CN108398494A - The screening of the more unrestrained sheep physiological period breeding GAP-associated protein GAPs in Xinjiang and identification method - Google Patents
The screening of the more unrestrained sheep physiological period breeding GAP-associated protein GAPs in Xinjiang and identification method Download PDFInfo
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Abstract
The invention discloses screening and identification methods that unrestrained sheep physiological period more than a kind of Xinjiang breeds GAP-associated protein GAP, belong to biotechnology, this approach includes the following steps：The acquisition of step 1, the more unrestrained sheep physiological period ovaries in Xinjiang：Step 2, protein extraction；The drafting of step 3, Bradford protein quantifications and standard curve；Step 4, sample label and mixed in equal amounts；The HPLC classifications of C18 chromatographic columns under step 5, high pH condition；Step 6, mass spectrometry procedure 7, data processing.The present invention is marked by iTRAQ reagents, and labeled peptide fragment carries out high-efficient liquid phase chromatogram HPLC separation and Mass Spectrometer Method MS, identifies 4847 albumen altogether, wherein the protein 47 0 of expression significant difference.
Description
Technical field
The invention belongs to biotechnologies, specifically, being related to a kind of more unrestrained sheep physiological period breeding phases in Xinjiang
Close screening and the identification method of albumen.
Background technology
More wave sheep have the good characteristics of long-term heat, throughout the year can heat, by long and short sunshine rule influenced compared with
It is small, it can be bred by " 1 year two tire " or " 2 years triplets ".And most of kind sheep is seasonal oestrus, by sunshine rule
It is affected, it is general only in summer and autumn heat, it can only be bred by 1 year tire.In scale, intensive culture trend
Under, where the breeding potential that excavates domestic animal to greatest extent is the pursuit of economic benefit, and to significantly limit sheep numerous for seasonal oestrus
Grow the performance of potential.From the point of view of Literature Consult and domain expert report, at present to the long-term heat of sheep and seasonal oestrus
Molecular genetic mechanism is also not very clear, only has to the major physiological approach of seasonal oestrus and substantially understands.No matter right and wrong
The virus of cell biological, or there is cyto-architectural prokaryotes and eucaryote, inhereditary material plays a role final
Executor is protein, and function and the activity of protein determine the movable all aspects of organism.
Isotope labelling is opposite and absolute quantitation (isobaric tags for relative and absolute
Quantitation, iTRAQ) technology have extraordinary accuracy and repeatability, answered extensively in molecular biology field
With.The technology is developed by Applied biosystems (Applied Biosystems Incorporation, ABI), is
A method of new, powerful opposite and absolute quantitation research.ITRAQ reagents are made of three parts：Reporter group (4
Mark experiment relative molecular mass is respectively 114,115,116 and 117；8 mark experiment relative molecular masses are respectively 113,114,
115,116,117,118,119 and 121)；(4 mark experiment relative molecular masses are respectively 31,30,29 and 28 to mass balance group；
24) and peptide reaction marking agent radical 8 mark experiment relative molecular masses are respectively 32,31,30,29,28,27,26 and, form 4
Kind or 8 kinds of relative molecular masses are 145 equivalent dystopy label.ITRAQ quantitative approach can be in a tandem mass spectrometry experiment
In compare 4 relative amounts at most up to the albumen in 8 samples simultaneously, have the characteristics that high efficiency, high precision.But mesh
Before, which is applied to the screening and identification of more unrestrained sheep physiological period ovary GAP-associated protein GAPs not yet.
Invention content
It is an object of the invention to make up blank in the prior art, it is proposed that a kind of more unrestrained sheep physiological periods in Xinjiang
Breed screening and the identification method of GAP-associated protein GAP.This method using isotope labelling is opposite and absolute quantitation to wave sheep more than Xinjiang not
GO functional analyses, KEGG path analysis and COG analyses are identified and made with physiological periods ovarian protein.
Its technical solution is as follows：
A kind of more unrestrained sheep physiological periods in Xinjiang breed screening and the identification method of GAP-associated protein GAPs, include the following steps：
The acquisition of step 1, the more unrestrained sheep physiological period ovaries in Xinjiang：
By estrus synchronization, detection of oestrus and pregnant identification technology, by unrestrained sheep more than dioestrus, oestrus and the gestational period
(raising of Tarim University's animal experiment station) takes out ovary immediately after butchering, and PBS buffer solution is used in combination to rinse (PH7.4) tissue table
Face, input liquid nitrogen is rapidly frozen rapidly later, and is taken back -80 DEG C of laboratory and saved backup.
Step 2, protein extraction：
It takes dioestrus, oestrus and the gestational period more unrestrained sheep ovaries to take 0.1g respectively, the protein lysate of 0.4mL is added
(8.0M ureas, 50mM NH4HCO3,1 × protease inhibitors) carries out ultrasonication, is set as each 2s, is spaced 10s, altogether
5min, whole operation carry out on ice.After ultrasonication object places 20min on ice, 4 DEG C, 10,000 × g, 30min is centrifuged,
It takes supernatant to be transferred to new pipe, then dispense protein extract and is saved backup in -80 DEG C of refrigerators.
The drafting of step 3, Bradford protein quantifications and standard curve
Albumen is quantified using Bradford methods, the bovine serum albumin(BSA) of known content (BSA) is first made into difference
The protein solution of concentration measures the absorbance value (OD values) after its colour developing, according to linear between OD values and albumen concentration respectively
Relationship draws out the standard curve of albumen concentration, then measures the OD values after the colour developing of testing protein solution, bent with reference to BSA standards
The concentration of line computation testing protein.All operations step is completed in room temperature state.
BSA8 μ g, 10 μ g, 12 μ g, 14 μ g, 16 μ g, 18 μ g are taken to be made into the protein solution of various concentration, and measure suction respectively
Light value, if correlation R2<0.95, standard curve cannot be used for calculating, and re-start experiment.
Protein solution is configured according to calculating, total volume is 20 μ L；Prepare to be detected sample 1.0uL simultaneously, be mixed with 19uL water,
Detection ELISA Plate, 2 multiple holes of each pattern detection are added.200 μ L Bradford working solutions are added in each multiple holes, and slight inhale is beaten
Mixing.It is protected from light, the static placement 15min of room temperature.It is detected with microplate reader, Detection wavelength is set in 595nm.It is derived according to standard curve
Go out to detect the total amount of albumen, and calculates respective concentration.Step 4, sample label and mixed in equal amounts
Each group sample peptide fragment takes about 100 μ g respectively, according to AB companies kit：iTRAQ Reagent-8plex
Multiplex Kit (AB SCIEX) specification is marked, and a small amount of sample is taken to mix after label, carries out LC-MS/MS mass spectrums
Detection, checks iTRAQ labeling effciencies.
The HPLC classifications of C18 chromatographic columns under step 5, high pH condition
Step 6, mass spectral analysis
Capillary high performance liquid chromatography
Every part of sample is detached using a nanoliter flow velocity HPLC liquid phase systems Easy nLC 1000.Buffer solution：A liquid is
0.1% aqueous formic acid, B liquid are 0.1% formic acid acetonitrile solution.Chromatographic column is balanced with 95% A liquid.Sample is by autosampler
Mass spectrum pre-column C18trap column (3 μm of 100 μ m 2cm of C18) are loaded to, then through analytical column C18column (3 μ of C18
M75 μ m 15cm) separation, flow velocity 300nl/min.
Mass Spectrometric Identification
Every part of sample uses Orbitrap Fusion lumos mass spectrograph mass spectrographs after capillary high performance liquid chromatography detaches
(Thermo Scientific) is analyzed by mass spectrometry.
Step 7, data processing
Raw mass spectrum data
Mass spectral analysis initial data is RAW files, with software software Sequest and Proteome Discoverer
(Thermo Scientific) carries out searching library Qualitative and quantitative analysis.
Database
This uses database：protein.selected.fasta
Search library
RAW files are committed to Sequest servers by Proteome Discoverer when searching library, selection has been built
Then the database stood carries out database search.
Further, step 5 is specially：
1. the 100 μ l mobile phase As of sample after label is drained are redissolved, vortex oscillation, 14,000g centrifugation 20min are drawn
Supernatant loading；
2. preparing 60 blank 1.5ml centrifuge tubes, it is labeled as 1-60 successively, for collecting isolated component 1-60；
3. flow velocity 0.7ml/min；
4. since the 5th minute, collect successively in every 1.5 minutes eluates to 1-60 centrifuge tubes；
5. vacuum refrigeration centrifugal drying；
6. the sample after drying is molten with the FA weights of 5ul 0.5%, and be collected into 60 components are merged into 6 groups
Point；
7. by the component 14,000g after merging, 10min is centrifuged, draws supernatant loading；It is general to draw 10ul.
Beneficial effects of the present invention are：
The present invention is marked by iTRAQ reagents, and labeled peptide fragment carries out high performance liquid chromatography (HPLC) separation and mass spectrum
It detects (MS), identifies 4847 albumen altogether, wherein the protein 47 0 of expression significant difference.Oestrus compared with dioestrus,
Upregulated protein 18；Down-regulation protein 102.Dioestrus is compared with the gestational period, upregulated protein 60, down-regulation protein 20；Hair
The feelings phase compared with the gestational period, upregulated protein 24, down-regulation protein 50；There is follistatin-related in upregulated protein
protein 1isoform X2[Ovis aries]、cleavage stimulation factor subunit 1isoform
X1、integrin alpha-M precursor、cyclin-Y-like protein 1isoform X1、cyclin-
Histone H2B type 2-F, cyclin-Y isoform in dependent kinase 16isoform X1 down-regulation proteins
The regulation and control of generation, the heat of the albumen such as X1, heat shock protein beta-6isoform X1 and hormone, the production of zygote
It gives birth to related with functions such as apoptosis, the attached plant of body early embryo and embryonic developments.
Specific implementation mode
Technical scheme of the present invention is described in more detail With reference to embodiment.
A kind of more unrestrained sheep physiological periods in Xinjiang breed screening and the identification method of GAP-associated protein GAPs, include the following steps：
The acquisition of step 1, the more unrestrained sheep physiological period ovaries in Xinjiang：
By estrus synchronization, detection of oestrus and pregnant identification technology, by unrestrained sheep more than dioestrus, oestrus and the gestational period
(raising of Tarim University's animal experiment station) takes out ovary immediately after butchering, and PBS buffer solution is used in combination to rinse (PH7.4) tissue table
Face, input liquid nitrogen is rapidly frozen rapidly later, and is taken back -80 DEG C of laboratory and saved backup.
Step 2, protein extraction：
It takes dioestrus, oestrus and the gestational period more unrestrained sheep ovaries to take 0.1g respectively, the protein lysate of 0.4mL is added
(8.0M ureas, 50mM NH4HCO3,1 × protease inhibitors) carries out ultrasonication, is set as each 2s, is spaced 10s, altogether
5min, whole operation carry out on ice.After ultrasonication object places 20min on ice, 4 DEG C, 10,000 × g, 30min is centrifuged,
It takes supernatant to be transferred to new pipe, then dispense protein extract and is saved backup in -80 DEG C of refrigerators.
The drafting of step 3, Bradford protein quantifications and standard curve
Albumen is quantified using Bradford methods, the bovine serum albumin(BSA) of known content (BSA) is first made into difference
The protein solution of concentration measures the absorbance value (OD values) after its colour developing, according to linear between OD values and albumen concentration respectively
Relationship draws out the standard curve of albumen concentration, then measures the OD values after the colour developing of testing protein solution, bent with reference to BSA standards
The concentration of line computation testing protein.All operations step is completed in room temperature state.
8 μ g of BSA, 10 μ g, 12 μ g, 14 μ g, 16 μ g, 18 μ g are taken to be made into the protein solution of various concentration, and measure suction respectively
Light value, if correlation R2<0.95, standard curve cannot be used for calculating, and re-start experiment.
Protein solution is configured according to calculating, total volume is 20 μ L；Prepare to be detected sample 1.0uL simultaneously, be mixed with 19uL water,
Detection ELISA Plate, 2 multiple holes of each pattern detection are added.200 μ L Bradford working solutions are added in each multiple holes, and slight inhale is beaten
Mixing.It is protected from light, the static placement 15min of room temperature.It is detected with microplate reader, Detection wavelength is set in 595nm.It is derived according to standard curve
Go out to detect the total amount of albumen, and calculates respective concentration.Step 4, sample label and mixed in equal amounts
Each group sample peptide fragment takes about 100 μ g respectively, according to AB companies kit：iTRAQ Reagent-8plex
Multiplex Kit (AB SCIEX) specification is marked, label information such as table 1.A small amount of sample is taken to mix after label, into
Row LC-MS/MS Mass Spectrometer Methods, check iTRAQ labeling effciencies.
1 sample labeling information of table
The HPLC classifications of C18 chromatographic columns under step 5, high pH condition
1. the 100 μ l mobile phase As of sample after label is drained are redissolved, vortex oscillation, 14,000g centrifugation 20min are drawn
Supernatant loading；
2. preparing 60 blank 1.5ml centrifuge tubes, it is labeled as 1-60 successively, for collecting isolated component 1-60；
3. flow velocity 0.7ml/min detaches gradient such as table 2；
2 high pH reversed phase chromatography separations gradient of table
4. since the 5th minute, collect successively in every 1.5 minutes eluates to 1-60 centrifuge tubes；
5. vacuum refrigeration centrifugal drying；
6. the sample after drying is molten with the FA weights of 5ul 0.5%, and be collected into 60 components are merged into 6 groups
Point；
7. by the component 14,000g after merging, 10min is centrifuged, draws supernatant loading；It is general to draw 10ul.
Step 6, mass spectral analysis
Capillary high performance liquid chromatography
Every part of sample is detached using a nanoliter flow velocity HPLC liquid phase systems Easy nLC 1000.Buffer solution：A liquid is
0.1% aqueous formic acid, B liquid are 0.1% formic acid acetonitrile solution.Chromatographic column is balanced with 95% A liquid.Sample is by autosampler
Mass spectrum pre-column C18trap column (3 μm of 100 μ m 2cm of C18) are loaded to, then through analytical column C18column (3 μm of C18
75 μ m 15cm) separation, flow velocity 300nl/min.Related fluid phase gradient is as follows：
3 liquid chromatogram gradient parameter of table
Mass Spectrometric Identification
Every part of sample uses Orbitrap Fusion lumos mass spectrograph mass spectrographs after capillary high performance liquid chromatography detaches
(Thermo Scientific) is analyzed by mass spectrometry.
Step 7, data processing
Raw mass spectrum data
Mass spectral analysis initial data is RAW files, with software software Sequest and Proteome Discoverer
(Thermo Scientific) carries out searching library Qualitative and quantitative analysis.
Database
This uses database：protein.selected.fasta
Search library
RAW files are committed to Sequest servers by Proteome Discoverer when searching library, selection has been built
Then the database stood carries out database search.Relevant parameter such as the following table 4.
Table 4 searches library parameter
The foregoing is only a preferred embodiment of the present invention, protection scope of the present invention is without being limited thereto, it is any ripe
Those skilled in the art are known in the technical scope of present disclosure, the letter for the technical solution that can be become apparent to
Altered or equivalence replacement are each fallen in protection scope of the present invention.
Claims (2)
1. screening and the identification method of a kind of more unrestrained sheep physiological period breeding GAP-associated protein GAPs in Xinjiang, which is characterized in that including
Following steps：
The acquisition of step 1, the more unrestrained sheep physiological period ovaries in Xinjiang：
By estrus synchronization, detection of oestrus and pregnant identification technology, after unrestrained sheep more than dioestrus, oestrus and the gestational period is butchered
Ovary is taken out immediately, PBS buffer solution is used in combination to rinse PH7.4 tissue surfaces, and input liquid nitrogen is rapidly frozen rapidly later, and band
- 80 DEG C of laboratory is gone back to save backup；
Step 2, protein extraction：
It takes dioestrus, oestrus and the gestational period more unrestrained sheep ovaries to take 0.1g respectively, the protein lysate of 0.4mL is added, it is described
The ingredient of protein lysate is：8.0M ureas, 50mM NH4HCO3,1 × protease inhibitors carry out ultrasonication, are set as
Each 2s, is spaced 10s, total 5min, and whole operation carries out on ice；After ultrasonication object places 20min on ice, 4 DEG C, 10,
000 × g centrifuges 30min, supernatant is taken to be transferred to new pipe, then dispenses protein extract and is saved backup in -80 DEG C of refrigerators；
The drafting of step 3, Bradford protein quantifications and standard curve
Albumen is quantified using Bradford methods, the bovine serum albumin(BSA) BSA of known content is first made into various concentration
Protein solution measures the absorbance value OD values after its colour developing respectively, according to the linear relationship between OD values and albumen concentration, draws
Go out the standard curve of albumen concentration, then measure the OD values after the colour developing of testing protein solution, is calculated with reference to BSA standard curves to be measured
The concentration of albumen；All operations step is completed in room temperature state；
8 μ g of BSA, 10 μ g, 12 μ g, 14 μ g, 16 μ g, 18 μ g are taken to be made into the protein solution of various concentration, and measure extinction respectively
Value, if correlation R2<0.95, standard curve cannot be used for calculating, and re-start experiment；
Protein solution is configured according to calculating, total volume is 20 μ L；Prepare to be detected sample 1.0uL simultaneously, mixes, be added with 19uL water
Detect ELISA Plate, 2 multiple holes of each pattern detection；200 μ L Bradford working solutions are added in each multiple holes, and slight inhale beats mixing；
It is protected from light, the static placement 15min of room temperature；It is detected with microplate reader, Detection wavelength is set in 595nm；Inspection is derived according to standard curve
The total amount of albumen is surveyed, and calculates respective concentration；Step 4, sample label and mixed in equal amounts
Each group sample peptide fragment takes 100 μ g respectively, according to AB companies kit：iTRAQ Reagent-8plex Multiplex
Kit specifications are marked, and a small amount of sample is taken to mix after label, carry out LC-MS/MS Mass Spectrometer Methods, check iTRAQ label effects
Rate；
The HPLC classifications of C18 chromatographic columns under step 5, high pH condition
Step 6, mass spectral analysis
Capillary high performance liquid chromatography
Every part of sample is detached using a nanoliter flow velocity HPLC liquid phase systems Easy nLC 1000；Buffer solution：A liquid is 0.1% first
Aqueous acid, B liquid are 0.1% formic acid acetonitrile solution；Chromatographic column is balanced with 95% A liquid；Sample is loaded to by autosampler
Mass spectrum pre-column C18trap 3 μm of 100 μ m 2cm of column, C18, then through analytical column C18column, 3 μm of 75 μ m of C18
15cm is detached, flow velocity 300nl/min；
Mass Spectrometric Identification
Every part of sample uses Orbitrap Fusion lumos mass spectrograph mass spectrographs after capillary high performance liquid chromatography detaches
Thermo Scientifi are analyzed by mass spectrometry；
Step 7, data processing
Raw mass spectrum data
Mass spectral analysis initial data is RAW files, is searched with software with software Sequest and Proteome Discoverer
Library Qualitative and quantitative analysis；
Database
This uses database：protein.selected.fasta
Search library
RAW files are committed to Sequest servers by Proteome Discoverer when searching library, selection has built up
Database, then carry out database search.
2. screening and the identification method of the more unrestrained sheep physiological period breeding GAP-associated protein GAPs in Xinjiang according to claim 1,
It is characterized in that, step 5 is specially：
1) the 100 μ l mobile phase As of sample after draining label are redissolved, vortex oscillation, and 14,000g centrifugation 20min draw supernatant
Loading；
2) prepare 60 blank 1.5ml centrifuge tubes, 1-60 is labeled as successively, for collecting isolated component 1-60；
3) flow velocity 0.7ml/min；
4) it since the 5th minute, collects successively in every 1.5 minutes eluates to 1-60 centrifuge tubes；
5) vacuum refrigeration centrifugal drying；
6) sample after drying is molten with the FA weights of 5ul 0.5%, and be collected into 60 components are merged into 6 components；
7) by the component 14,000g after merging, 10min is centrifuged, draws supernatant loading 10ul.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109991324A (en) * | 2019-01-28 | 2019-07-09 | 新疆农业科学院农产品贮藏加工研究所 | A kind of method of the more unrestrained sheep mutton of identification |
| CN110398558A (en) * | 2019-07-23 | 2019-11-01 | 甘肃农业大学 | Method based on tibetan sheep testis differential protein before and after the sexal maturity of DIA technology mining |
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| CN105467022A (en) * | 2014-09-05 | 2016-04-06 | 天士力制药集团股份有限公司 | Analysis method for neuron injury related differentially expressed protein |
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- 2018-02-02 CN CN201810106686.0A patent/CN108398494B/en not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105467022A (en) * | 2014-09-05 | 2016-04-06 | 天士力制药集团股份有限公司 | Analysis method for neuron injury related differentially expressed protein |
Non-Patent Citations (3)
| Title |
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| LINA WANG ET AL.: "Legumain: A Biomarker for Diagnosis and Prognosis of Human Ovarian Cancer", 《JOURNAL OF CELLULAR BIOCHEMISTRY》 * |
| 贾建磊 等: "不同电导率水对绵羊卵巢蛋白提取及双向电泳影响的研究", 《2014年全国养羊生产与学术研讨会议论文集》 * |
| 陈潇飞 等: "iTRAQ技术及其在动物蛋白质组学中的研究进展", 《黑龙江畜牧兽医》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109991324A (en) * | 2019-01-28 | 2019-07-09 | 新疆农业科学院农产品贮藏加工研究所 | A kind of method of the more unrestrained sheep mutton of identification |
| CN109991324B (en) * | 2019-01-28 | 2022-03-11 | 新疆农业科学院农产品贮藏加工研究所 | Method for identifying Duolang mutton |
| CN110398558A (en) * | 2019-07-23 | 2019-11-01 | 甘肃农业大学 | Method based on tibetan sheep testis differential protein before and after the sexal maturity of DIA technology mining |
| CN110398558B (en) * | 2019-07-23 | 2022-04-01 | 甘肃农业大学 | Method for excavating sexual maturity anterior-posterior Tibetan sheep testis differential protein based on DIA technology |
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| Publication number | Publication date |
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| CN108398494B (en) | 2019-11-15 |
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