CN112858692B - BeSO 4 Method for detecting and analyzing differential expression protein of lung tissue of infected rat - Google Patents
BeSO 4 Method for detecting and analyzing differential expression protein of lung tissue of infected rat Download PDFInfo
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Classifications
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
Abstract
BeSO 4 The method for detecting and analyzing the differential expression protein of the lung tissue of the infected rat comprises the following steps: 1. obtaining rat lungOrganizing; 2. preparing a sample; 3. protein reduction treatment; 4. protein alkylation treatment; 5. carrying out proteolysis; 6. TMT labeling is carried out on the protein; 7. performing LC-MS/MS detection on the sample; 8. GO and KEGG enrichment analysis; 9. PPI network diagram analysis. The invention utilizes TMT marked quantitative proteomics technology and bioinformatics analysis technology to realize rapid and high-flux detection of BeSO on one hand 4 On the other hand, the protein expression condition of the infected rat lung tissue realizes the rapid and accurate screening of the key protein, namely BeSO, from the rat lung tissue 4 The occurrence mechanism of the lung cell injury indicates the research direction, and has great guiding significance for diagnosing and treating beryllium lung.
Description
Technical Field
The invention relates to the field of experimental research of pulmonary diseases, in particular to a BeSO 4 A method for detecting and analyzing differential expression protein of lung tissue of infected rats.
Background
Beryllium (Be) is an off-white alkaline earth metal with a high melting point and high electrical and thermal conductivity, is mainly used in three forms of Beryllium metal, beryllium alloy and Beryllium oxide, and is widely used in aerospace, information technology, medical industry and nuclear industry.
Beryllium was listed as a class i carcinogen by the international cancer research institution as early as 1993 and exposure to beryllium compounds can cause acute beryllium disease, beryllium sensitization, chronic beryllium disease, lung cancer disease. Beryllium exposure (i.e., exposure to environments containing beryllium compounds) can damage many organs throughout the body. The lung is a main target organ for beryllium damage, and the beryllium damage to the lung can cause inflammation of a respiratory system, granuloma of lung tissues and interstitial fibrosis of the lung, so that the structure and the function of the lung tissues are damaged, and a beryllium lung is formed. A more serious problem is that after stopping the exposure of beryllium for many years, beryllium still exists in the lungs of the individual, causing persistent damage to the lungs.
At present, the toxicological mechanism of the beryllium compound caused lung injury is not completely elucidated, and the treatment aiming at the beryllium lung is only symptomatic and supportive treatment. Therefore, deep understanding of the toxicological mechanism of beryllium compounds to cause lung injury is particularly important for controlling "beryllium lung".
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a BeSO 4 Method for detecting and analyzing differential expression protein of lung tissue of infected rat, which explores BeSO from molecular field 4 The biomarker of the lung tissue of the infected rat has guiding significance for the subsequent exploration of the toxic action mechanism of beryllium compounds and diagnosis and treatment of beryllium lung.
The technical scheme of the invention is as follows: beSO 4 The method for detecting and analyzing the differential expression protein of the lung tissue of the infected rat comprises the following steps:
s01, obtaining rat lung tissue:
a. 10 healthy male rats are selected, and are randomly divided into a control group of 5 groups and a contaminated group of 5 groups, and the treated rats are respectively fed with water normally and are fed for 6 weeks, and 0.25ml of BeSO is instilled in the contaminated group at the 2 nd week 4 The solution was introduced into the rat trachea, and the control group instilled 0.25ml of sterilized ultrapure water into the rat trachea at week 2;
b. the method comprises the steps that 10 rats in the contaminated group and the control group are anesthetized by diethyl ether, blood is collected by an abdominal aorta blood sampling method, and after the rats die, lungs are taken respectively for subsequent use;
s02, sample preparation:
a. weighing 50mg of each lung, respectively cutting, putting into different sterile mortar bodies, and adding 300ul 8M urea lysate and 3ul protease inhibitor into each sterile mortar body;
b. respectively grinding lung tissues in 10 sterile mortar bodies into meat emulsion in a grinding instrument, and standing for 30min to enable protein in the lung tissues to fully generate cleavage reaction;
c. respectively collecting 10 samples into 10 centrifuge tubes, respectively carrying out centrifugal treatment, and respectively collecting supernatant in the 10 centrifuge tubes after the centrifugal treatment, wherein the supernatant contains proteins in lung tissues;
d. measuring the protein concentration in 10 parts of supernatant, taking the solution containing 150ug protein from 10 parts of supernatant according to the protein concentration of each part of supernatant, and transferring the solution into the inner tube of different ultrafiltration tubes;
s03, protein reduction treatment:
a. adding 10mM DTT solution into the inner tubes of 10 ultrafiltration tubes respectively, supplementing the solution amount in each ultrafiltration tube to 500ul, centrifuging each ultrafiltration tube, and discarding the filtrate in the outer tube of the ultrafiltration tube after centrifuging;
b. repeating the step b for at least two times, so that the lysate is fully replaced by the DTT;
c. the 10 ultrafiltration tubes are placed in an incubator at 37 ℃ for incubation for 1h in a dark place, so that the protein in the inner tube of the ultrafiltration tubes and DTT fully undergo reduction reaction;
s04, protein alkylation treatment:
a. IAA solution with the concentration of 20mM is respectively added into the inner tubes of 10 ultrafiltration tubes, and the solution quantity in each ultrafiltration tube is complemented to 500ul; centrifuging each ultrafiltration tube, and discarding filtrate in the outer tube of the ultrafiltration tube after centrifuging;
b. repeating the step a at least twice, so that the DTT is fully replaced by the IAA;
c. placing 10 ultrafiltration tubes in an incubator at 37 ℃ in a dark place for 1h, so that protein in the inner tube of the ultrafiltration tubes and IAA fully undergo alkylation reaction;
s05, proteolysis:
a. adding TEAB solution with the concentration of 100mM into the inner tubes of 10 ultrafiltration tubes respectively, and supplementing the solution quantity in each ultrafiltration tube to 450ul; centrifuging each ultrafiltration tube, and discarding filtrate in the outer tube of the ultrafiltration tube after centrifuging;
b. repeating the step a at least twice, so that the IAA is fully replaced by the TEAB;
c. transferring the solution in the inner tubes of the 10 ultrafiltration tubes to 10 EP tubes in a one-to-one correspondence manner, respectively adding trypsin into each EP tube, and carrying out enzymolysis reaction on each EP tube at 37 ℃ for 14-16h;
in this step, the amount of trypsin required to be added per 100ug of protein was 1mg;
s06, carrying out TMT labeling on the protein:
marking solution samples in 10 EP pipes after enzymolysis by using TMT labels, using one label for each pipe sample, placing each EP pipe in an incubator at 37 ℃ for marking reaction for 1h, respectively adding 8ul of hydroxylamine solution with 5% mass concentration into each EP pipe after the marking reaction is finished, and standing for 15min to terminate the marking reaction;
s07, carrying out LC-MS/MS detection on the sample:
a. mixing 5-tube samples of the contamination group into a tube, namely a tube A, mixing 5-tube samples of the control group into a tube B, concentrating and drying the solutions in the tube A and the tube B respectively, adding 200ul of 0.1% FA into the tube A and the tube B respectively to dissolve, dry and concentrate the concentrated samples, and then respectively dividing the solutions in the tube A and the tube B to obtain 45-tube fractional samples, concentrating and evaporating all the fractional samples for later test;
b. the 45-tube concentrated evaporated samples of the contamination group are respectively named as C 1 、C 2 、C 3 ·····C 45 At C 1 - C 15 Adding 20ul of 0.1% FA into the concentrated and evaporated sample for dissolving, and then using C 1 Dissolving the C in the solution of the number 16 Number and C 31 Sample number C 2 Dissolving the C in the solution of the number 17 Number and C 32 Sample number C 3 Dissolving the C in the solution of the number 18 Number and C 33 Sample number, and so on, with C 15 Sequentially dissolving C in solution 30 Number and C 45 Sample number, finally obtaining 15 tubes of solution of the contamination group, wherein each tube of solution contains 3 tubes of concentrated and evaporated substance of the sample;
c. the 45-tube concentrated evaporated samples of the control group were designated as D respectively 1 、D 2 、D 3 ·····D 45 At D 1 - D 15 Adding 20ul of 0.1% FA into the concentrated and evaporated sample for dissolving, and then using D 1 Dissolving the solutions D in sequence 16 Number and D 31 Sample number C 2 Dissolving the solutions D in sequence 17 Number and C 32 Sample number D 3 Dissolving the solutions D in sequence 18 Number and D 33 Sample number, so on, with D 15 Sequentially dissolving D in solution 30 Number and D 45 Sample number, finally obtaining 15 tubes of solutions of a control group, wherein each tube of solution contains 3 tubes of concentrated and evaporated substances of the sample;
d. performing on-machine detection on 30-tube solutions of the contamination group and the control group in a mass spectrometer, and searching by using a UniProt human database aiming at detection data to obtain original data containing protein types, abundance information and relative molecular weight;
s08, GO and KEGG enrichment analysis:
a. screening the original data by Persue software according to the standards of P value < 0.05 and FC < 1.2 to find out the differential expression protein;
b. performing GO enrichment analysis and KEGG enrichment analysis on the differential expression protein based on two software packages of R language 'clusterifier' and 'org.Hs.eg.db', obtaining biological functions, distribution positions and molecular functions of the differential expression protein through GO enrichment analysis, and obtaining the differential expression protein to participate in BeSO through KEGG enrichment analysis 4 Signaling pathways that cause lung cell damage;
c. screening the differentially expressed proteins associated with the signaling pathway, referred to as associated differentially expressed proteins, from the differentially expressed proteins based on the results of the KEGG enrichment analysis; screening proteins participating in the signal pathway from related differentially expressed proteins by combining with a pathway diagram corresponding to the signal pathway;
s09, PPI network diagram analysis:
a. inputting the related differential expression protein into a String online database for searching, and storing a search result as a TSV format file;
b. visual analysis is carried out on the TSV format file through Cytoscape software, and a PPI network diagram composed of related differential expression proteins is drawn;
c. screening for key proteins from proteins involved in the signaling pathway, which have a correlation with at least 2 proteins, in combination with the PPI network map.
The invention further adopts the technical scheme that: in the S01 step, beSO 4 The concentration of the solution was 12ml/kg.
The invention further adopts the technical scheme that: in the step S01, the rat is SD rat.
The invention further adopts the technical scheme that: in step S02, the sterile mortar is left to stand in a refrigerator at 4 ℃.
The invention further adopts the technical scheme that: in step S03, proteins smaller than 3Kda are enabled to enter the outer tube of the ultrafiltration tube through centrifugal treatment, and proteins larger than 3Kda are trapped in the inner tube of the ultrafiltration tube.
The invention further adopts the technical scheme that: in step S03, ultrafiltration Guan Jing is placed in an incubator at 37 ℃.
The invention further adopts the technical scheme that: in step S05, 1mg of trypsin is required to be added per 100ug of protein.
The invention further adopts the technical scheme that: in step S06, the EP tube is left to stand in an incubator at 37 ℃.
Compared with the prior art, the invention has the following advantages:
1. on the one hand, the rapid and high-throughput detection of BeSO is realized by utilizing TMT marked quantitative proteomics technology and bioinformatics analysis technology 4 On the other hand, the protein expression condition of the infected rat lung tissue realizes the rapid and accurate screening of the key protein (the key protein and BeSO) 4 Associated with injury to rat lung cells), beSO 4 The occurrence mechanism of the lung cell injury indicates the research direction, and has great guiding significance for diagnosing and treating beryllium lung.
2. The TMT marked quantitative proteomics technology has the characteristics of high sensitivity, high flux, strong separation capability and wide application range, can obtain quantitative information of the same peptide segment among different samples, and lays a foundation for further obtaining quantitative data of protein.
The invention is further described below with reference to the drawings and examples.
Drawings
FIG. 1 is a volcanic diagram of a differentially expressed protein in step S08;
FIG. 2 is a cluster map of differentially expressed proteins in step S08;
FIG. 3 is a graph of GO-Cellular Component analysis in step S08;
FIG. 4 is a graph of GO-Biological Process analysis in step S08;
FIG. 5 is a graph of GO-Molecular Function analysis in step S08;
FIG. 6 is a KEGG enrichment analysis chart in step S08;
FIG. 7 is a diagram of RNA transport signal pathways in step S08;
FIG. 8 is a PPI network diagram of the related differentially expressed proteins in step S09.
Detailed Description
Example 1:
BeSO 4 the method for detecting and analyzing the differential expression protein of the lung tissue of the infected rat comprises the following steps:
s01, obtaining rat lung tissue:
a. 10 healthy male rats are selected, and are randomly divided into a control group of 5 groups and a contaminated group of 5 groups, and the treated rats are respectively fed with water normally and are fed for 6 weeks, and 0.25ml of BeSO is instilled in the contaminated group at the 2 nd week 4 The solution was introduced into the rat trachea, and the control group instilled 0.25ml of sterilized ultrapure water into the rat trachea at week 2;
b. 10 rats in the contaminated group and the control group are anesthetized by diethyl ether, blood is collected by an abdominal aorta blood sampling method, and after the rats die, lungs are taken for subsequent use.
In this step, beSO 4 The concentration of the solution was 12ml/kg.
In this step, the rat variety was SD rat.
S02, sample preparation:
a. weighing 50mg of each lung, respectively cutting, placing into different sterile mortar bodies (10 sterile mortar bodies), and adding 300ul 8M urea lysate and 3ul protease inhibitor into each sterile mortar body;
b. respectively grinding lung tissues in 10 sterile mortar bodies into meat emulsion in a grinding instrument, and respectively standing in a refrigerator at 4 ℃ for 30min to enable proteins in the lung tissues to fully undergo cleavage reaction;
c. respectively collecting 10 samples into 10 centrifuge tubes, respectively carrying out centrifugal treatment, and respectively collecting supernatant in the 10 centrifuge tubes after the centrifugal treatment, wherein the supernatant contains proteins in lung tissues;
d. the protein concentration in 10 parts of the supernatant was measured, and based on the protein concentration in each part of the supernatant, the amount of the solution containing 150ug of protein was taken out of 10 parts of the supernatant and transferred to the inner tube of different ultrafiltration tubes (the number of ultrafiltration tubes was 10).
In this step, the temperature of the centrifugation was 4℃and the rotational speed was 12000rmp/min for 10min.
S03, protein reduction treatment:
a. adding 10mM DTT solution into the inner tubes of 10 ultrafiltration tubes respectively, supplementing the solution amount in each ultrafiltration tube to 500ul, centrifuging each ultrafiltration tube, and discarding the filtrate in the outer tube of the ultrafiltration tube after centrifuging;
b. repeating the step b for at least two times, so that the lysate is fully replaced by the DTT;
c. 10 ultrafiltration tubes are placed in an incubator at 37 ℃ for 1h, so that the protein in the inner tube of the ultrafiltration tubes and DTT fully undergo reduction reaction.
In this step, proteins less than 3Kda were allowed to enter the outer tube of the ultrafiltration tube by centrifugation, and proteins greater than 3Kda were trapped in the inner tube of the ultrafiltration tube.
In this step, the temperature of the centrifugation was 4℃and the rotational speed was 12000rmp/min for 15min.
S04, protein alkylation treatment:
a. IAA solution with the concentration of 20mM is respectively added into the inner tubes of 10 ultrafiltration tubes, and the solution quantity in each ultrafiltration tube is complemented to 500ul; centrifuging each ultrafiltration tube, and discarding filtrate in the outer tube of the ultrafiltration tube after centrifuging;
b. repeating the step a at least twice, so that the DTT is fully replaced by the IAA;
c. and (3) placing 10 ultrafiltration tubes in an incubator at 37 ℃ in a static manner, and standing for 1h in a dark place to enable the protein in the inner tube of the ultrafiltration tubes to fully react with IAA in an alkylation manner.
In this step, the temperature of the centrifugation was 4℃and the rotational speed was 14000rmp/min for 15min.
S05, proteolysis:
a. adding TEAB solution with the concentration of 100mM into the inner tubes of 10 ultrafiltration tubes respectively, and supplementing the solution quantity in each ultrafiltration tube to 500ul; centrifuging each ultrafiltration tube, and discarding filtrate in the outer tube of the ultrafiltration tube after centrifuging;
b. repeating the step a at least twice, so that the IAA is fully replaced by the TEAB;
c. transferring the solutions in the inner tubes of the 10 ultrafiltration tubes to 10 EP tubes in a one-to-one correspondence manner, respectively adding trypsin into each EP tube, and carrying out enzymolysis reaction on each EP tube at 37 ℃ for 14-16h.
In this step, the amount of trypsin required to be added is 1mg per 100ug of protein.
In this step, the temperature of the centrifugation was 4℃and the rotational speed was 14000rmp/min for 15min.
S06, carrying out TMT labeling on the protein:
a. the labeling reaction is carried out by using TMT labels (model of label product is Thermo Scientific ™ TMT ™) to label solution samples in 10 EP pipes after enzymolysis, one label is used for each pipe sample, each EP pipe is placed in an incubator at 37 ℃ for carrying out the labeling reaction for 1h, and after the labeling reaction is finished, 8ul of hydroxylamine solution with 5% mass concentration is respectively added into each EP pipe, and the mixture is kept stand for 15min to terminate the labeling reaction.
In this step, the EP tube was left to stand in an incubator at 37 ℃.
S07, carrying out LC-MS/MS detection on the sample:
a. mixing 5-tube samples of the contamination group into a tube, namely a tube A, mixing 5-tube samples of the control group into a tube B, concentrating and drying the solutions in the tube A and the tube B respectively, adding 200ul of 0.1% FA into the tube A and the tube B respectively to dissolve, dry and concentrate the concentrated samples, and then respectively dividing the solutions in the tube A and the tube B to obtain 45-tube fractional samples, concentrating and evaporating all the fractional samples for later test;
b. the 45-tube concentrated evaporated samples of the contamination group are respectively named as C 1 、C 2 、C 3 ·····C 45 At C 1 - C 15 Adding 20ul of 0.1% FA into the concentrated and evaporated sample for dissolving, and then using C 1 Dissolving the C in the solution of the number 16 Number and C 31 Sample number C 2 Dissolving the C in the solution of the number 17 Number and C 32 Sample number C 3 Dissolving the C in the solution of the number 18 Number and C 33 Sample number, and so on, with C 15 Sequentially dissolving C in solution 30 Number and C 45 Sample number, finally obtaining 15 tubes of solution of the contamination group, wherein each tube of solution contains 3 tubes of concentrated and evaporated substance of the sample;
c. the 45-tube concentrated evaporated samples of the control group were designated as D respectively 1 、D 2 、D 3 ·····D 45 At D 1 - D 15 Adding 20ul of 0.1% FA into the concentrated and evaporated sample for dissolving, and then using D 1 Dissolving the solutions D in sequence 16 Number and D 31 Sample number C 2 Dissolving the solutions D in sequence 17 Number and C 32 Sample number D 3 Dissolving the solutions D in sequence 18 Number and D 33 Sample number, so on, with D 15 Sequentially dissolving D in solution 30 Number and D 45 Sample number, finally obtaining 15 tubes of solutions of a control group, wherein each tube of solution contains 3 tubes of concentrated and evaporated substances of the sample;
d. performing on-machine detection on 30-tube solutions of the infected group and the control group in a mass spectrometer, searching by using a UniProt human database according to detection data, and extracting quantitative data of infected proteins of the infected group and uninfected proteins of the control group into an excel table, wherein the data in excel is original data, and the original data comprises protein types, abundance information and relative molecular weight information.
In the step, the steps b and c are not sequenced.
S08, GO and KEGG enrichment analysis:
a. according to the standard that P value is less than 0.05 and FC is more than 1.2, screening the original data through Persue software to find 422 differential expression proteins, wherein 197 differential expression proteins are remarkably up-regulated, and 225 differential expression proteins are remarkably down-regulated;
b. performing GO enrichment analysis and KEGG enrichment analysis on the 422 differential expression proteins based on two software packages of R language 'clusterifier' and 'org.Hs.eg.db', obtaining biological functions, distribution positions and molecular functions of the differential expression proteins through GO enrichment analysis, and obtaining the differential expression proteins to participate in BeSO through KEGG enrichment analysis 4 Signaling pathways that cause lung cell damage (regulate RNA transport);
c. screening out the 422 differentially expressed proteins (8 in total, UBE21, NUP85, NUP88, RNPS1, NUP155, EIF2B4, EIF2B3, respectively) associated with the signaling pathway (modulating RNA transport) based on the results of the KEGG enrichment analysis, referred to as associated differentially expressed proteins; and 5 proteins participating in the signaling pathway (regulating RNA transportation) are screened from related differentially expressed proteins by combining with a corresponding pathway diagram of the signaling pathway (RNA transportation signaling pathway diagram).
In this step, referring to fig. 1-2, the distribution of differentially expressed proteins between the affected group and the control group is more intuitively shown by volcanic image (made by graphpad prism 7 software) and cluster thermal image (made by omicshare cloud platform).
In this step, referring to FIGS. 3-5, the GO enrichment analysis results show that the differentially expressed proteins are concentrated in cell components mediating ribosomes, mitochondria and Golgi apparatus, and participate in RNA operation, RNA transport and RNA localization, and the differentially expressed proteins are involved in two molecular functions of ribonucleoside binding and nuclear pore structure components.
In this step, see FIG. 6, which shows the results of the KEGG enrichment analysis for BeSO 4 Toxic effects on rat lung cells are achieved by modulating RNA transductionA signal path.
In this step, referring to FIG. 7, an RNA transport signal pathway diagram shows that, among 8 related differentially expressed proteins, the pathway of action of NUP85 and NUP88 is the process of the nuclear pore complex (Nuclear Pore Complex, NPC); the pathway of action of EIF2B4 and EIF2B3 is the process of translation initiation factors (Translation Initiation Factors, EIFs); the route of action of RNPS1 is the process of Exon Junction Complex (EJC).
S09, PPI network diagram analysis:
a. inputting 8 related differential expression proteins into a String online database for searching, and storing the searching result as a TSV format file;
b. visual analysis is carried out on the TSV format file through Cytoscape software, and a PPI network diagram consisting of 8 related differential expression proteins is drawn;
c. 3 key proteins (NUP 85, NUP88, RNPS 1) were selected from 5 proteins involved in the RNA transport signal pathway, which have a correlation with at least 2 proteins at the same time, in combination with the PPI network map.
In this step, referring to FIG. 8, among 8 related differentially expressed proteins, 3 proteins, NUP85, NUP88, RNPS1, are involved in the RNA transport signal pathway and have a correlation with at least 2 proteins, and thus are identified as key proteins.
The method combines the proteomics technology and the bioinformatics analysis technology, and explores the BeSO from the molecular field 4 The biomarker of the lung tissue of the infected rat has guiding significance for the subsequent exploration of the toxic action mechanism of beryllium compounds and diagnosis and treatment of beryllium lung. Proteomics is the science of taking proteome as a research object and researching the composition, the expression level and the modification mode of the proteome, and tandem mass spectrometry tag technology is a reliable technology in proteomics, and can carry out relative quantitative and identification analysis on proteins, polypeptides, nucleic acids and other biological macromolecules. The interaction relationship between proteins can be analyzed by adopting a related bioinformatics analysis method (GO and KEGG enrichment analysis), so that the functions of the proteins can be further explored. Thereby generating lifeThe research of the mechanism and action rules of pathological process provides a new thought, helps to reveal a molecular network related to the beryllium lung disease, and can provide a new thought for the early prevention work of the beryllium lung disease, the diagnosis, the treatment and the prognosis development of the disease.
Claims (8)
1. BeSO 4 The method for detecting and analyzing the differential expression protein of the lung tissue of the infected rat is characterized by comprising the following steps: the method comprises the following steps:
s01, obtaining rat lung tissue:
a. 10 healthy male rats are selected, and are randomly divided into a control group of 5 groups and a contaminated group of 5 groups, and the treated rats are respectively fed with water normally and are fed for 6 weeks, and 0.25ml of BeSO is instilled in the contaminated group at the 2 nd week 4 The solution was introduced into the rat trachea, and the control group instilled 0.25ml of sterilized ultrapure water into the rat trachea at week 2;
b. the method comprises the steps that 10 rats in the contaminated group and the control group are anesthetized by diethyl ether, blood is collected by an abdominal aorta blood sampling method, and after the rats die, lungs are taken respectively for subsequent use;
s02, sample preparation:
a. weighing 50mg of each lung, respectively cutting, putting into different sterile mortar bodies, and adding 300ul 8M urea lysate and 3ul protease inhibitor into each sterile mortar body;
b. respectively grinding lung tissues in 10 sterile mortar bodies into meat emulsion in a grinding instrument, and standing for 30min to enable protein in the lung tissues to fully generate cleavage reaction;
c. respectively collecting 10 samples into 10 centrifuge tubes, respectively carrying out centrifugal treatment, and respectively collecting supernatant in the 10 centrifuge tubes after the centrifugal treatment, wherein the supernatant contains proteins in lung tissues;
d. measuring the protein concentration in 10 parts of supernatant, taking the solution containing 150ug protein from 10 parts of supernatant according to the protein concentration of each part of supernatant, and transferring the solution into the inner tube of different ultrafiltration tubes;
s03, protein reduction treatment:
a. adding 10mM DTT solution into the inner tubes of 10 ultrafiltration tubes respectively, supplementing the solution amount in each ultrafiltration tube to 500ul, centrifuging each ultrafiltration tube, and discarding the filtrate in the outer tube of the ultrafiltration tube after centrifuging;
b. repeating the step b for at least two times, so that the lysate is fully replaced by the DTT;
c. placing 10 ultrafiltration tubes in an incubator at 37 ℃ for 1h, so that the protein in the inner tube of the ultrafiltration tubes and DTT fully undergo a reduction reaction;
s04, protein alkylation treatment:
a. IAA solution with the concentration of 20mM is respectively added into the inner tubes of 10 ultrafiltration tubes, and the solution quantity in each ultrafiltration tube is complemented to 500ul; centrifuging each ultrafiltration tube, and discarding filtrate in the outer tube of the ultrafiltration tube after centrifuging;
b. repeating the step a at least twice, so that the DTT is fully replaced by the IAA;
c. placing 10 ultrafiltration tubes in an incubator at 37 ℃ in a dark place for 1h, so that protein in the inner tube of the ultrafiltration tubes and IAA fully undergo alkylation reaction;
s05, proteolysis:
a. adding TEAB solution with the concentration of 100mM into the inner tubes of 10 ultrafiltration tubes respectively, and supplementing the solution quantity in each ultrafiltration tube to 500ul; centrifuging each ultrafiltration tube, and discarding filtrate in the outer tube of the ultrafiltration tube after centrifuging;
b. repeating the step a at least twice, so that the IAA is fully replaced by the TEAB;
c. transferring the solution in the inner tubes of the 10 ultrafiltration tubes to 10 EP tubes in a one-to-one correspondence manner, respectively adding trypsin into each EP tube, and carrying out enzymolysis reaction on each EP tube at 37 ℃ for 14-16h;
s06, carrying out TMT labeling on the protein:
marking solution samples in 10 EP pipes after enzymolysis by using TMT labels, using one label for each pipe sample, placing each EP pipe in an incubator at 37 ℃ for marking reaction for 1h, respectively adding 8ul of hydroxylamine solution with 5% mass concentration into each EP pipe after the marking reaction is finished, and standing for 15min to terminate the marking reaction;
s07, carrying out LC-MS/MS detection on the sample:
a. mixing 5-tube samples of the contamination group into a tube, namely a tube A, mixing 5-tube samples of the control group into a tube B, concentrating and drying the solutions in the tube A and the tube B respectively, adding 200ul of 0.1% FA into the tube A and the tube B respectively to dissolve, dry and concentrate the concentrated samples, and then respectively dividing the solutions in the tube A and the tube B to obtain 45-tube fractional samples, concentrating and evaporating all the fractional samples for later test;
b. the 45-tube concentrated evaporated samples of the contamination group are respectively named as C 1 、C 2 、C 3 ·····C 45 At C 1 - C 15 Adding 20ul of 0.1% FA into the concentrated and evaporated sample for dissolving, and then using C 1 Dissolving the C in the solution of the number 16 Number and C 31 Sample number C 2 Dissolving the C in the solution of the number 17 Number and C 32 Sample number C 3 Dissolving the C in the solution of the number 18 Number and C 33 Sample number, and so on, with C 15 Sequentially dissolving C in solution 30 Number and C 45 Sample number, finally obtaining 15 tubes of solution of the contamination group, wherein each tube of solution contains 3 tubes of concentrated and evaporated substance of the sample;
c. the 45-tube concentrated evaporated samples of the control group were designated as D respectively 1 、D 2 、D 3 ·····D 45 At D 1 - D 15 Adding 20ul of 0.1% FA into the concentrated and evaporated sample for dissolving, and then using D 1 Dissolving the solutions D in sequence 16 Number and D 31 Sample number C 2 Dissolving the solutions D in sequence 17 Number and C 32 Sample number D 3 Dissolving the solutions D in sequence 18 Number and D 33 Sample number, so on, with D 15 Sequentially dissolving D in solution 30 Number and D 45 Sample number, finally obtaining 15 tubes of solutions of a control group, wherein each tube of solution contains 3 tubes of concentrated and evaporated substances of the sample;
d. performing on-machine detection on 30-tube solutions of the contamination group and the control group in a mass spectrometer, and searching by using a UniProt human database aiming at detection data to obtain original data containing protein types, abundance information and relative molecular weight;
s08, GO and KEGG enrichment analysis:
a. screening the original data by Persue software according to the standards of P value < 0.05 and FC < 1.2 to find out the differential expression protein;
b. performing GO enrichment analysis and KEGG enrichment analysis on the differential expression protein based on two software packages of R language 'clusterifier' and 'org.Hs.eg.db', obtaining biological functions, distribution positions and molecular functions of the differential expression protein through GO enrichment analysis, and obtaining the differential expression protein to participate in BeSO through KEGG enrichment analysis 4 Signaling pathways that cause lung cell damage;
c. screening the differentially expressed proteins associated with the signaling pathway, referred to as associated differentially expressed proteins, from the differentially expressed proteins based on the results of the KEGG enrichment analysis; screening proteins participating in the signal pathway from related differentially expressed proteins by combining with a pathway diagram corresponding to the signal pathway;
s09, PPI network diagram analysis:
a. inputting the related differential expression protein into a String online database for searching, and storing a search result as a TSV format file;
b. visual analysis is carried out on the TSV format file through Cytoscape software, and a PPI network diagram composed of related differential expression proteins is drawn;
c. screening key proteins from proteins involved in the signal pathway, wherein the key proteins have correlation with at least 2 proteins simultaneously, and the key proteins are NUP85, NUP88 and RNPS1, by combining with the PPI network diagram.
2. BeSO as claimed in claim 1 4 The method for detecting and analyzing the differential expression protein of the lung tissue of the infected rat is characterized by comprising the following steps: in the S01 step, beSO 4 The concentration of the solution was 12ml/kg.
3. BeSO as claimed in claim 2 4 The method for detecting and analyzing the differential expression protein of the lung tissue of the infected rat is characterized in that: in the step S01, the rat is SD rat.
4. A BeSO according to claim 3 4 The method for detecting and analyzing the differential expression protein of the lung tissue of the infected rat is characterized by comprising the following steps: in step S02, the sterile mortar is left to stand in a refrigerator at 4 ℃.
5. BeSO as claimed in claim 4 4 The method for detecting and analyzing the differential expression protein of the lung tissue of the infected rat is characterized by comprising the following steps: in step S03, proteins smaller than 3Kda are enabled to enter the outer tube of the ultrafiltration tube through centrifugal treatment, and proteins larger than 3Kda are trapped in the inner tube of the ultrafiltration tube.
6. BeSO as claimed in claim 5 4 The method for detecting and analyzing the differential expression protein of the lung tissue of the infected rat is characterized by comprising the following steps: in step S03, ultrafiltration Guan Jing is placed in an incubator at 37 ℃.
7. BeSO as claimed in claim 6 4 The method for detecting and analyzing the differential expression protein of the lung tissue of the infected rat is characterized by comprising the following steps: in step S05, 1mg of trypsin is required to be added per 100ug of protein.
8. BeSO as claimed in claim 7 4 The method for detecting and analyzing the differential expression protein of the lung tissue of the infected rat is characterized by comprising the following steps: in step S06, the EP tube is left to stand in an incubator at 37 ℃.
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| KR20070018987A (en) * | 2004-07-01 | 2007-02-14 | 패러다임 테라퓨틱스 리미티드 | Use of the receptor GPR86 |
| WO2006099569A2 (en) * | 2005-03-11 | 2006-09-21 | National Jewish Medical And Research Center | Methods and compositions for beryllium-induced disease |
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