CN109613268A - Differential protein system and its screening technique and application in a kind of Erectile Dysfunction patients serum - Google Patents
Differential protein system and its screening technique and application in a kind of Erectile Dysfunction patients serum Download PDFInfo
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Abstract
The present invention provides a kind of Erectile Dysfunction patients serum differential protein systems, belong to proteomics research technical field；The Erectile Dysfunction patients serum differential protein system includes 27 species diversity albumen, and the albumen in the differential protein system can generate certain influence to people's penile erectile function.It lays a good foundation for the screening of detection, Erectile Dysfunction drug for above-mentioned differential protein system further progress Erectile Dysfunction illness and the further research for carrying out Erectile Dysfunction disease mechanism and study on prevention, provides foundation.
Description
Technical field
The invention belongs to proteomics research technical field more particularly to a kind of Erectile Dysfunction patients serums
Middle differential protein system and its screening technique and application.
Background technique
Erectile Dysfunction (Erectile Dysfunction, ED) refers to cannot reaching or cannot tieing up for duration
It holds and adequately erects to obtain satisfied sexual life, disease time was at least more than 6 months.With population in the world Aging Problem
Aggravation, the disease incidence of Erectile Dysfunction shows an increasing trend year by year, at present to Erectile Dysfunction patient's function
The effective treatment method restored is few, and means are more single, are that Erectile Dysfunction Erectile Dysfunction patient controls
Difficult maximum deficiency is treated, therefore is current hot and difficult issue to the study on prevention of Erectile Dysfunction.
It is more to cause the cause of disease that Erectile Dysfunction occurs, mainly includes psychological, organic, mixed type three classes.
It is psychological mainly due to a lack of or acceptance error sex education, by mental wound, the influence of the mental diseases such as anxiety-depression, husband
The uncoordinated equal generation of wife's relationship；Organic is mainly because of penile disease, the lesion of local vascular, nerve, other systemic diseases
Deng generation；Mixed type is mainly generated because of psychological and organic collective effect.Most of penile erectile function barriers at present
Hindering patient is Combination, and the means for treating Erectile Dysfunction are more single, are broadly divided into oral drugs and operation is controlled
Treat two kinds.
And it is less for the research of the Erectile Dysfunction on protein level, currently without a kind of efficient maturation
Method.
Summary of the invention
In view of this, the purpose of the present invention is to provide differential protein bodies in a kind of Erectile Dysfunction patients serum
System and screening technique and application.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical schemes:
A kind of Erectile Dysfunction patients serum differential protein system, the albumen system include albumen shown in table 1.
1 Erectile Dysfunction patients serum's differential protein system of table
Preferably, the albumen system includes albumen shown in table 2.
2 Erectile Dysfunction patients serum's differential protein preferred system of table
The present invention provides the proteosomes to tie up to the application in preparation detection Erectile Dysfunction kit.
It is tied up to the present invention provides the proteosome and prepares answering in Erectile Dysfunction drug screening kit
With.
The present invention provides the proteosomes to tie up in preparation prevention and/or treatment Erectile Dysfunction drug
Using.
The present invention also provides a kind of screening techniques of differential protein in Erectile Dysfunction patients serum, including with
Lower step:
1) separation and Extraction protein is sample protein matter from Erectile Dysfunction patient serum sample；From normal person
Separation and Extraction protein is reference protein matter in blood serum sample；
2) respectively enzymolysis step 1) in obtain sample protein matter and reference protein matter obtain sample peptide fragment and control peptide fragment；
3) sample peptide fragment is marked respectively with iTRAQ reagent and compare the control that peptide fragment obtains the sample peptide fragment and label of label
Peptide fragment；
4) the control peptide fragment for the sample peptide fragment and label for detecting the label respectively using LC-MS obtains sample peptide fragment
Mass Spectrometer Method data and control peptide fragment Mass Spectrometer Method data；
5) analytical procedure 4) obtain sample peptide fragment Mass Spectrometer Method data and control peptide fragment Mass Spectrometer Method data obtain
Sample protein data and reference protein data；
6) the sample protein data are compared with reference protein data, in the sample protein data with compare egg
The different albumen of expression quantity is differential protein in white data.
Preferably, differential protein described in step 6) includes the differential protein of expression quantity up-regulation and the difference that expression quantity is lowered
Albumen；The expression quantity of the differential protein of the expression quantity up-regulation in the sample is greater than 1.2 with the ratio of the expression quantity in compareing；
The expression quantity of the differential protein that the expression quantity is lowered in the sample with the ratio of the expression quantity in compareing less than 0.833, nothing
The expression quantity of the albumen of differential expression in the sample is with the ratio of the expression quantity in compareing between 1.2 to 0.833.
Preferably, 3 biology repetitions are arranged in Erectile Dysfunction patient serum sample in step 1).
Preferably, if a certain albumen the Erectile Dysfunction patient serum sample two or three biology
Expression quantity up-regulation is shown as in repeating, then the albumen is the differential protein of expression quantity up-regulation；If a certain albumen is vigorous in the penis
It rises in two or three biology repetition of dysfunction blood serum sample and shows as expression quantity downward, then the albumen is expression
Measure the differential protein lowered；If a certain albumen is repeated in a biology of the Erectile Dysfunction patient serum sample
In show as expression quantity up-regulation or lower, and other 2 biology repeat in show as indifference, then the albumen be expression quantity
Up-regulation or the differential protein lowered.
Beneficial effects of the present invention: differential protein system packet in Erectile Dysfunction patients serum provided by the invention
27 species diversity albumen are included, the albumen in the differential protein system can generate certain influence to people's penile erectile function.Needle
To the detection of above-mentioned differential protein system further progress Erectile Dysfunction illness, Erectile Dysfunction drug
Screening and further carry out Erectile Dysfunction disease mechanism research lay a good foundation with study on prevention, provide according to
According to.
Detailed description of the invention
Fig. 1 is that the SDS of protein integrity detects figure；
Fig. 2 is the canonical plotting of protein quantification；
Fig. 3 is the iTRAQ overhaul flow chart of Erectile Dysfunction patient candidate serum differential protein；
Fig. 4 is that Erectile Dysfunction patient candidate serum differential protein MRM verifies flow chart.
Specific embodiment
The present invention provides a kind of Erectile Dysfunction patients serum differential protein system, the albumen system includes
Albumen shown in table 1.
In the present invention, differential protein system includes 27 species diversity albumen in the Erectile Dysfunction patients serum,
In the albumen system 27 in differential protein be by MRM verifying Erectile Dysfunction patients serum and normal person's blood
The albumen of difference is implicitly present in clear, the differential protein has great meaning for the further investigation of Erectile Dysfunction
Justice.
Further, the present invention also provides a kind of preferred Erectile Dysfunction patients serum differential protein bodies
It is that the expression quantity variation of the albumen and the albumen in the preferred albumen system is shown in Table 3.
The expression quantity of protein classes and the albumen in 3 albumen system of table changes
In the present invention, the differential protein includes the differential protein of expression quantity up-regulation and the differential protein that expression quantity is lowered；
The expression quantity of the differential protein of the expression quantity up-regulation in the sample is greater than 1.2 with the ratio of the expression quantity in compareing；
The expression quantity of the differential protein that the expression quantity is lowered in the sample with the ratio of the expression quantity in compareing less than 0.833, nothing
The expression quantity of the albumen of differential expression in the sample is with the ratio of the expression quantity in compareing between 1.2 to 0.833.
It include 86 kinds of albumen in the preferred differential protein system in the present invention, wherein 48 kinds of protein expression up-regulations, 38
There were significant differences with the expressing quantity in normal human serum for the downward of kind protein expression, for the depth of Erectile Dysfunction
Entering research has greater significance.
The present invention also provides the proteosomes to tie up to the application in preparation detection Erectile Dysfunction kit.
Protein in heretofore described albumen system is the differential protein of Erectile Dysfunction patient and normal person；It is described
Albumen system can characterize Erectile Dysfunction to a certain extent；Therefore it can be used as detection Erectile Dysfunction
Testing index.
It is tied up to the present invention also provides the proteosome and prepares answering in Erectile Dysfunction drug screening kit
With.The expression quantity variation of albumen and Erectile Dysfunction tight association in albumen system of the present invention, can be as yin
The index of stem erectile dysfunction drug screening.
The present invention also provides the proteosomes to tie up in preparation prevention and/or treatment Erectile Dysfunction drug
Application.In the present invention, the expression quantity that the drug is preferably capable adjusting albumen in above-mentioned albumen system revert to normal person
The drug of corresponding expressing quantity range in serum.
The present invention also provides a kind of screening techniques of differential protein in Erectile Dysfunction patients serum, including with
Lower step: 1) separation and Extraction protein is sample protein matter from Erectile Dysfunction patient serum sample；From normal person
Separation and Extraction protein is reference protein matter in blood serum sample；2) respectively enzymolysis step 1) in obtain sample protein matter and control
Protein obtains sample peptide fragment and control peptide fragment；3) sample peptide fragment and control peptide fragment is marked to be marked respectively with iTRAQ reagent
Sample peptide fragment and label control peptide fragment；4) the sample peptide fragment of the label and pair of label are detected respectively using LC-MS
The Mass Spectrometer Method data of sample peptide fragment and the Mass Spectrometer Method data of control peptide fragment are obtained according to peptide fragment；5) analytical procedure 4) obtain sample
The Mass Spectrometer Method data of product peptide fragment and the Mass Spectrometer Method data of control peptide fragment obtain sample protein data and reference protein data；6)
The sample protein data are compared with reference protein data, in the sample protein data with table in reference protein data
It is differential protein up to different albumen is measured.
Present invention separation and Extraction protein from Erectile Dysfunction patient serum sample is sample protein matter；From just
Separation and Extraction protein is reference protein matter in ordinary person's blood serum sample.The present invention is from Erectile Dysfunction patient serum sample
Middle separation and Extraction protein preferably includes: the high-abundance proteins 1.1) removed in serum, obtains low-abundance protein system；1.2)
Reducing agent is added into the low-abundance protein system, opens the space structure of protein in low-abundance protein system, and uses iodine
Maintain the space structure of the protein in the open state for acetamide；1.3) space structure in the step 1.2) is in
The albumen of opening state is quantified.In the present invention, high-abundance proteins preferably use in the place to go serum in step 1.1)
ProteoMiner Protein Enrichment Kit is carried out, and concrete operations are referring to kit specification.The present invention removes blood
The reason of albumen of high abundance, is that there are very big influences on low-abundance protein identification for the albumen of high abundance in serum in clear, and
Most protein markers concentrate in middle low-abundance protein.By the enrichment of low-abundance protein, the shadow of high-abundance proteins is reduced
It rings, is conducive to the discovery of low-abundance protein marker.The present invention is after the step 1.1), by the low-abundance protein system of acquisition
It mixes and is handled with reducing agent, open the space structure of protein in low-abundance protein system；The reducing agent is preferably two
Sulphur threitol, final concentration of the reducing agent in the low-abundance protein system is preferably 8~12mM, more preferably 10mM；
The temperature of the processing is preferably 53~59 DEG C, and more preferably 56 DEG C, the time of the processing is preferably 40~80min, more excellent
It is selected as 60min.After the space structure of protein, iodoacetamido is added in opening low-abundance protein system in the present invention thereto
Amine；Final concentration of the iodo-acetamide in the low-abundance protein system is preferably 50~60mM, preferably 55mM；This
System after addition iodo-acetamide is preferably placed under dark surrounds and stands after the iodo-acetamide is added by invention
1h；The effect of heretofore described iodo-acetamide is to be alkylated histidine and cysteine in system, keeps protein empty
Between structure be maintained at the state being opened.The present invention is after the protein steric structure is maintained at the state being opened, to egg
White matter is quantified；The quantitative method is preferably bradford method.In the present invention, separates and mention from normal human sera samples
Take the method for protein consistent with the method for the separation and Extraction protein from Erectile Dysfunction patient serum sample.
The present invention digests the sample protein matter and reference protein after obtaining sample protein matter and reference protein matter respectively
Matter obtains sample peptide fragment and control peptide fragment.Before enzymatic hydrolysis of the present invention, preferably further include to the protein carry out ultrafiltration from
Heart washing；It is carried out after preferably cleaning solution is mixed with sample protein matter, the cleaning solution is preferably mass concentration 50%TEAB
Buffer.The molecular cut off of heretofore described ultrafiltration centrifuge washing is preferably 10KD, the centrifugation of the ultrafiltration centrifuge washing
Power is preferably 13000~15000g, more preferably 14000g, and the time of the ultrafiltration centrifuge washing is preferably 35~45min, more
Preferably 40min；The temperature of the ultrafiltration centrifuge washing is preferably 3~5 DEG C, and more preferably 4 DEG C.Heretofore described ultrafiltration from
Heart washing is preferred to be repeated 2~4 times.In the present invention, the enzyme used that digests is preferably trypsase；The trypsase
It is preferably 3.3:(90~110 with sample protein matter/reference protein matter mass ratio), more preferably 3.3:100.Institute in the present invention
The temperature for stating enzymatic hydrolysis is preferably 35~39 DEG C, and more preferably 37 DEG C；The time of the enzymatic hydrolysis is preferably 22~28h, more preferably
24h；In the present invention, the enzymatic hydrolysis preferably carries out in a water bath.The present invention preferably uses mass concentration after the enzymatic hydrolysis
50%TEAB buffer redissolves protein and obtains sample peptide fragment.The preparation method and sample peptide fragment of heretofore described control peptide fragment
Unanimously, details are not described herein.
The present invention marks sample peptide fragment and right after obtaining the sample peptide fragment and control peptide fragment, with iTRAQ reagent respectively
The sample peptide fragment of label and the control peptide fragment of label are obtained according to peptide fragment.The present invention is not particularly limited the method for the label,
Using this field conventional labels method, in specific implementation process of the invention, preferably using iTRAQ labelling kit into
Line flag.The present invention does not have special limitation to the source of the iTRAQ labelling kit, using the iTRAQ of this field routine
Labelling kit.Kit used in specific implementation process of the present invention is 4 mark iTRAQ reagents, iTRAQ Reagents-
4plex kits；The producer of the kit is SCIEX；It, can be to protein digestion including the isotope reagent of 4 kinds of equivalent
Peptide fragment is marked afterwards, to can carry out accurate identification to peptide fragment in conjunction with the methods of tandem mass spectrum and quantify；The label
Concrete operations are referring to kit specification.
The present invention detects the sample peptide fragment of the label and pair of label using LC-MS after the marker respectively
The Mass Spectrometer Method data of sample peptide fragment and the Mass Spectrometer Method data of control peptide fragment are obtained according to peptide fragment.
In the present invention, pre-separation is carried out to the label peptide fragment in the system after the label using high performance liquid chromatography, is obtained
To the sample peptide fragment of label and the control peptide fragment of label.The present invention is not particularly limited the equipment for carrying out high performance liquid chromatography,
Using conventional high-performance liquid chromatograph.In specific implementation process of the present invention, preferably using SCX strong cat ion exchange column point
From using 20%~30% gradient eluent (10mM KH in pH=3.0 phosphate buffer2PO4, 2M KCl, 25%
ACN, pH=3), elution requirement and gradient reference instrument and consumptive material concrete operations illustrate, peptide fragment after elution label.
The present invention after peptide fragment, purifies peptide fragment after the label, obtains peptide fragment after purification after being marked；This hair
Desalting processing is carried out to the peptide fragment after label using C18 reverse-phase chromatography in bright specific implementation process.
The present invention carries out Mass Spectrometer Method after obtaining peptide fragment after purification, to the peptide fragment after purification, obtains the original text of mass spectrum
Part；The equipment of the Mass Spectrometer Method is preferably Q-Exactive mass spectrograph.In the present invention, the parameter setting of the Mass Spectrometer Method is excellent
Choosing is shown in Table 4.
The parameter setting of 4 Mass Spectrometer Method of table
The present invention analyzes the Mass Spectrometer Method data and control peptide fragment of the sample peptide fragment of acquisition after obtaining the mass spectrum
Mass Spectrometer Method data obtain sample protein data and reference protein data；In the present invention, the analysis includes that mass-spectrogram sieves
Mass-spectrogram search and protein quantification after choosing, screening.In the present invention, the mass-spectrogram screening preferably uses PD
(Proteome Discoverer 1.3, thermo) software；The mass-spectrogram screening parameter setting of the PD software is shown in Table 5.
The mass-spectrogram screening parameter of 5 PD software of table is arranged
| Parent ion mass range | 350-6000Da |
| Minimum peak number in second order ms figure | 10 |
| Signal-to-noise ratio S/N threshold value | 1.5 |
In the present invention, the mass-spectrogram search after the screening is preferably carried out using mascot software；Described search
Parameter setting is preferably shown in Table 6.
The parameter setting that table 6 is searched for
The present invention carries out quantifying for albumen after described search.In the present invention, the quantitative of the albumen preferably uses PD
Software carries out.7 are shown in Table using the parameter setting that PD software carries out the protein quantification.
The parameter setting of 7 protein quantification of table
The present invention obtains sample protein data and reference protein data after the protein quantification；By the sample protein
Data are compared with reference protein data, the albumen different from expression quantity in reference protein data in the sample protein data
For differential protein.In the present invention, the Erectile Dysfunction patient serum sample is preferable to provide 3 biology and repeats,
When analyzing expressing quantity variation, determine that expressing quantity up-regulation or the standard lowered are preferably as follows: if a certain albumen is in institute
It states in two or three biology repetition of Erectile Dysfunction patient serum sample and shows as expression quantity up-regulation, then the egg
The white differential protein for expression quantity up-regulation；If a certain albumen at two of the Erectile Dysfunction patient serum sample or
Three biology show as expression quantity downward in repeating, then the albumen is the differential protein that expression quantity is lowered；If a certain albumen exists
Expression quantity up-regulation is shown as in the biology repetition of the Erectile Dysfunction patient serum sample or is lowered, and
Other 2 biology shows as indifference in repeating, then the albumen is the differential protein that expression quantity is raised or lowered.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
1.1 research objects select No.1 Hospital Attached to Xinjiang Medical Univ.'s in December, 2016 in May, 2015-and the 4th attached
Belong to the outpatient service of andrology, hospital and impotence patient 90, normal healthy people 30 made a definite diagnosis that are hospitalized.The diagnostic criteria of impotence is referring to 1993
Subject to the definition of year National Institutes of Health.Impotence patient is included in standard: (1) it is married, live together, living conditions it is good；
(2) male of the age at 20~60 years old；(3) patient is without serious organic disease and spirit, the nervous system disease；(4) spouse without
Serious organic disease can sufficiently cooperate requirement of experiment.The exclusion criteria of impotence patient: (1) the device matter type impotence patient made a definite diagnosis,
Such as wound, venous fistula type, arteriosity, tunica albuginea damage type；(2) medicine type impotence, such as hypertension agents, estrogen；(3) patient closes
And have cardiovascular, liver kidney and hemopoietic system severe primary disease, mental disease or it is not able to cooperate person；(4) spouse is tight with whole body
Weight organic disease, such as liver failure, renal failure, heart failure.Normal person's standard is through physical examination and electrocardiogram
(ECG), Chest X-rays, blood urine routine, blood glucose, hepatic and renal function etc. check, are not in the mood for, liver, brain, lung, kidney, endocrine, the main device such as immune
The organic disease of official's system, penile erectile function is normal, there is normal sexual life, index >=22 IIEF-5.
The classification of 1.2 impotence weights and 5 (international of sexual desire evaluation criteria application international index of erectile function
Index of erectile function 5items, IIEF-5) questionnaire table progressive functional assessment, content includes: pair
It is inserted into success rate after the degree of confidence of erection function, erection, maintains satisfaction after erectile condition, success rate of sexual intercourse, sexual intercourse.
For each problem of IIEF-5 according to 1~5 point of evaluation score, total score is 25 points.Evaluation criteria: wherein it is for 5~7 points of IIEF table integral
Severe impotence, 8~11 points are moderate impotence, and 12~21 points are slight impotence, and >=22 points are no impotence.Using international male's sexual desire
Scale -4 (malesexualfunction4items, MSF-4) questionnaire table carries out sexual desire assessment, and content includes: subjective
To the hope of sexual life, the satisfaction of erection function, console oneself or sexual intercourse after climax reach satisfaction, ejaculation be satisfied with journey
Degree.Each problem presses 1~4 point of evaluation score, and total score is 16 points.The early fasting blood before the meal of acquisition is simultaneously centrifugated serum, protects
In the presence of to be measured in -80 DEG C of low temperature refrigerators.
1.3 iTRAQ detect impotence patient serum candidate serum differential protein
1.3.1 Protein Extraction
(1) ProteoMiner (brand: BIO-RAD, article No.: 163-3007, specification: 10/box, kit full name:
ProteoMiner Protein Enrichment Kit) remove high-abundance proteins in serum；Specific steps are said referring to kit
Bright book:
A) it is centrifuged pillar, 1000g is centrifuged 30~60s, removes the liquid storage in pillar, abandons the waste liquid in collecting pipe.
B) bottom cover is loaded onto, 250 μ l wash buffer, and top cover is added.5min is shaken on earthquake device, makes column material
It sufficiently mixes with wash buffer, and sufficiently elutes.
C) bottom cover is removed, pillar is put into 1000g in collecting pipe and is centrifuged 30~60s, abandons waste liquid.
D) b)-c is repeated) step
E) bottom cover is covered, the column material of 200 μ l is activated good at this time.
F) sample needs not precipitate, and 10000g centrifugation 10min goes to precipitate.
G) 250 μ l samples are added, top cover is added, room temperature concussion 2h on earthquake device.
H) bottom cover is removed, 1000g is centrifuged 30~60s, abandons waste liquid.
I) add bottom cover, 250 μ lwashbuffer are added, in addition top cover, shakes 5min.
J) it goes bottom cover 1000g to be centrifuged 30~60s, abandons waste liquid.
K) i)-j is repeated) twice.It is added after wash buffer and is got to the column material suction on wall in solution every time with rifle.
L) 250 μ l ddH are added2O。
M) it covers, shakes 1min.
N) top cover and bottom cover are gone, 1000g is centrifuged 30~60s, abandons waste liquid.
O) bottom on is added 60 μ l Elution Buffer (4M urea, 1% (w/v) CHAPS, 5% acetic acid).
P) upper cover, whirlpool shake 15min.
Q) top cover and bottom cover are gone, 1000g is centrifuged 1min.
R) o)-q is repeated) step one is to twice.
S) -20 DEG C of haemocyanins saved after enrichment or directly progress next step experiment.
(2) DTT is added in supernatant (DTT-- dithiothreitol (DTT), reducing agent open protein steric structure, opened disulfide bond)
To final concentration 10mM.56 DEG C of water-bath 1h.After taking-up, being rapidly added IAM, (IAM-- iodo-acetamide, makes histidine and cysteine
Alkylation, makes protein steric structure be maintained at the state being opened.) to final concentration 55mM, darkroom stands 1h.
(3) quantitative, albumen is quantified using bradford method.
Bradford quantitative work process:
A) measurement of standard curve
Prepare: protein standard liquid storage (2 μ g/ μ L of BSA original liquid concentration) being taken out and is restored to room temperature；10 times are diluted to 0.2 μ g/
μ L, as protein standard solution.
Bradford, which measures working solution and takes out, restores room temperature；
Prepare lysate identical with sample and distilled water according to measurement dosage.
B) 96 hole enzyme mark versions are numbered, reagent is added according to the form below 8, mixes, is placed at room temperature for 0.5min and is put into spectrophotometer
It is measured.
Volume is added in 8 96 hole elisa Plates reagent of table
C) measurement of sample
96 hole enzyme mark versions are numbered, reagent and sample is added by table 9, mixes, is placed at room temperature for 0.5min and is put into spectrophotometric
Meter is measured.
9 sample of table measures reagent and volume is added
D) data processing
In Excel worksheet, using absorbance as ordinate y, reactive protein amount (μ g) is abscissa x, and it is bent to draw standard
Line returns out linear equation y=a (x)+b, linearly dependent coefficient r square value, using the average value of sample measurement result as measurement
Value y (absorbance value, simple for number), reactive protein amount (μ g) x value of sample to be tested is calculated with the linear equation returned out.
Quantitation curves are as shown in Figure 1.
Quantitative result is as shown in table 10, wherein 1: normal group (N group)；2: erectile dysfunction group (ED1 group)；3: erection function
Energy obstacle group (ED2 group) 4: erectile dysfunction group (ED3 group)
10 protein quantification result of table
SDS detects protein integrity as shown in Fig. 2, albumen is complete, no degradation.If albumen has degradation, under cannot continuing
The experiment of one step needs to separate again.
1.3.2 the digestion of protein
(1) each sample takes 100 μ g albumen volumes, is added in 10K super filter tube, 14000g, 4 DEG C, is centrifuged 40min, abandons
Waste liquid.
(2) 200ul 50%TEAB, 14000g, 4 DEG C is added, is centrifuged 40min, abandons waste liquid.
(3) it repeats the above steps twice.
(4) Trypsin of 1 μ g/ μ l is added, the enzyme of 3.3 μ g is added in every 100 μ g protein substrate, and 37 DEG C of water-baths are for 24 hours.Freeze
Then dry digestive juice redissolves peptide fragment using TEAB (water: TEAB=1:1) every 30 μ l of pipe.
1.3.3 the label of peptide fragment
(1) labelled reagent is balanced to room temperature.
(2) every pipe labelled reagent (100 μ g albumen of reagent label of a unit.) 70 μ l isopropanols of middle addition, it mixes
1min, centrifugal drying to tube bottom.
(3) mixed labelled reagent is added in peptide fragment, the different size of isotope labelling of different samples.
(4) it after mixing, gets rid of to tube bottom, is stored at room temperature 2h.
(5) vacuum drains sample after label.
1.3.4 HPLC pre-separation
Use conventional liquid phase, strong cation exchange chromatography column (SCX)
(1) reagent and preparation of samples
Sample: sample is that 10 times are diluted with A liquid after label, and phosphoric acid tune pH to 3.0,15000g is centrifuged 10min, takes supernatant.
The ingredient of A liquid: 25%ACN, 10mM KH2PO4, with phosphoric acid tune pH value to 3.0
The ingredient of B liquid: 25%ACN, 2M KCL, 10mM KH2PO4, with phosphoric acid tune pH value to 3.0.
(2) separation method is set
(3) loading is run according to the separation gradient set.This pre-separation is mixed into B liquid since 31min, and 38min is opened
Beginning appearance, 1min collect 1 component, collect 26 components altogether, are distributed according to chromatogram, the less part of appearance are merged, most
It is merged into 16 components eventually and carries out subsequent processing.Specific gradient is shown in Table 11.
11 HPLC gradient of table
| Time(min) | B |
| 0.01 | Start (0%) |
| 45 | 0% |
| 46 | 5% |
| 66 | 30% |
| 71 | 50% |
| 76 | 50% |
| 81 | 100% |
| 91 | 100% |
| 91.01 | Stop |
1.3.5 the purifying of peptide fragment
Using C18 reverse-phase chromatography desalination, steps are as follows:
(1) 1ml methanol activates column material.
(2) 5%ACN is balanced.
(3) sample dissolved column with 1ml MilliQ water.
(4) 5% acetonitrile of 1ml washes column desalination.
(5) the pure acetonitrile elution of 2*500 μ l.
(6) low-temperature centrifugation drains acetonitrile.
(7) 0.1% formic acid redissolve peptide fragment after purification.
1.3.6 Mass Spectrometer Method
By the component of 16 pre-separations after purification, machine testing is gone up respectively.
Q-Exactive mass spectrograph detects peptide segment signal, and parameter is as shown in table 4.
The elution requirement and gradient parameter of NanoLC:
A liquid ingredient: water, 0.1%FA
B liquid ingredient: acetonitrile, 0.1%FA
Chromatographic column: Type C 18, specification 100mm*75mm, aperture 300A, 5 μm of partial size
Flow velocity: 400nl/min
Gradient is shown in Table 12.
The gradient of 12 NanoLC of table
| Time(min) | B |
| 0 | 5% |
| 10 | 5% |
| 40 | 30% |
| 45 | 60% |
| 48 | 80% |
| 55 | 80% |
| 58 | 5% |
| 65 | 5% |
| 65.01 | Stop |
1.3.7 protein quantification and qualification
(1) scanning of the mass spectrum finishes, and obtains mass spectrum original document
(2) after mass spectrum original document being input to PD (Proteome Discoverer 1.3, thermo) software, software
Screening extraction can be carried out to mass spectrogram.
Mass-spectrogram screening parameter is as shown in table 5.
(3) spectrogram after PD is extracted scans for (parameter of search is shown in Table 6), after search, PD with MASCOT software
Software according to mascot search result and the first step screening after spectrogram carry out protein quantification (protein quantification is completed by PD software,
7) quantitative parameter is shown in Table.
The parameter setting of 7 protein quantification of table
Wherein, Protein ratio Type: for quantitative peptide fragment obtaining value method, median refers to taking middle position
Method.
Minimum peptides: for quantitative least unique peptide number
Normalisation method: antidote, median, which refers to choosing in one group of sample, can all quantify egg
White median is corrected.
(4) obtained analysis data are retrieved in people (uniprot20160315) database, obtains penile erectile function
Impaired patients serum candidate's differential protein.
The screening technique of 2 Erectile Dysfunction patients serum's candidate's differential proteins and list
The screening technique of impotence patient serum candidate's differential protein albumen:
The each group differential protein that will be obtained by iTRAQ joint LC-MS-MS technology, wherein ED group 90 are divided into 3 groups, ED1
Group, ED2 group and ED3 group, N group are normal group, and specific screening technique is, ED each group compared with N histone matter, obtain ED1/N,
The differential protein of ED2/N and ED3/N；This three groups of differential proteins are counted, the type and quantity of three groups of common albumen are found,
In three groups jointly up-regulation (or lower) be in up-regulated expression (or downward) or two groups up-regulation (or downward) be up-regulated expression (under
Mileometer adjustment reaches) or three groups in one group of expression it is variant, remaining two groups of indifference, with this group expression be final expression.
86 kinds of candidate albumens wherein 48 kinds of albumen up-regulated expressions are obtained altogether, and 38 kinds of albumen lower expression；The name of specific albumen
Claim and expression quantity situation is shown in Table 2.
Embodiment 2
The haemocyanin of ED each group and Normal group, the extraction of albumen and purification process and parameter are extracted with embodiment 1,
Peptide fragment after purification is identified through mass spectrograph.
1, peptide fragment after purification is redissolved, each sample takes 2 μ g to be uniformly mixed, and 2 μ g of mixing sample is taken to carry out Mass Spectrometer Method
(QE mass spectrograph, shotgun method).
2, scanning of the mass spectrum finishes, and the mass spectral results that shotgun is obtained imported into Thermofisher Proteome
Discoverer 1.4, software carry out data retrieval, obtain Identification of Fusion Protein result.
3, QE the MS detection parameters and its liquid phase parameter are shown in the ITRAQ parameter of embodiment 1.
4, according to qualification result, the comprehensive default target to be originated from iTRAQ carries out the foundation of MRM method.
5, the peptide fragment that target proteins meet MRM standard is extracted with Skyline software, with uniport_human_9606_
20180824 databases are background, analyze extracted peptide fragment whether be target proteins specific peptide fragment.
The MRM of experiment sample is detected
1, experiment sample each takes 2 μ g to carry out corresponding target MRM detection respectively, and in triplicate.
2,6500 mass spectrograph detection parameters and its liquid phase parameter see the table below
It is as follows that MRM verifies mass spectrometry parameters:
Mass spectrograph: ABSCIEX6500
Nanoliter liquid phase: 425 nano LC of Eksigent
MRM is quantitative-nanoliter liquid-phase chromatographic column and mobile phase
MRM quantifies-nanoliter liquid phase gradient
MRM quantifies-Nano-ESI source parameters
MRM quantifies -6500 mass spectrometer parameters
MRM quantifies-target peptide fragment detection parameters
* for the peptide fragment of different mass-to-charge ratioes, fragmentation amount: CE (collisionenergy) is calculated according to following formula: | CE |
=(slope) * (m/z)+(intercept)
3, testing result is corresponding target signal response diagram.
4, corresponding integrating peak areas result is that MRM quantifies original value.
5, it is corrected by the external standard between sample.Correction reference is the immediate external standard peptide fragment of each peptide fragment RT.
6, mean value is taken between three repetitions after correcting, as peptide fragment quantitative values.
7, the peptide fragment quantitative values of each albumen, take mean value as protein quantification value.
8, protein quantification value compares analysis and obtains the ratio value of MRM quantitative comparison.
9, MRM result and iTRAQ result are compared, discovery has 27 kinds of candidate differential protein verification results and ITRAQ result
Unanimously, 13 be the results are shown in Table.
13 MRM result of table and iTRAQ result uniformity comparison
As can be seen from the above embodiments, differential protein system packet in Erectile Dysfunction patients serum provided by the invention
27 species diversity albumen are included, the albumen in the differential protein system is the differential protein verified by MRM, for above-mentioned difference egg
The detection of lean type system further progress Erectile Dysfunction illness, the screening of Erectile Dysfunction drug and into one
The research that step carries out Erectile Dysfunction disease mechanism is laid a good foundation with study on prevention, provides foundation.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of Erectile Dysfunction patients serum differential protein system, which is characterized in that the albumen system include with
Lower albumen:
2. albumen system according to claim 1, which is characterized in that the albumen system includes following albumen:
3. proteosome of any of claims 1 or 2 ties up to the application in preparation detection Erectile Dysfunction kit.
4. proteosome of any of claims 1 or 2 ties up to the application prepared in Erectile Dysfunction drug screening kit.
5. proteosome of any of claims 1 or 2 ties up to preparation prevention and/or treats answering in Erectile Dysfunction drug
With.
6. the screening technique of differential protein in a kind of Erectile Dysfunction patients serum, comprising the following steps:
1) separation and Extraction protein is sample protein matter from Erectile Dysfunction patient serum sample；From normal human serum
Separation and Extraction protein is reference protein matter in sample；
2) respectively enzymolysis step 1) in obtain sample protein matter and reference protein matter obtain sample peptide fragment and control peptide fragment；
3) sample peptide fragment is marked respectively and is compareed peptide fragment with iTRAQ reagent obtain the sample peptide fragment of label and the control peptide of label
Section；
4) the control peptide fragment for the sample peptide fragment and label for detecting the label respectively using LC-MS obtains the matter of sample peptide fragment
It composes detection data and compares the Mass Spectrometer Method data of peptide fragment；
5) analytical procedure 4) obtain sample peptide fragment Mass Spectrometer Method data and control peptide fragment Mass Spectrometer Method data obtain sample
Protein data and reference protein data；
6) the sample protein data are compared with reference protein data, in the sample protein data with reference protein number
It is differential protein according to the different albumen of middle expression quantity.
7. screening technique according to claim 6, which is characterized in that differential protein described in step 6) includes on expression quantity
The differential protein that the differential protein and expression quantity of tune are lowered；The expression quantity of the differential protein of expression quantity up-regulation in the sample with
The ratio of expression quantity in control is greater than 1.2；The expression quantity of the differential protein that the expression quantity is lowered in the sample with right
The ratio of expression quantity according in is less than 0.833, the expression quantity of the albumen that indifference schedule reaches in the sample and the expression in compareing
The ratio of amount is between 1.2 to 0.833.
8. screening technique according to claim 6 or 7, which is characterized in that Erectile Dysfunction patient in step 1)
Blood serum sample is arranged 3 biology and repeats.
9. screening technique according to claim 8, which is characterized in that if a certain albumen is in the Erectile Dysfunction
Two or three biology of patient serum sample show as expression quantity up-regulation in repeating, then the albumen is the difference of expression quantity up-regulation
M-band；If a certain albumen table in two or three biology of the Erectile Dysfunction patient serum sample repeat
It is now lowered for expression quantity, then the albumen is the differential protein that expression quantity is lowered；If a certain albumen hinders in the penile erectile function
Hinder and show as expression quantity up-regulation in the biology repetition of patient serum sample or lower, and is repeated in other 2 biology
In show as indifference, then the albumen be expression quantity raise or lower differential protein.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101164586A (en) * | 2006-10-18 | 2008-04-23 | 山西太行药业股份有限公司 | Medicinal composition for treating sexual impotence and preparation process thereof |
| CN106645748A (en) * | 2016-12-16 | 2017-05-10 | 新疆医科大学 | Mammalian differential serum protein and differentially expressed protein system of penile smooth muscle tissue of abnormal and phlegmatic type of impotence syndrome and screening method and application |
| CN106916217A (en) * | 2017-03-17 | 2017-07-04 | 华南农业大学 | Using the method for iTRAQ technical research Chronic Infection of Toxoplasma Mice brain tissues differential expression protein groups |
| US20180299461A1 (en) * | 2015-04-30 | 2018-10-18 | DH Technologies Development Pte Ltd. | Identification of Glycosylation Forms |
| CN108872599A (en) * | 2018-07-09 | 2018-11-23 | 清紫生物科技（深圳）有限公司 | PDLIM3 (PDZ and LIM domain 3) is used as the application of stomach cancer marker |
| CN108918868A (en) * | 2018-07-19 | 2018-11-30 | 中国医学科学院北京协和医院 | It is a kind of detect osteoarthritis marker and its application |
| CN109142565A (en) * | 2018-07-27 | 2019-01-04 | 重庆早柒天生物科技股份有限公司 | The screening technique of premature rupture of fetal membranes pregnant woman's vaginal fluid differential protein based on iTRAQ technology |
-
2019
- 2019-01-09 CN CN201910020695.2A patent/CN109613268B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101164586A (en) * | 2006-10-18 | 2008-04-23 | 山西太行药业股份有限公司 | Medicinal composition for treating sexual impotence and preparation process thereof |
| US20180299461A1 (en) * | 2015-04-30 | 2018-10-18 | DH Technologies Development Pte Ltd. | Identification of Glycosylation Forms |
| CN106645748A (en) * | 2016-12-16 | 2017-05-10 | 新疆医科大学 | Mammalian differential serum protein and differentially expressed protein system of penile smooth muscle tissue of abnormal and phlegmatic type of impotence syndrome and screening method and application |
| CN106916217A (en) * | 2017-03-17 | 2017-07-04 | 华南农业大学 | Using the method for iTRAQ technical research Chronic Infection of Toxoplasma Mice brain tissues differential expression protein groups |
| CN108872599A (en) * | 2018-07-09 | 2018-11-23 | 清紫生物科技（深圳）有限公司 | PDLIM3 (PDZ and LIM domain 3) is used as the application of stomach cancer marker |
| CN108918868A (en) * | 2018-07-19 | 2018-11-30 | 中国医学科学院北京协和医院 | It is a kind of detect osteoarthritis marker and its application |
| CN109142565A (en) * | 2018-07-27 | 2019-01-04 | 重庆早柒天生物科技股份有限公司 | The screening technique of premature rupture of fetal membranes pregnant woman's vaginal fluid differential protein based on iTRAQ technology |
Non-Patent Citations (2)
| Title |
|---|
| ESTHER SOK HWEE CHEOW 等: "Myocardial Injury Is Distinguished from Stable Angina by a Set of Candidate Plasma Biomarkers Identified Using iTRAQ/MRM-Based Approach", 《JOURNAL OF PROTEOME RESEARCH》 * |
| 马文静 等: "基于iTRAQ标记结合IPA生物分析平台筛选异常黏液质型阳痿病证大鼠血清蛋白质组学研究", 《中国男科学杂质》 * |
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