CN109633175B - Human serum differential protein composition of Italian saxophone tablets acting on penile erectile dysfunction and screening method and application thereof - Google Patents
Human serum differential protein composition of Italian saxophone tablets acting on penile erectile dysfunction and screening method and application thereof Download PDFInfo
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Abstract
The invention provides a human serum differential protein combination of an Italian saxophone tablet acting on penile erectile dysfunction, belonging to the technical field of screening of drug action target protein, wherein the differential protein combination comprises 45 proteins. The invention obtains the expression change of the in vivo protein of the imazak tablet acting on the ED patient based on the screening, analysis and verification of the serum differential protein marker of the ED patient treated by the imazak tablet, and carries out systematic research on the molecular mechanism of the involved protein and the involved signal conduction path, thereby further defining the mechanism of the medicine, guiding the clinical application of the medicine and simultaneously serving the secondary development of the medicine. In addition, after the differential protein combination is obtained, the invention can intervene on upstream and downstream proteins through pertinently researching the signal path of the target protein, thereby achieving the purpose of treating impotence.
Description
Technical Field
The invention relates to the technical field of screening of drug action target protein, in particular to a human serum differential protein composition of an Yimusaka tablet acting on penile erectile dysfunction and a screening method and application thereof.
Background
Erectile Dysfunction (ED) refers to the inability or repetition of a sustained or repeated penile erection sufficient to complete a satisfactory sexual life, and also refers to the inability of the penis to continuously obtain or maintain a sufficient erection to complete a satisfactory intercourse for at least 6 months or more. ED is a common sexual dysfunction in men, mainly affects men over 40 years old, and the prevalence rate increases with age, and according to research, 52% of men over 40-70 years old suffer from erectile dysfunction in different degrees. In recent years, the stress on the aspects of life, work, psychology and the like is gradually increased, and the prevalence rate of ED is gradually increased and the ED is younger. ED is a very common disease in men and factors associated with ED include: unhealthy lifestyle, metabolic syndrome, psychological and vascular factors, and the like. Normal penile erection is a complex process of cavernous artery dilation, smooth muscle relaxation, and venous occlusion of the penis, and defects in any one of these links may lead to ED.
The Yimuzake tablet is recorded in Sihuiyai Zamu of medical science, mainly comprises musk, nux vomica, frankincense, bull penis, galangal and the like, and the formula takes the musk as a main drug and has the functions of tonifying kidney and strengthening yang; semen Strychni and Olibanum as adjuvants have effects of benefiting kidney and invigorating yang; the bull penis is used as an adjuvant drug for replenishing vital essence and inducing astringency, and the galangal rhizome is used as a guiding drug for harmonizing the drug properties and warming the kidney. It is the traditional proved recipe for Uygur nationality to treat premature ejaculation and impotence. The medicine has better treatment effect on impotence, spermatorrhea and the like caused by kidney-yang deficiency and unconsolidation of kidney qi, improves the sense, the comorbid sense and the holding power of a reproductive system while tonifying the reproductive system to control organs, brain and kidney, thereby enhancing the pleasure feeling of a human body in the aspect of sexual life and avoiding the phenomenon of premature ejaculation caused by overexcitation.
At present, the action mechanism of the Yimusaka tablet is not clear, and after the Yimusaka tablet acts on an ED patient, the expression of proteins in the patient is changed, the molecular mechanism of the related proteins and the involved signal conduction path are not clear, so that the Yimusaka tablet cannot be well clinically applied.
Disclosure of Invention
The invention aims to provide a human serum differential protein combination of an Italian saxophone tablet acting on penile erectile dysfunction, a screening method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a human serum differential protein combination of an Italian saxophone tablet acting on penile erectile dysfunction, which comprises the following 45 proteins:
the invention provides application of the protein combination in the scheme in preparing a kit for detecting the effect of the Italian saxophone tablets on treating the penile erectile dysfunction.
The invention also provides application of the protein combination in screening medicines for treating penile erectile dysfunction.
The invention also provides application of the protein combination in preparing a medicament for treating penile erectile dysfunction.
The invention also provides a screening method of the protein combination in the scheme, which comprises the following steps:
1) collecting fasting blood of a normal person group, a pre-treatment penile erection dysfunction group and a post-treatment penile erection dysfunction group of the Yimusake tablet respectively, and centrifuging to obtain serum;
2) removing the high-abundance protein in the serum obtained in the step 1), and extracting the low-abundance protein in the serum;
3) carrying out enzymolysis on the low-abundance protein in the serum obtained in the step 2) to obtain a peptide segment;
4) carrying out isotope labeling on the peptide fragment obtained in the step 3), and mixing the labeled peptide fragment with the group of samples to obtain a label;
5) separating the marked peptide segment in the marker in the step 4) by adopting high performance liquid chromatography to obtain a marked peptide segment;
6) purifying the marked peptide segment in the step 5) to obtain a purified peptide segment;
7) performing mass spectrometry detection on the purified peptide fragment obtained in the step 6) to obtain a mass spectrometry original file;
8) screening the original mass spectrum file by adopting protome scanner 1.3 software to obtain a spectrogram to be processed;
9) searching a spectrogram to be processed by adopting MASCOT software, and carrying out quantitative analysis to obtain analysis data;
10) and (3) retrieving the analysis data in the step 9) in a person database to obtain the Italian saxophone medicine target protein.
Preferably, the post-imox tablet treatment penile erectile dysfunction group is a patient administered an oral treatment with an imox tablet.
Preferably, the time of the oral treatment is 12-18 d; the dosage of the oral treatment is 0.8-1.2 g/d.
Preferably, the isotopically labeled reagent of step 4) is an iTRAQ reagent.
Preferably, the parameters of the screening in step 8) include: mass of parent ion, minimum peak number in secondary mass spectrogram and signal-to-noise ratio; the mass of the parent ions is 350-6000 Da; and the minimum peak number in the secondary mass spectrogram is 9-11.
Preferably, the search parameter in step 9) includes: fixed modification, variable modification, primary quality deviation, secondary quality deviation, maximum allowable miscut count, enzyme type and database; the fixed modification is Carbammidomethyl (C); the variable modifications are iTRAQ 4plex (k) and iTRAQ 4; the first-level mass deviation is 14-16 ppm; the secondary mass deviation is 18-22 mmu; the maximum allowable leaky tangent number is 1; the type of the enzyme is Trypsin; the database is uniprot 20160315.
The invention has the beneficial effects that: the invention provides a human serum differential protein combination of an Italian saxophone tablet acting on penile erectile dysfunction, which comprises 45 proteins. The invention obtains the effect of the imazak tablet and the expression change of protein in the body of an ED patient, the molecular mechanism of the related protein and the involved signal conduction path based on the screening, analysis and verification of the blood serum difference protein marker of the ED patient treated by the imazak tablet, and carries out systematic research, thereby further defining the mechanism of the medicine, guiding the clinical application of the medicine and simultaneously serving the secondary development of the medicine. In addition, after the differential protein combination is obtained, the invention can intervene on upstream and downstream proteins through pertinently researching the signal path of the target protein, thereby achieving the purpose of treating impotence.
Description of the drawings:
FIG. 1 shows a standard curve for protein quantification in example 1;
FIG. 2 shows the SDS-detected protein mass gel in example 1.
FIG. 3 is an iTRAQ assay for human serum proteins.
Detailed Description
The invention provides a human serum differential protein combination of an Italian saxophone tablet acting on penile erectile dysfunction, which comprises the following 45 proteins, and is shown in a table 1.
TABLE 1 human serum albumin combinations of imazak tablets for penile erectile dysfunction
In the present invention, the expression of the combination of proteins is shown in Table 2. Wherein the expression condition before drug intervention is the result of comparing the treated anterior negative stem erectile dysfunction group (ED group) with the normal human group (N group); the expression of the drug after the intervention is the result of comparing the treatment of anterior penis erectile dysfunction (ED group) with the treatment of post-penile erectile dysfunction (EDY group) of the Yimusaka tablet.
TABLE 2 expression of human serum differential protein combinations of Italian saxophone tablets on penile erectile dysfunction before and after intervention
The invention provides application of the protein combination in the scheme in preparing a kit for detecting the effect of the Italian saxophone tablets on treating the penile erectile dysfunction.
The invention also provides application of the protein combination in screening medicines for treating penile erectile dysfunction.
The invention also provides application of the protein combination in preparing a medicament for treating penile erectile dysfunction.
The invention also provides a screening method of the protein combination in the scheme, which comprises the following steps:
1) collecting fasting blood of a normal person group, a pre-treatment penile erection dysfunction group and a post-treatment penile erection dysfunction group of the Yimusake tablet respectively, and centrifuging to obtain serum;
2) removing the high-abundance protein in the serum obtained in the step 1), and extracting the low-abundance protein in the serum;
3) carrying out enzymolysis on the low-abundance protein in the serum obtained in the step 2) to obtain a peptide segment;
4) carrying out isotope labeling on the peptide fragment obtained in the step 3), and mixing the labeled peptide fragment with the group of samples to obtain a label;
5) separating the marked peptide segment in the marker in the step 4) by adopting high performance liquid chromatography to obtain a marked peptide segment;
6) purifying the marked peptide segment in the step 5) to obtain a purified peptide segment;
7) performing mass spectrometry detection on the purified peptide fragment obtained in the step 6) to obtain a mass spectrometry original file;
8) screening the original mass spectrum file by adopting protome scanner 1.3 software to obtain a spectrogram to be processed;
9) searching a spectrogram to be processed by adopting MASCOT software, and carrying out quantitative analysis to obtain analysis data;
10) and (3) retrieving the analysis data in the step 9) in a person database to obtain the Italian saxophone medicine target protein.
The method comprises the steps of respectively collecting fasting blood of a normal person group (N group), a treatment anterior penis erectile dysfunction group (ED group) and an Italian saxophone tablet treatment post-penis erectile dysfunction group (EDY group), and centrifuging to obtain serum, wherein the fasting blood is collected by comparing an impotence patient with a normal person to obtain candidate differential protein of the impotence patient, namely the candidate differential protein before drug intervention, and comparing the patient after the Italian saxophone tablet drug treatment with the impotence patient to obtain candidate differential protein after drug intervention; the collection amount of the fasting blood is preferably 5 mL; the centrifugal temperature is preferably 1-5 ℃, and more preferably 4 ℃; the rotating speed of the centrifugation is preferably 3000-3500 rpm, and more preferably 3200 rpm; the centrifugation time is preferably 5-10 min, and more preferably 8 min; the centrifugation frequency is preferably 1-2 times; the serum is preferably stored at-80 ℃.
In the present invention, the diagnostic criteria for penile erectile dysfunction (impotence) is defined by reference to national institute of health in 1993; the sexual impotence weight grading and sexual desire assessment standard uses an international erection function index 5 (international erection function 5items, IIEF-5) questionnaire to perform sexual function assessment, and comprises the following contents: the degree of confidence in erectile function, the insertion success rate after erection, the maintenance of the erection state, the success rate of sexual intercourse, and the satisfaction after sexual intercourse. Each IIEF-5 question is scored according to 1-5 points, and the total score is 25 points. Evaluation criteria: wherein the IIEF table score is 5-7 for severe impotence, 8-11 for moderate impotence, 12-21 for mild impotence, and not less than 22 for no impotence. Sexual desire assessment was performed using the International Male sexual desire Table-4 (MSF-4) questionnaire, which included: subjectively on desire for sexual life, satisfaction with erectile function, satisfaction with orgasm after masturbation or intercourse, and satisfaction with ejaculation. Each question is scored according to 1-4 points, and the total score is 16 points.
In the invention, the number of the normal people is preferably 25-35, and more preferably 30; the normal person is examined by physical examination and Electrocardiogram (ECG), chest X-ray, hematuria routine, blood sugar, liver and kidney functions and the like, organic lesions of main organ systems such as heart, liver, brain, lung, kidney, endocrine, immunity and the like are absent, the penile erection function is normal, the sexual life is normal, and the IIEF-5 index is more than or equal to 22.
In the invention, the number of the patients in the group with penile erectile dysfunction is preferably 50-70, and more preferably 60.
In the invention, the object of the penile erection dysfunction group treated by the Italian saxophone tablet is the patient of the penile erection dysfunction group treated by the Italian saxophone tablet in the scheme; the treatment modality is preferably oral treatment; the time of the oral treatment is preferably 12-18 d, and more preferably 15 d; the dosage of the oral treatment is preferably 0.8-1.2 g/d, and more preferably 1 g/d.
After obtaining serum, removing high-abundance protein in the serum, and extracting low-abundance protein in the serum; the method for removing the high-abundance Protein in the serum is not particularly limited, the conventional method in the field is adopted, and in the specific implementation process of the invention, the high-abundance Protein in the serum is removed by adopting a ProteMiner Protein Enrichment Kit (brand: BIO-RAD, cat number: 163-3007, specification: 10 pieces/Kit). The reason for removing the high-abundance protein in the serum is that the high-abundance protein in the serum has great influence on the identification of the low-abundance protein, and most protein markers are concentrated in the low-abundance protein. Through the enrichment of the low-abundance protein, the influence of the high-abundance protein is reduced, and the discovery of the low-abundance protein marker is facilitated. The method for extracting the low-abundance protein in the serum is not particularly limited, and the conventional method for extracting the serum protein in the field can be adopted.
After the low-abundance protein is extracted and obtained, the method preferably further comprises the steps of carrying out quantitative determination on the low-abundance protein; the method for quantitative determination is not particularly limited, and the conventional protein quantitative determination method in the field is adopted, and the bradford method is adopted to perform the quantitative determination on the low-abundance protein in the specific implementation process of the invention. The quantitative determination of the invention is used for verifying the extraction effect of the low-abundance protein.
After the low-abundance protein is quantified, carrying out enzymolysis on the low-abundance protein in serum to obtain a peptide segment; the method for enzymolysis is not particularly limited, and the conventional method for enzymolysis of protein in the field can be adopted.
After a peptide fragment is obtained, carrying out isotope labeling on the peptide fragment, and mixing the labeled peptide fragment and a group of samples to obtain a label; the labeling method is not particularly limited, the conventional labeling method in the field is adopted, and in the specific implementation process of the invention, an iTRAQ labeling kit is adopted for labeling. The source of the iTRAQ labeling kit of the present invention is not particularly limited, and commercially available iTRAQ labeling kits known to those skilled in the art may be used. The Kit used in the specific implementation process of the invention is Reagent-8Plex M mu Ltiplex Kit (Applied Biosystem)8 standard iTRAQ Reagent, wherein 8 isotope reagents (reporter group mass 113-121, not 120) with the same quantity are included, and the peptide fragment after proteolysis can be labeled, so that the peptide fragment can be accurately identified and quantified by combining methods such as tandem mass spectrometry and the like.
After a marker is obtained, separating a marked peptide segment in the marker by adopting high performance liquid chromatography to obtain a marked peptide segment; the invention has no special limitation on the equipment for carrying out the high performance liquid chromatography, and can be carried out by adopting a conventional liquid phase instrument. In the specific implementation process of the invention, SCX strong cation exchange column is used for separation, and the separation is carried out in phosphate buffer solution with pH being 3Eluting with a gradient of 20% to 30% (10mM KH)2PO42M KCl, 25% ACN, pH 3), and eluting under specific conditions and with specific operation instructions of gradient reference instrument and consumable, and eluting the labeled peptide fragment.
After the marked peptide segment is obtained, purifying the marked peptide segment to obtain a purified peptide segment; in the specific implementation process of the invention, C18 reverse phase chromatography is adopted to desalt the marked peptide section.
After obtaining the purified peptide fragment, carrying out mass spectrum detection on the purified peptide fragment to obtain a mass spectrum original file; the mass spectrum detection equipment is preferably a Q-active mass spectrometer; the parameters of the mass spectrometric detection are seen in table 3.
TABLE 3 parameters of Mass Spectrometry measurements
After obtaining a mass spectrum original file, screening the mass spectrum original file by adopting protome discover 1.3 software to obtain a spectrogram to be processed; the parameters of the screen are shown in Table 4.
Table 4 parameters of mass spectrometry screening
After a spectrogram to be processed is obtained, MASCOT software is adopted to search the spectrogram to be processed, and quantitative analysis is carried out to obtain analysis data; the parameters of the search are seen in table 5; see table 6 for the quantitative analysis parameters.
TABLE 5 parameters for MASCOT software search of spectra to be processed
TABLE 6 parameters for quantitative analysis of spectra to be processed by MASCOT software
Protein quantitative value types: peptide fragment value taking method for quantification, mean refers to the method of taking the median position.
Minimum unique peptide number: the minimum unique peptide number used for quantification.
The normalization method comprises the following steps: the mean refers to the median of all quantifiable proteins in a group of samples selected for correction.
After the analysis data are obtained, the analysis data are searched in a person database to obtain the Italian Saka medicine target protein; the people database is uniprot 20160315.
After the Isuzkake medicine target protein is obtained, the upstream and downstream proteins can be intervened by pertinently researching the signal path of the target protein, so that the purpose of treating impotence is achieved.
The human serum albumin difference combination of the invention for treating erectile dysfunction and the screening method and application thereof will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the invention.
Example 1 screening method of human serum albumin combination acting on penile erectile dysfunction by Italian saxophone tablets
The subjects selected 60 cases (before treatment) of impotence patients diagnosed in the outpatient clinic and in the hospitalization of the first and fourth subsidiary hospitals of the Xinjiang medical university at 5-2016-12 months in 2015, and were treated orally with 15-day Yimusake tablets (1.0 g/day), and 30 normal healthy persons. The normal people are examined by physical examination and Electrocardiogram (ECG), chest X-ray, hematuria routine, blood sugar, liver and kidney functions and the like, organic lesions of main organ systems such as heart, liver, brain, lung, kidney, endocrine, immunity and the like are absent, the penile erection function is normal, the sexual life is normal, and the IIEF-5 index is more than or equal to 22. The standard for diagnosing impotence is defined by the national institute of health in 1993. Inclusion criteria for impotence patients: (1) the conditions of married, living and living are good; (2) males between 20-60 years of age; (3) patients without serious organic disease and mental and nervous system disease; (4) the mate has no serious organic diseases and can fully meet the experimental requirements. Exclusion criteria for impotence patients: (1) patients with sexual impotence with confirmed physical diagnosis, such as trauma, venous fistula type, arterial type, and leukodermic lesion type; (2) medicinal impotence, such as hypertension, estrogen, etc.; (3) patients with serious primary diseases of cardiovascular, liver and kidney and hemopoietic system, psychosis or incoordination; (4) the spouse has severe organic diseases of the whole body, such as liver failure, kidney failure, heart failure and the like. The recovery standard of the patient with the Ixak tablet is as follows: there was an increase in MSF-4 score and IIEF-5 score before and after treatment.
The sexual impotence grade and sexual desire assessment criteria was performed using the international erectile function index 5 (international index of choice function 5items, IIEF-5) questionnaire, which included: the degree of confidence in erectile function, the insertion success rate after erection, the maintenance of the erection state, the success rate of sexual intercourse, and the satisfaction after sexual intercourse. Each IIEF-5 question is scored according to 1-5 points, and the total score is 25 points. Evaluation criteria: wherein the IIEF table score is 5-7 for severe impotence, 8-11 for moderate impotence, 12-21 for mild impotence, and not less than 22 for no impotence. Sexual desire assessment was performed using the International Male sexual desire Table-4 (MSF-4) questionnaire, which included: subjectively on desire for sexual life, satisfaction with erectile function, satisfaction with orgasm after masturbation or intercourse, and satisfaction with ejaculation. Each question is scored according to 1-4 points, and the total score is 16 points.
The invention combines various groups of differential proteins obtained by iTRAQ and LC-MS-MS technology, wherein 60 cases before drug intervention are ED groups, 60 cases after drug intervention are EDY groups, and a normal group is N groups; comparing the proteins of the EDY group and the ED group to obtain the EDY/ED differential protein; and (4) counting the types and the quantity of the common proteins of the ED/N and the EDY/ED differential proteins to obtain the drug target protein of the Yimusaka tablet acting on the impotence patient.
The specific operation process is as follows:
1. collecting fasting blood before breakfast, immediately placing whole blood in 4 ℃ environment for 30-40 min, centrifuging at 3000-3500 rpm for 5-10 min, centrifuging the obtained supernatant at 4 ℃ again at 3000-3500 rpm for 30-40 min to obtain serum, separating the serum, and storing in a-80 ℃ low-temperature refrigerator for testing.
2. Protein extraction
ProteoMiner (brand: BIO-RAD, cat # 163-3007, specification: 10 pieces/box, Kit full name: ProteoMinerProtein Enrichment Kit) removes high-abundance proteins in serum.
a) The column was centrifuged, 1000g for 50s, the stock solution was removed from the column and the tube was discarded.
b) And (3) installing a bottom cover, adding 250 mu L washbuffer, covering a top cover, and vibrating on a vibrator for 5min to ensure that the column material is fully and uniformly mixed with the washbuffer and is fully eluted.
c) Remove the bottom cap, put the column into the collection tube for 1000g and centrifuge for 50s, discard the waste liquid.
d) Repeating steps b) to c)
e) The bottom cap was closed and 200. mu.L of the column was activated.
f) The sample needs to be free of precipitate and centrifuged at 10000g for 10min to remove the precipitate.
g) Add 250. mu.L of sample, add the cap, and shake on a shaker at room temperature for 2 h.
h) Remove the bottom cover, centrifuge for 50s at 1000g, discard the waste liquid.
i) Add bottom cap, add 250 μ Lwashbuffer, add top cap, shake for 5 min.
j) Remove the bottom cover 1000g, centrifuge for 50s, discard the waste liquid.
k) Repeat i) -j) twice, and pipette the wall column into solution with a gun after each wash buffer addition.
L) Add 250. mu.L of ddH2O。
m) capping and shaking for 1 min.
n) removing the top cover and the bottom cover, centrifuging for 50s at 1000g, and discarding the waste liquid.
o) Add 60. mu.L of ElutionBuffer (4M Urea, 1% (w/v) CHAPS, 5% acetic acid) to the bottom.
p) cover the device, and shake the device for 15min by vortex.
q) remove top and bottom caps, 1000g centrifuge for 1 min.
r) repeating the steps o) -q) one to two times.
s) -20 ℃ to store the enriched serum protein or directly to the next experiment.
(2) DTT was added to the supernatant to a final concentration of 10 mM. Water bath at 56 deg.c for 1 hr. After removal, IAM was added rapidly to a final concentration of 55mM and allowed to stand in the dark for 1 h.
(3) Quantification, protein quantification using Bradford method, Bradford quantification protocol as follows:
a) determination of the Standard Curve
Preparing: taking out a protein standard stock solution (the concentration of BSA stock solution is 2 mu g/mu L) and restoring to room temperature; diluted 10-fold to 0.2. mu.g/. mu.L as protein standard solution.
Taking out the working solution for Bradford measurement and recovering the working solution to room temperature;
the same lysate and distilled water as the sample were prepared according to the measurement amounts.
b) Numbering 96-hole enzyme label plates, adding reagents according to the table 7, uniformly mixing, standing at room temperature for 0.5min, and placing into a spectrophotometer for determination.
TABLE 7 enzyme-linked plate sample-adding reagent
c) Determination of samples
Numbering 96-hole enzyme label plate, adding reagent and sample according to Table 8, mixing, standing at room temperature for 0.5min, and placing into spectrophotometer for determination.
TABLE 8 enzyme linked plate sample adding reagent and sample
d) Data processing
In the Excel worksheet, a standard curve is drawn with the absorbance as the ordinate y and the amount of the reaction protein (μ g) as the abscissa x, the linear equation y ═ a (x) + b and the square value of the linear correlation coefficient r are regressed, the average value of the measurement results of the sample is used as the measurement value y, the amount of the reaction protein (μ g) x of the sample to be measured is calculated by using the regressed linear equation, and the quantitative standard curve is shown in fig. 1.
The quantitative results are shown in Table 9. Wherein, AY 1: normal group (N group) AY 2: erectile dysfunction group (ED group); AY 3: yimuzake tablet medicine intervention group (EDY group)
TABLE 9 quantitative measurement results of protein
Protein quality gel diagram for SDS assay see fig. 2, and the results in fig. 2 show that all groups of proteins are not degraded and have better protein integrity.
3. Digestion of proteins
(1) The protein volume of 100 ug of each sample was added to a 10K ultrafiltration tube at 14000g and 4 ℃ and centrifuged for 40min, and the waste liquid was discarded.
(2) 200 μ L of 50% TEAB, 14000g, was added, centrifuged at 4 ℃ for 40min, and the waste liquid was discarded.
(3) The above steps are repeated twice.
(4)1 μ g/μ L of Trypsin was added, 3.3 μ g of enzyme was added per 100 μ g of protein substrate, and water bath was carried out at 37 ℃ for 24 h. The digest was lyophilized and then reconstituted with 30 μ L of TEAB (water: TEAB ═ 1:1) per tube.
4. Labelling of peptide fragments
(1) The labeling reagent was equilibrated to room temperature.
(2) Add 70. mu.L of isopropanol to each tube of labeling reagent, mix well for 1min, centrifuge (room temperature, 1000g, 10 sec) and spin to the bottom of the tube.
(3) The mixed labeling reagent was added (70. mu.L of labeling reagent was added to 30. mu.L of peptide solution) to the peptide fragments, and different samples were labeled with isotopes of different sizes.
(4) After mixing, the mixture is thrown to the bottom of the tube and stands for 2 hours at room temperature.
(5) The labeled sample was vacuum drained. (open the sample tube, place in the sample holder of the vacuum extractor, cover the instrument lid, click on, vacuum spin dry for 30 min.)
HPLC preseparation
Using an Agilent conventional liquid phase, strong cation exchange chromatography column (SCX)
(1) Reagent and sample preparation
Sample preparation: after labeling, the sample was diluted 10 times with solution A, adjusted to pH 3.0 with phosphoric acid, centrifuged at 15000g for 10min, and the supernatant was removed.
The components of the solution A: 25% ACN, 10mM KH2PO4Adjusting pH to 3.0 with phosphoric acid
The components of the solution B: 25% ACN, 2M KCL, 10mM KH2PO4The pH was adjusted to 3.0 with phosphoric acid.
(2) Setting separation method
(3) The samples were loaded and run according to the separation gradient of table 10. The pre-separation starts to mix in the liquid B from 31min, starts to generate peaks at 38min, collects 1 component at 1min, collects 30 components in total, combines the parts with less peaks according to chromatogram distribution, finally combines the parts into 16 components and carries out subsequent treatment.
TABLE 10 elution gradient of the liquid phase
6. Purification of peptide fragments
Desalting by C18 reverse phase chromatography, the steps were as follows:
(1)1mL of methanol activated the column.
(2) 5% ACN balance.
(3) The sample was dissolved through the column with 1 mM MILLIQ water.
(4)1mL of 5% acetonitrile was used to wash the column for desalting.
(5)2 x 500 μ L of pure acetonitrile.
(6) And centrifuging at low temperature and pumping out acetonitrile.
(7) And (3) re-dissolving the purified peptide fragment with 0.1% formic acid.
7. Mass spectrometric detection
And (3) respectively carrying out machine detection on 16 components subjected to pre-separation and purification. Setting mass spectrum parameters and liquid phase parameters. The Q-active mass spectrometer detects the peptide fragment signals, and the detection parameters are shown in Table 3.
Elution conditions and gradient parameters of NanoLC:
the component of the solution A: water, 0.1% FA
And B, liquid component: acetonitrile, 0.1% FA
A chromatographic column: type C18, specification 100mm 75mm, pore diameter 300A, particle size 5 μm
Flow rate: 400nl/min
See table 11 for elution gradients.
TABLE 11 liquid elution gradient
8. Qualitative and quantitative analysis of proteins
(1) And obtaining a mass spectrum original file after the mass spectrum scanning is finished.
(2) After inputting the mass spectrum raw file into PD (protein resolver 1.3, thermo) software, the software will screen the mass spectrum. See table 4 for mass spectrum screening parameters.
(3) And searching the spectrogram extracted by PD by using mascot, and after the search is finished, carrying out quantitative analysis by PD software according to the mascot search result and the spectrogram screened in the first step. See table 5 for search parameters and table 6 for quantitative analysis parameters.
9. The obtained analysis data is searched in a human (uniprot20160315) database to obtain the serum difference protein of the patient with the Yimusaka tablets for treating the impotence, namely the clinical Yimusaka drug target protein. See table 2 for specific experimental results.
The embodiment shows that the Yimuzak tablets act on the human serum differential protein combination for erectile dysfunction, can be used for pertinently researching the signal path of the target protein and intervening the upstream and downstream proteins of the target protein, and achieves the purpose of treating impotence.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. An application of human serum differential protein combination of an Italian saxophone tablet acting on penile erectile dysfunction in the preparation of a kit for detecting the effect of the Italian saxophone tablet on penile erectile dysfunction treatment;
the protein combination consists of the following 45 proteins;
2. an application of an imosake tablet acting on a human serum differential protein combination for treating penile erectile dysfunction in screening drugs for treating penile erectile dysfunction;
the protein combination consists of the following 45 proteins;
3. an application of an imosake tablet acting on a human serum differential protein combination for treating penile erectile dysfunction in the preparation of a medicament for treating penile erectile dysfunction;
the protein combination consists of the following 45 proteins;
4. the use according to any one of claims 1 to 3, wherein the screening method for the combination of proteins comprises the following steps:
1) collecting fasting blood of a normal person group, a pre-treatment penile erection dysfunction group and a post-treatment penile erection dysfunction group of the Yimusake tablet respectively, and centrifuging to obtain serum;
2) removing the high-abundance protein in the serum obtained in the step 1), and extracting the low-abundance protein in the serum;
3) carrying out enzymolysis on the low-abundance protein in the serum obtained in the step 2) to obtain a peptide segment;
4) carrying out isotope labeling on the peptide fragment obtained in the step 3), and mixing the labeled peptide fragment with the group of samples to obtain a label;
5) separating the marked peptide segment in the marker in the step 4) by adopting high performance liquid chromatography to obtain a marked peptide segment;
6) purifying the marked peptide segment in the step 5) to obtain a purified peptide segment;
7) performing mass spectrometry detection on the purified peptide fragment obtained in the step 6) to obtain a mass spectrometry original file;
8) screening the original mass spectrum file by adopting protome scanner 1.3 software to obtain a spectrogram to be processed;
9) searching a spectrogram to be processed by adopting MASCOT software, and carrying out quantitative analysis to obtain analysis data;
10) and (3) retrieving the analysis data in the step 9) in a person database to obtain the Italian saxophone medicine target protein.
5. The use according to claim 4, wherein the post-Italian-saxophone-treatment penile erectile dysfunction group is a patient given an oral treatment with Italian-saxophone.
6. The use according to claim 5, wherein the oral treatment is carried out for a period of 12-18 days; the dosage of the oral treatment is 0.8-1.2 g/d.
7. The use of claim 4, wherein the isotopically-labelled reagent of step 4) is an iTRAQ reagent.
8. The use according to claim 4, wherein the parameters of the screening in step 8) include: mass of parent ion, minimum peak number in secondary mass spectrogram and signal-to-noise ratio; the mass of the parent ions is 350-6000 Da; and the minimum peak number in the secondary mass spectrogram is 9-11.
9. The application according to claim 4, wherein the parameters of the search in step 9) include: fixed modification, variable modification, primary quality deviation, secondary quality deviation, maximum allowable miscut count, enzyme type and database; the fixed modification is Carbammidomethyl (C); the variable modifications are iTRAQ 4plex (k) and iTRAQ 4; the first-level mass deviation is 14-16 ppm; the secondary mass deviation is 18-22 mmu; the maximum allowable leaky tangent number is 1; the type of the enzyme is Trypsin; the database is uniprot 20160315.
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