned materials can be prepared by conventional method.
The specific operation method of the content comprises the following steps:
1) taking an isolated plasma sample, melting overnight at 4 ℃ before detection, centrifuging at 2500rpm for 10min, diluting with a sample diluent by 1:200 times, and detecting;
2) preparing a standard substance: centrifuging 2 parts of standard substance for 30S, fully dissolving the standard substance with 1ml of sample diluent (S8), repeatedly blowing and beating for at least 5 times by using a pipette, taking 7 centrifuge tubes (S1-S7) of 1.5ml, adding 250 mu l of sample diluent into each centrifuge tube, sucking 250 mu l of standard substance S8 into the first centrifuge tube (S7), lightly blowing and beating, and uniformly mixing; sucking 250. mu.l from S7 into a second EP tube (S6), and gently pipetting and mixing; performing multiple dilution on the standard product by analogy; s1 is a sample diluent;
3) the method comprises the following operation steps:
a) aiming at the set standard substance hole and the set sample hole to be detected, 100 mu l of the standard substance or the sample to be detected is respectively added into each hole, the mixture is gently shaken and uniformly mixed, a plate paste is pasted, and the mixture is incubated at 37 ℃ for 2.5 hours;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 1.5 hours at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a sticking film, and incubating for 1 hour at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 20 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction;
4) data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample.
Second detection groupThe method comprises the following steps: a perforated plate coated with a translipoprotein-C3 antibody, a translipoprotein-C3 pure product as a standard substance, a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the standard product of the translipoprotein-C3 is 0,15.6, 31.25, 62.5,125,250,500 and 1000 ng/ml;
the biotin labeled antibody is a biotinylated anti-translipoprotein-C3 antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M phosphate buffer pH 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M phosphate buffer pH7.20.05% thimerosal;
the sample diluent is 85% sodium chloride, 5% disodium hydrogen phosphate, 4% sodium dihydrogen phosphate, 5% goat serum, 1% Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid;
the above-mentioned materials can be prepared by conventional method.
The specific operation method of the content comprises the following steps:
1) the plasma sample is melted overnight at 4 ℃ before detection, centrifuged at 2500rpm for 10min, diluted 1:400 times with the sample diluent and detected.
2) Preparing a standard substance: after 2 portions of the standard were centrifuged for 30 seconds, the standard was sufficiently dissolved with 1ml of the sample diluent (S8), repeatedly pipetting at least 5 times using a pipette, taking 7 sample centrifuge tubes of 1.5ml (S1-S7), adding 150 μ l of each sample diluent, sucking 150 μ l of the standard S8 into the first centrifuge tube (S7), gently pipetting and mixing. Pipette 150. mu.l from S7 into a second EP tube (S6) and gently pipette the mixture. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) and setting a standard sample hole and a sample hole to be detected. Adding 50 mul of standard substance or sample to be tested with corresponding concentration into each hole;
b) immediately adding 50 mul of horse radish peroxidase labeled avidin, adding 50 mul of biotin labeled antibody according to the same sequence, slightly shaking and uniformly mixing, attaching a plate paste, and incubating for 50 minutes at 37 ℃;
c) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
d) adding 100 mul of substrate solution into each hole, and developing for 15 minutes at 37 ℃ in a dark place;
e) adding 50 mul of stop solution into each hole to stop the reaction;
f) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction.
4) Data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample.
Third detection groupThe method comprises the following steps: a porous plate coated with a phospholipid transfer protein antibody, a phospholipid transfer protein pure product serving as a standard substance, a biotin labeled antibody, horseradish peroxidase labeled avidin, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the phospholipid transfer protein standard substance is
0,2.34,4.68,9.375,18.75,37.5,75,150ng/ml；
The biotin labeled antibody is a biotinylated anti-phospholipid transfer protein antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the washing solution comprises: NaCl 9.0g, Tween 206 ml 0.5%, distilled water to 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The above-mentioned materials can be prepared by conventional method.
The specific operation method of the content comprises the following steps:
1) the plasma sample is melted overnight at 4 ℃ before detection, centrifuged at 2500rpm for 10min, diluted 1:200 times with the sample diluent and detected.
2) Preparing a standard substance: centrifuging 2 parts of standard substance for 30S, fully dissolving the standard substance with 1ml of sample diluent (S8), repeatedly blowing and beating for at least 5 times by using a pipette, taking 7 centrifuge tubes (S1-S7) of 1.5ml, adding 250 mu l of sample diluent into each centrifuge tube, sucking 250 mu l of standard substance S8 into the first centrifuge tube (S7), lightly blowing and beating, and uniformly mixing; sucking 250. mu.l from S7 into a second EP tube (S6), and gently pipetting and mixing; performing multiple dilution on the standard product by analogy; s1 is a sample diluent;
3) the method comprises the following operation steps:
a) aiming at the set standard substance hole and the set sample hole to be detected, 100 mu l of the standard substance or the sample to be detected is respectively added into each hole, the mixture is gently shaken and uniformly mixed, a plate paste is pasted, and the mixture is incubated at 37 ℃ for 2.5 hours;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 2 hours at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a sticking film, and incubating for 50 minutes at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 20 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction;
3) data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample.
Fourth detection groupThe method comprises the following steps: porous plate coated with glutathione peroxidase antibody, glutathione peroxidase pure product as standard substance, biotin-labeled antibody, horseradish peroxidase-labeled avidin, biotin-labeled antibody diluent, and horseradish peroxidase-labeled avidin diluentRelease solution, sample diluent, washing solution, substrate solution and stop solution, wherein,
the concentration of the glutathione peroxidase standard substance is
0,15.6,31.2,62.5,125,250,500,1000μIU/ml；
The biotin labeled antibody is a biotinylated anti-glutathione peroxidase antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M Phosphate Buffered Saline (PBS) ph 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is 86% of sodium chloride, 4.5% of disodium hydrogen phosphate, 3.5% of sodium dihydrogen phosphate, 5% of goat serum, 1% of Proclin-300 and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 20 (0.5%) 5ml, adding distilled water to 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The above-mentioned materials can be prepared by conventional method.
The specific operation method of the content comprises the following steps:
1) the plasma sample is melted overnight at 4 ℃ before detection, centrifuged at 2500rpm for 10min, diluted 1:2000 times with the sample diluent and detected.
2) Preparing a standard substance: after 2 standard samples are centrifuged for 30S, the standard samples are fully dissolved by 1ml of sample diluent (S8), at least 5 times of blowing and beating are carried out repeatedly by using a pipette, 7 sample centrifuge tubes (S1-S7) of 1.5ml are taken, 250 mu l of sample diluent is added into each centrifuge tube, 250 mu l of the standard sample S8 is sucked into the first centrifuge tube (S7), and the mixture is lightly blown and beaten and mixed evenly. Aspirate 250. mu.l from S7 into a second EP tube (S6) and gently pipette. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) aiming at the set standard substance hole and the set sample hole to be detected, 100 mu l of the standard substance or the sample to be detected is respectively added into each hole, the mixture is gently shaken and uniformly mixed, a plate paste is pasted, and the mixture is incubated at 37 ℃ for 2.5 hours;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 2.5 hours at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a sticking film, and incubating for 50 minutes at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 20 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction;
3) data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample.
Fifth detection groupThe method comprises the following steps: a perforated plate coated with the titin, a pure product of the titin as a standard substance, a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the standard product of the titin is 0,23.5,47,94,187.5,375,750,1500 pg/ml;
the biotin labeled antibody is a biotinylated anti-titin antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M Phosphate Buffered Saline (PBS) ph 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is 85% sodium chloride, 5% disodium hydrogen phosphate, 4% sodium dihydrogen phosphate, 5% goat serum, 1% Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The above-mentioned materials can be prepared by conventional method.
The specific operation method of the content comprises the following steps:
1) plasma samples were thawed overnight at 4 ℃ and centrifuged at 2500rpm for 10min prior to testing.
2) Preparing a standard substance: after 2 standard samples are centrifuged for 30S, the standard samples are fully dissolved by 1ml of sample diluent (S8), at least 5 times of blowing and beating are carried out repeatedly by using a pipette, 7 sample centrifuge tubes (S1-S7) of 1.5ml are taken, 250 mu l of sample diluent is added into each centrifuge tube, 250 mu l of the standard sample S8 is sucked into the first centrifuge tube (S7), and the mixture is lightly blown and beaten and mixed evenly. Aspirate 250. mu.l from S7 into a second EP tube (S6) and gently pipette. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) aiming at the set standard substance hole and the set sample hole to be detected, 100 mu l of the standard substance or the sample to be detected is respectively added into each hole, the mixture is gently shaken and uniformly mixed, a plate paste is pasted, and the mixture is incubated at 37 ℃ for 2.5 hours;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 2 hours at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a sticking film, and incubating for 50 minutes at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 20 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction;
3) data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample.
The invention has the beneficial effects that:
the invention uses modern biological technology and bioinformatics analysis to carry out preliminary research on the detection and functions of cholesteryl ester transfer protein, translipoprotein-C3, phospholipid transfer protein, glutathione peroxidase and adiponectin, the cholesteryl ester transfer protein with high concentration, glutathione peroxidase and adiponectin, the translipoprotein-C3 with low concentration and the phospholipid transfer protein have the purpose of predicting the first new atrial fibrillation, and can be applied to the preparation of detection preparations for early intervention; meanwhile, the hierarchical analysis depending on the concentration of the five proteins can also be applied to the preparation of a new atrial fibrillation screening kit, and has better evaluation efficiency and potential huge application value on new atrial fibrillation.
Drawings
FIG. 1 is a graph of the proteomics of the interaction of networks containing cholesteryl ester transfer protein, translipoprotein-C3, phospholipid transfer protein, glutathione peroxidase, and titin;
FIG. 2 shows the content of cholesteryl ester transfer protein, translipoprotein-C3, phospholipid transfer protein, glutathione peroxidase, and titin in patients with and without atrial fibrillation measured by ELISA, wherein the ratio of CETP: a cholesteryl ester transfer protein; APO-C3: translipoprotein-C3; PLTP: phospholipid transfer proteins; GPX 3: glutathione peroxidase; TTN: and (3) myoglobin.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description is made with reference to the accompanying drawings and the detailed description.
Example 1
1. Collection of human plasma samples
Among patients who received the first CABG, patients who developed postoperative atrial fibrillation were the experimental group, and patients who maintained sinus rhythm were the control group. All patients (experimental and control) were blood sampled in the morning prior to surgery, fasting for >10 h. Each patient collected 2ml of whole blood from the peripheral vein, immediately centrifuged at 2500rpm for 10 minutes, and the upper plasma was separated. And dividing the separated plasma into a plurality of equal parts, placing the equal parts in a plasma collecting pipe, and placing the equal parts in a refrigerator at the temperature of minus 80 ℃ for freezing storage to be detected.
2. Proteomics detection
1) Plasma proteome detection
Proteins were detected in two sets of each mixed sample using iTRAQ combined with multidimensional liquid chromatography-mass spectrometry (LC-MS/MS) (Applied Biosystems, Foster City, CA).
2) Sample preparation
a) A high-abundance protein Removal Kit (ProteExtractTM Albumin/IgG Removal Kit, CALBIOCHEM, USA) was purchased to remove the high-abundance protein.
b) 5 volumes of acetone were added and the precipitate was allowed to settle at-20 ℃ for 1 hour. Centrifugation was carried out at 10000g for 10 minutes at 4 ℃ to collect the precipitate.
c) And (3) putting the dried powder solution into a sample lysate, and fully dissolving the protein in a constant-temperature water bath at 30 ℃.
d) The solution was centrifuged at 15000g for 15min at room temperature, the supernatant was collected and centrifuged again to remove the impurities sufficiently.
e) The supernatant is the total protein solution of the tissue, and the protein concentration is measured, subpackaged and stored at-80 ℃ for later use or directly used for iTRAQ analysis.
3) Sample quantification
According to the quantitative principle, the sample: BCA (bicinchoninic acid) and cupric sulfate
And other reagents are mixed together to form the apple green, namely the BCA working reagent. Under alkaline conditions, when the BCA is combined with protein, the protein reduces Cu2+ into Cu +, one Cu + chelates two BCA molecules, the working reagent forms a purple compound from the original apple green, the water-soluble compound shows the maximum light absorption at 562nm, and the light absorption and the protein concentration have good linear relation in a wide range, so the protein concentration can be calculated according to the light absorption value.
Proteins in the sample were quantified according to the BCA method. The experimental procedure was as follows:
a) and (3) making a standard curve graph by taking the concentration of the standard protein as an abscissa and the OD562 as an ordinate, and solving a regression equation.
b) The OD562 values were used to prepare a standard curve of BSA concentration.
c) Diluting a sample to be detected with a proper volume by 10 times, detecting the absorbance of the sample, calculating the average OD562 of the sample, substituting the average OD562 into a regression equation, finally calculating the detected concentration of the sample to be detected, and multiplying the detected concentration by 10 to obtain the real concentration of the sample.
4) SDS-PAGE electrophoretic sample detection experiment
a) Mu.g of each sample was taken and separated by 12% SDS-PAGE.
b) The separated gel was stained by Coomassie blue staining. The specific operation is as follows: fixing for 2 hours; dyeing for 12 hours; and washing with water until the background is clear.
c) The stained gel was scanned using an ImageScanner scanner in grayscale
Mode, optical density value is 300 dpi.
5) Reductive alkylation and enzymolysis of protein
The method comprises the following specific steps:
a) after the protein is quantified, 100 mu g of each sample is taken, precooled acetone with 5 times of volume is adopted for precipitation, and the solution is placed at the temperature of minus 20 ℃ for 1 hour to fully precipitate the protein.
b) The resulting precipitate was centrifuged at 12,000rpm for 10 minutes at 4 ℃ and vacuum-freeze-dried.
c) The protein precipitate was dissolved well with 50. mu.l of the Dissolution Buffer in the iTRAQ kit, and 4. mu.l of Reducing Reagent was added and reacted at 60 ℃ for 1 hour.
d) Mu.l of Cysteine-Blocking Reagent was added, the reaction was carried out at room temperature for 10 minutes, the protein solution after the reductive alkylation was added to a 10K ultrafiltration tube, the mixture was centrifuged at 12,000rpm for 20 minutes, and the bottom solution of the collection tube was discarded.
e) Add 100. mu.l of Dissolution Buffer, centrifuge at 12,000rpm for 20 minutes, discard the bottom solution of the collection tube, repeat 3 times.
f) The collection tube was replaced with a new one, and 50. mu.l of sequencing grade trypsin solution at a concentration of 50 ng/. mu.l was added to the ultrafiltration tube and reacted at 37 ℃ for 12 hours.
g) Centrifuging at 12,000rpm for 20 min, collecting peptide fragments after enzymolysis, adding 50 μ l of precipitation Buffer into an ultrafiltration tube, centrifuging at 12000rpm for 20 min, collecting tube bottom solution, and combining with the previous solution.
6) Protein labeling and mass spectrometry pilot experiments
a) Taking out the iTRAQ reagent from the refrigerator, and balancing to room temperature;
b) centrifuging the iTRAQ reagent to the bottom of the tube;
c) dissolving iTRAQ reagent with 150 μ l isopropanol;
d) transferring 50 mu l of sample (100 mu g of enzymolysis product) into a new centrifuge tube, adding the iTRAQ reagent, and reacting at room temperature for 2 hours;
e) the reaction was stopped by adding 100. mu.l of water;
f) mixing all the marked samples, carrying out vortex oscillation, and centrifuging to the bottom of the tube;
g) and (5) freezing and drying the sample in vacuum, and reserving the sample for iTRAQ separation and identification.
7)2D-LC-MSMS analysis reversed phase chromatographic separation
a) The freeze-dried sample was dissolved with 110. mu.L of mobile phase A solution;
b) peptide fragment separation was performed on an Agilent 1200HPLC, and the column chromatography, Agilent, was purchased with the specific parameters: protecting the core: analytical Guard Column 4.6 x 12.5mm 5-micron separation Column: Narrow-Bore 2.1 × 150mm 5 μm, detection wavelength: ultraviolet 210nm and 280 nm; flow rate: 0.3ml/min, non-linear binary gradient.
The time table of the flow ratio of peptide fragment chromatographic separation is shown in table 1.
TABLE 1
Figure BDA0002679812530000161
c) Discard 0-5 minutes, collect 1 tube every 4.5 minutes for 6-45 minutes, collect 1 tube for 46-50 minutes, total 10 tubes of sample solution, then freeze dry each tube of solution thoroughly. And obtaining a one-dimensional separation chromatogram.
Reverse chromatography-TripleTOF analysis
a) The lyophilized polypeptide sample after cation exchange separation was redissolved in Nano-RPLC Buffer A.
b) On-line Nano-RPLC liquid chromatography in Eksigent nanolC-UltraTMThe 2D system (AB SCIEX) was performed and the dissolved sample was loaded onto a C18 pre-column (100. mu. m.times.3 cm, C18,3 μm,
Figure BDA0002679812530000171
) Then, the flow rate is maintained to flush and desalt for 10 min.
c) The analytical column is a C18 reverse phase chromatographic column (75 μm x 15cm C18-3 μm
Figure BDA0002679812530000172
ChromXP eksingent), the gradient used in the experiment was such that mobile phase B rose from 5% to 35% within 70 min.
d) The mass spectrum adopts a TripleTOF5600 system (AB SCIEX) combined with a nanoliter spray III ion source (AB SCIEX, USA), the spray voltage is 2.5kV, the air curtain pressure is 30PSI, the atomization pressure is 5PSI, the heater temperature is 150 ℃, the mass spectrum scanning mode is in an Information-Dependent acquisition working mode (IDA, Information Dependent Analysis), the single-spectrum scanning time of the primary TOF-MS is 250MS, at most 35 secondary spectra with charges of 2+ to 5+ and a single-second count greater than 150 are acquired in each IDA cycle, the cycle time of each cycle is fixed to 2.5 seconds, the collision cell energy is set to be suitable for Collision Induced Dissociation (CID) of all precursor ions, the dynamic exclusion is set to be 18 seconds, and is approximately equal to the half-peak width of the chromatogram.
8) Database retrieval
The data processing is carried out by adopting Software of Protein Pilot Software v.5.0(AB SCIEX, USA) containing Paragon algorithm, and the database used in the experiment is a human database which is derived from Uniprot. The protein identification is mainly realized by matching experimental tandem mass spectrum data with theoretical mass spectrum data obtained by database simulation, so that a protein identification result is obtained. The parameters are set as follows:
proteomic mass spectrometry search parameters
Sample Type:iTRAQ 8plex(Peptide Labeled)
Cys.Alkylation:Iodoacetamide
Digestion:Trypsin
Instrument:TripleTOF 5600
Database:Homo Sapiens.fasta
Search Effort:Thorough
User Modified Parameter Files:No
The proteomics detected the interaction of the network containing adiponectin, alanyl membrane aminopeptidase and dopamine β -hydroxylase is shown in figure 1.
Example 2
Enzyme-linked immunosorbent assay (ELISA)
The cholesterol ester transfer protein, the translipoprotein-C3, the phospholipid transfer protein, the glutathione peroxidase and the titin were verified in newly collected plasma samples by ELISA. The experimental principle is as follows: the antibody of the protein to be detected is pre-embedded at the bottom of the 96-well plate, and the protein to be detected in the 96-well plate can be combined with the antibody after the standard substance and the sample are added. After removal of unbound substrate, biotin-conjugated antibody to the protein to be tested is added. Washing, an anti-biotin conjugated horseradish peroxidase labeled antibody (HRP) was added, and unbound HRP was removed by washing. The color developing agent was added, and after the reaction was terminated, the absorbance of the liquid was measured.
The cholesterol ester transfer protein was verified to be present,comprises the following steps:
multi-well plate coated with cholesteryl ester transfer protein antibody
Pure cholesteryl ester transfer protein as standard substance
Biotin-labeled antibody
Horse radish peroxidase labeled avidin
Biotin labeled antibody diluent
Horse radish peroxidase labeled avidin diluent
Sample diluent
Cleaning solution
Substrate solution
Stopping liquid
Wherein
The concentration of the cholesteryl ester transfer protein standard substance is
0,0.78,1.56,3.12,6.25,12.5,25,50ng/ml；
The biotin labeled antibody is a biotinylated anti-cholesteryl ester transfer protein antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M phosphate buffer pH 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M phosphate buffer pH7.20.05% thimerosal;
the sample diluent is 86% of sodium chloride, 4.5% of disodium hydrogen phosphate, 3.5% of sodium dihydrogen phosphate, 5% of goat serum, 1% of Proclin-300 and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The specific operation method comprises the following steps:
1) taking an isolated plasma sample, melting overnight at 4 ℃ before detection, centrifuging at 2500rpm for 10min, diluting with a sample diluent by 1:200 times, and detecting;
2) preparing a standard substance: centrifuging 2 parts of standard substance for 30S, fully dissolving the standard substance with 1ml of sample diluent (S8), repeatedly blowing and beating for at least 5 times by using a pipette, taking 7 centrifuge tubes (S1-S7) of 1.5ml, adding 250 mu l of sample diluent into each centrifuge tube, sucking 250 mu l of standard substance S8 into the first centrifuge tube (S7), lightly blowing and beating, and uniformly mixing; sucking 250. mu.l from S7 into a second EP tube (S6), and gently pipetting and mixing; performing multiple dilution on the standard product by analogy; s1 is a sample diluent;
3) the method comprises the following operation steps:
a) aiming at the set standard substance hole and the set sample hole to be detected, 100 mu l of the standard substance or the sample to be detected is respectively added into each hole, the mixture is gently shaken and uniformly mixed, a plate paste is pasted, and the mixture is incubated at 37 ℃ for 2.5 hours;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 1.5 hours at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a sticking film, and incubating for 1 hour at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 20 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction;
4) data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample.
Verification of Translipoprotein-C3Comprising:
multi-well plate coated with translipoprotein-C3 antibody
Pure product of translipoprotein-C3 as standard substance
Biotin-labeled antibody
Horse radish peroxidase labeled avidin
Biotin labeled antibody diluent
Horse radish peroxidase labeled avidin diluent
Sample diluent
Cleaning solution
Substrate solution
Stopping liquid
Wherein:
the concentration of the standard product of the translipoprotein-C3 is 0,15.6, 31.25, 62.5,125,250,500 and 1000 ng/ml;
the biotin labeled antibody is a biotinylated anti-translipoprotein-C3 antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M phosphate buffer pH 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M phosphate buffer pH7.20.05% thimerosal;
the sample diluent is 85% sodium chloride, 5% disodium hydrogen phosphate, 4% sodium dihydrogen phosphate, 5% goat serum, 1% Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The specific operation method comprises the following steps:
1) the plasma sample is melted overnight at 4 ℃ before detection, centrifuged at 2500rpm for 10min, diluted 1:400 times with the sample diluent and detected.
2) Preparing a standard substance: after 2 portions of the standard were centrifuged for 30 seconds, the standard was sufficiently dissolved with 1ml of the sample diluent (S8), repeatedly pipetting at least 5 times using a pipette, taking 7 sample centrifuge tubes of 1.5ml (S1-S7), adding 150 μ l of each sample diluent, sucking 150 μ l of the standard S8 into the first centrifuge tube (S7), gently pipetting and mixing. Pipette 150. mu.l from S7 into a second EP tube (S6) and gently pipette the mixture. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) and setting a standard sample hole and a sample hole to be detected. Adding 50 mul of standard substance or sample to be tested with corresponding concentration into each hole;
b) immediately adding 50 mul of horse radish peroxidase labeled avidin, adding 50 mul of biotin labeled antibody according to the same sequence, slightly shaking and uniformly mixing, attaching a plate paste, and incubating for 50 minutes at 37 ℃;
c) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
d) adding 100 mul of substrate solution into each hole, and developing for 15 minutes at 37 ℃ in a dark place;
e) adding 50 mul of stop solution into each hole to stop the reaction;
f) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction.
4) Data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample. The results are shown in FIG. 2.
Validation of phospholipid transfer proteinsComprising:
multi-well plate coated with phospholipid transfer protein antibody
Phospholipid transfer protein pure product as standard substance
Biotin-labeled antibody
Horse radish peroxidase labeled avidin
Cleaning solution
Substrate solution
Stopping liquid
Wherein:
the concentration of the phospholipid transfer protein standard substance is
0,2.34,4.68,9.375,18.75,37.5,75,150ng/ml；
The biotin labeled antibody is a biotinylated anti-phospholipid transfer protein antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the washing solution comprises: NaCl 9.0g, Tween 206 ml 0.5%, distilled water to 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The above-mentioned materials can be prepared by conventional method.
The specific operation method of the content comprises the following steps:
1) the plasma sample is melted overnight at 4 ℃ before detection, centrifuged at 2500rpm for 10min, diluted 1:200 times with the sample diluent and detected.
2) Preparing a standard substance: centrifuging 2 parts of standard substance for 30S, fully dissolving the standard substance with 1ml of sample diluent (S8), repeatedly blowing and beating for at least 5 times by using a pipette, taking 7 centrifuge tubes (S1-S7) of 1.5ml, adding 250 mu l of sample diluent into each centrifuge tube, sucking 250 mu l of standard substance S8 into the first centrifuge tube (S7), lightly blowing and beating, and uniformly mixing; sucking 250. mu.l from S7 into a second EP tube (S6), and gently pipetting and mixing; performing multiple dilution on the standard product by analogy; s1 is a sample diluent;
3) the method comprises the following operation steps:
a) aiming at the set standard substance hole and the set sample hole to be detected, 100 mu l of the standard substance or the sample to be detected is respectively added into each hole, the mixture is gently shaken and uniformly mixed, a plate paste is pasted, and the mixture is incubated at 37 ℃ for 2.5 hours;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 2 hours at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a sticking film, and incubating for 50 minutes at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 20 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction;
3) data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample. The results are shown in FIG. 2.
Validation of glutathione peroxidaseComprising:
multi-well plate coated with glutathione peroxidase antibody
Glutathione peroxidase pure product as standard substance
Biotin-labeled antibody
Horse radish peroxidase labeled avidin
Cleaning solution
Substrate solution
Stopping liquid
Wherein:
the concentration of the glutathione peroxidase standard substance is
0,15.6,31.2,62.5,125,250,500,1000μIU/ml；
The biotin labeled antibody is a biotinylated anti-glutathione peroxidase antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the washing solution comprises: NaCl 9.0g, Tween 206 ml 0.5%, distilled water to 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The above-mentioned materials can be prepared by conventional method.
The specific operation method of the content comprises the following steps:
1) the plasma sample is melted overnight at 4 ℃ before detection, centrifuged at 2500rpm for 10min, diluted 1:2000 times with the sample diluent and detected.
2) Preparing a standard substance: after 2 standard samples are centrifuged for 30S, the standard samples are fully dissolved by 1ml of sample diluent (S8), at least 5 times of blowing and beating are carried out repeatedly by using a pipette, 7 sample centrifuge tubes (S1-S7) of 1.5ml are taken, 250 mu l of sample diluent is added into each centrifuge tube, 250 mu l of the standard sample S8 is sucked into the first centrifuge tube (S7), and the mixture is lightly blown and beaten and mixed evenly. Aspirate 250. mu.l from S7 into a second EP tube (S6) and gently pipette. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) aiming at the set standard substance hole and the set sample hole to be detected, 100 mu l of the standard substance or the sample to be detected is respectively added into each hole, the mixture is gently shaken and uniformly mixed, a plate paste is pasted, and the mixture is incubated at 37 ℃ for 2.5 hours;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 2.5 hours at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a sticking film, and incubating for 50 minutes at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 20 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction;
3) data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample. The results are shown in FIG. 2.
Validation of titinComprising:
multiwell plate coated with titin antibody
The pure product of the titin is used as a standard product
Biotin-labeled antibody
Horse radish peroxidase labeled avidin
Cleaning solution
Substrate solution
Stopping liquid
Wherein:
the concentration of the standard product of the titin is 0,23.5,47,94,187.5,375,750,1500 pg/ml;
the biotin labeled antibody is a biotinylated anti-titin antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the washing solution comprises: NaCl 9.0g, Tween 206 ml 0.5%, distilled water to 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The above-mentioned materials can be prepared by conventional method.
The specific operation method of the content comprises the following steps:
1) plasma samples were thawed overnight at 4 ℃ and centrifuged at 2500rpm for 10min prior to testing.
2) Preparing a standard substance: after 2 standard samples are centrifuged for 30S, the standard samples are fully dissolved by 1ml of sample diluent (S8), at least 5 times of blowing and beating are carried out repeatedly by using a pipette, 7 sample centrifuge tubes (S1-S7) of 1.5ml are taken, 250 mu l of sample diluent is added into each centrifuge tube, 250 mu l of the standard sample S8 is sucked into the first centrifuge tube (S7), and the mixture is lightly blown and beaten and mixed evenly. Aspirate 250. mu.l from S7 into a second EP tube (S6) and gently pipette. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) aiming at the set standard substance hole and the set sample hole to be detected, 100 mu l of the standard substance or the sample to be detected is respectively added into each hole, the mixture is gently shaken and uniformly mixed, a plate paste is pasted, and the mixture is incubated at 37 ℃ for 2.5 hours;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 2 hours at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a sticking film, and incubating for 50 minutes at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 20 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction;
3) data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample. The results are shown in FIG. 2.
To summarize, by example 1, it was found that translipoprotein-C3 and phosphotransferase protein were down-regulated in plasma of patients with post-operative atrial fibrillation compared to the control group, and both proteins were enriched in the peroxisome proliferator-activated receptor-alpha (PPAR- α) pathway by enrichment assays. This suggests that there may be a down-regulation of the PPAR-alpha pathway in patients with post-operative atrial fibrillation. The PPAR- α pathway down regulation in atrial tissue shifts the energy metabolic pattern of the heart from fatty acid metabolism dominated to glucose metabolism dominated by inhibition of fatty acid β oxidation and promotion of carbohydrate metabolism. There may be down-regulation of fatty acid transporters on the membrane surface of atrial tissue, while there may be up-regulation of sugar transporters. Resulting in decreased lipid and increased glycogen accumulation in the atria. In addition, glycolysis of glucose in the cytosol to produce NADP can provide reducing equivalents to the mitochondrial electron transport chain via a shuttle mechanism, which, together with NADP produced by the tricarboxylic acid cycle in the mitochondria, increase the respiratory chain to produce oxygen radicals that can place the atrial tissue in an oxidatively stressed state and can damage ion channels on the membrane surface of the atrial muscle leading to atrial electrical remodeling. As described above, the down-regulation of PPAR- α may increase the susceptibility to arrhythmia by altering the energy metabolism pattern in the atria and increasing the production of oxygen radicals, which lead to metabolic and electrical remodeling in the atria. Patients in this group are prone to atrial fibrillation after receiving surgery. Glutathione is a short peptide consisting of glutamic acid, cysteine and glycine, and is present in various organs and cells of the body. It is an antioxidant, can protect the sulfhydryl in protein molecule from oxidation, protect the activity of sulfhydryl protein and enzyme, and has the functions of direct or indirect gene expression regulation, enzyme activity and metabolism regulation, cell protection, amino acid transfer, and immunologic function regulation. The balance of redox states ensures the smooth progression of a variety of biological processes, including cell signaling, transcription factor activity, chromatin remodeling, protein modification, membrane integrity and maintenance of mitochondrial function. The pathophysiological processes in various diseases involve a state of imbalance in redox state due to an increase in active oxygen. Such as atherosclerosis, neurodegeneration, cancer, heart failure, and the like. Systemic inflammatory responses and increased reactive oxygen species in the atria are thought to be responsible for atrial fibrillation, which can impair atrial contraction, disrupt energy metabolism of the cardiac fibers, and shorten the effective refractory period of the atria. Glutathione is oxidized to oxidized glutathione by glutathione peroxidase in the presence of hydrogen peroxide in mammalian cells and tissues and then reduced to reduced glutathione by the action of NADPH-dependent glutathione reductase. The reduction in glutathione potentially increases the susceptibility of the cell to adverse fates associated with oxidative stress. Glutathione peroxidase protein in plasma of patients with postoperative atrial fibrillation is up-regulated compared with that of patients in a control group, and the protein is enriched to a glutathione metabolic pathway through enrichment analysis. This indicates that the in vivo oxidative stress of patients with atrial fibrillation is higher before surgery, the glutathione metabolic pathway in vivo is activated, glutathione peroxidase is up-regulated, and the reserve of reduced glutathione may be reduced, resulting in insufficient antioxidant capacity in atrial tissue. After undergoing cardiac surgery, there is an increase in reactive oxygen species in the atria, and the reduced antioxidant capacity results in increased susceptibility to post-operative atrial fibrillation.
Referring to the results of proteomics for screening and diagnosing patients with post-operative atrial fibrillation in example 1, example 2, using the ELSIA method, again demonstrated that post-operative atrial fibrillation patients had higher plasma concentrations of cholesteryl ester transfer protein, glutathione peroxidase, and adiponectin, and lower plasma concentrations of translipoprotein-C3 and phospholipid transfer protein, compared to the control group. The results are in accordance with example 1, see FIG. 2.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims (4)

1. A protein composition characterized by: comprises cholesteryl ester transfer protein antibody, translipoprotein-C3 antibody, phospholipid transfer protein antibody, glutathione peroxidase antibody and titin.
2. Use of the protein composition of claim 1 for the preparation of new atrial fibrillation test formulations or medical devices.
3. Use of a protein composition according to claim 2, characterized in that: the medical apparatus is a new atrial fibrillation screening kit.
4. A new atrial fibrillation screening kit applying the multi-protein composition is characterized in that: comprises 5 protein detection groups:
first detection groupThe method comprises the following steps: a multi-hole plate coated with a cholesteryl ester transfer protein antibody, a pure cholesteryl ester transfer protein product serving as a standard substance, a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the cholesteryl ester transfer protein standard substance is 0,0.78,1.56,3.12,6.25,12.5,25 and 50 ng/ml;
the biotin labeled antibody is a biotinylated anti-cholesteryl ester transfer protein antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M phosphate buffer pH 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M phosphate buffer pH7.20.05% thimerosal;
the sample diluent is 86% of sodium chloride, 4.5% of disodium hydrogen phosphate, 3.5% of sodium dihydrogen phosphate, 5% of goat serum, 1% of Proclin-300 and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid;
second detection groupThe method comprises the following steps: a perforated plate coated with a translipoprotein-C3 antibody, a translipoprotein-C3 pure product as a standard substance, a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the standard product of the translipoprotein-C3 is 0,15.6, 31.25, 62.5,125,250,500 and 1000 ng/ml;
the biotin labeled antibody is a biotinylated anti-translipoprotein-C3 antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M phosphate buffer pH 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M phosphate buffer pH7.20.05% thimerosal;
the sample diluent is 85% sodium chloride, 5% disodium hydrogen phosphate, 4% sodium dihydrogen phosphate, 5% goat serum, 1% Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid;
third detection groupThe method comprises the following steps: a porous plate coated with a phospholipid transfer protein antibody, a phospholipid transfer protein pure product serving as a standard substance, a biotin labeled antibody, horseradish peroxidase labeled avidin, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the phospholipid transfer protein standard substance is 0,2.34,4.68,9.375,18.75,37.5 and 75,150 ng/ml;
the biotin labeled antibody is a biotinylated anti-phospholipid transfer protein antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the washing solution comprises: NaCl 9.0g, Tween 206 ml 0.5%, distilled water to 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid;
fourth detection groupThe method comprises the following steps: a porous plate coated with a glutathione peroxidase antibody, a glutathione peroxidase pure product serving as a standard substance, a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the glutathione peroxidase standard substance is 0,15.6,31.2 and 62.5,125,250,500,1000 mu IU/ml;
the biotin labeled antibody is a biotinylated anti-glutathione peroxidase antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M Phosphate Buffered Saline (PBS) ph 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is 86% of sodium chloride, 4.5% of disodium hydrogen phosphate, 3.5% of sodium dihydrogen phosphate, 5% of goat serum, 1% of Proclin-300 and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 20 (0.5%) 5ml, adding distilled water to 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid;
fifth detection groupThe method comprises the following steps: a perforated plate coated with the titin, a pure product of the titin as a standard substance, a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the standard product of the titin is 0,23.5,47,94,187.5,375,750,1500 pg/ml;
the biotin labeled antibody is a biotinylated anti-titin antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M Phosphate Buffered Saline (PBS) ph 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is 85% sodium chloride, 5% disodium hydrogen phosphate, 4% sodium dihydrogen phosphate, 5% goat serum, 1% Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
CN202010959218.5A 2019-12-04 2020-09-14 Protein composition, application thereof and new atrial fibrillation screening kit Pending CN112326970A (en)

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CN117711620A (en) * 2023-12-15 2024-03-15 南方医科大学南方医院 Protein-based atrial fibrillation occurrence risk prediction system and storage medium

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CN110208550A (en) * 2019-07-03 2019-09-06 贵州省临床检验中心 One kind marker relevant with risk of recurrence after Atrial fibrillation radiofrequency ablation is combined and its is applied
CN110221078A (en) * 2019-06-04 2019-09-10 中国农业科学院生物技术研究所 A kind of enzyme linked immunological kit of quantitative detection herbicide resistant protein Bar

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CN110221078A (en) * 2019-06-04 2019-09-10 中国农业科学院生物技术研究所 A kind of enzyme linked immunological kit of quantitative detection herbicide resistant protein Bar
CN110208550A (en) * 2019-07-03 2019-09-06 贵州省临床检验中心 One kind marker relevant with risk of recurrence after Atrial fibrillation radiofrequency ablation is combined and its is applied

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* Cited by examiner, † Cited by third party
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CN117711620A (en) * 2023-12-15 2024-03-15 南方医科大学南方医院 Protein-based atrial fibrillation occurrence risk prediction system and storage medium

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