CN108627585A - The method that significant difference expresses albumen in D-ALPHA-Hydroxypropionic acid fermentation process is identified based on iTRAQ protein sciences - Google Patents
The method that significant difference expresses albumen in D-ALPHA-Hydroxypropionic acid fermentation process is identified based on iTRAQ protein sciences Download PDFInfo
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Abstract
The present invention relates to the methods for identifying significant difference expression albumen in D lactic fermentation process based on iTRAQ protein sciences；Under optimal technological condition for fermentation D production of lactic acid is carried out using L.delbrueckii；Extraction and quantitative L.delbrueckii logarithmic phase intracellular total proteins under optimal technological condition for fermentation；To extract and quantitative intracellular total protein carry out enzymolysis and isotopic tag label；LC MS/MS detections are carried out to the peptide fragment for digesting and marking；Database retrieval is carried out to the mass spectrometric data of detection, carries out the qualitative and quantitative analysis of albumen；Differentially expressed protein analysis, the albumen of identification significant difference expression are carried out according to abundance to qualitative and Quantitative Western.By systems biology, L.delbrueckii is analyzed for the first time under different fermentations process conditions, and the intracellular protein group profile of height fermentation D lactic acid processes reaches the follow-up strain promotion of guidance and further fermentation condition optimization.
Description
Technical field
The invention belongs to lactobacillus system biology fields, are related to a kind of Lactobacillus delbrueckii (Lactobacillus
Delbrueckii, Laboratories Accession are bought in China General Microbiological culture presevation administrative center, bacterium numbering
CGMCC1.2624) under optimal technological condition for fermentation, using absolute and relative quantitative (the Isobaric tags of isotope labelling
For relative and absolute quantitation, abbreviation iTRAQ) proteomic techniques analysis intracellular protein group wheel
Exterior feature, the method for identifying significant difference expression albumen during D-ALPHA-Hydroxypropionic acid high-efficiency fermenting.
Background technology
D-ALPHA-Hydroxypropionic acid molecular formula CH3CHOHCOOH, relative molecular weight 90.08, No. CAS：10326-41-7, be it is a kind of the most
Common metabolite, being widely used in the industries such as food, chemical industry, agricultural and medicine is general, is three great tradition organic acids
One of.D-ALPHA-Hydroxypropionic acid is the precursor of a variety of chiral materials as a chiral centre, is widely used in pharmacy, higher effective and lower toxic pesticide
And the chiral synthesis in the fields such as herbicide, cosmetics.Another even more important purposes of D-ALPHA-Hydroxypropionic acid is as synthesizing polylactic acid
(PLA) starting monomer of material.A kind of renewable, recyclable emerging plastics that polylactic acid receives significant attention in recent years are
The potential substitute of petroleum-based plastics.
Protein is cell activities " executor ", in order to disclose cell activities from system in albumen level
Rule, in the genome times afterwards comprehensively, the concept of protein groups is come into being, and refers to the gene institute table of a cell or a tissue
The all protein reached.Traditional mode studied single protein cannot be satisfied the requirement of genome times afterwards comprehensively.
This is because：(1) generation of biological phenomena is often multifactor impact, necessarily involves multiple protein；(2) multiple albumen
The participation of matter is woven into network or parallel generation, or in cascade cause and effect；(3) when executing physiological function protein table
It is now various, dynamic and substantially stationary constant not as genome.Therefore to have to the complicated activity of life comprehensively and
Deep understanding must study protein in entirety, dynamic, the level of network.Based on the exhausted of isotope labelling
Pair and relative quantification proteomic techniques, abbreviation iTRAQ proteomic techniques, be the protein groups detection technique of rising in recent years,
Compared to traditional two-dimensional gel electrophoresis technology, having quantitative accurate, high throughput, (identification and quantification protein quantity is far super two-dimentional solidifying
Gel electrophoresis technology), highly sensitive (low-abundance protein can be detected) and detection range extensively (not only can be with identification and quantification cytoplasm egg
In vain, can also identification and quantification memebrane protein and nucleoprotein etc.) the advantages that.
At present in terms of the patent of invention of iTRAQ proteomic techniques is mainly used for disease treatment.For example, Nanning section
The full of beard wound et al. of city remittance Information technology Co., Ltd has invented a kind of side that iTRAQ technologies are applied to Bones and joints scale-model investigation
(a kind of iTRAQ marks the application in osteoarthritis to method.Publication number：CN107727728A), iTRAQ eggs are carried out to sample
White group analysis, Recognition Different albumen, the function and possible interaction that analysis differential protein plays in the grid of OA,
Screen candidate's OA markers.The invention carries out the confirmation of marker by crowd's sample, assesses the diagnosis of these OA candidate markers
The value of OA simultaneously analyzes itself and clinical correlation, propose they in terms of the clinical applications such as the prevention of OA, early diagnosis can
Row, and its meaning in crowd's bony articulation health early warning.Chen Ning of Beijing bio tech ltd Bang Fei et al. is invented
A kind of method (a kind of lung cancer and pulmonary nodule egg that iTRAQ protein sciences are applied to lung cancer and pulmonary nodule disease research
White characteristic spectrum and its construction method and application.Publication number：CN107449916A), the invention is respectively from lung cancer/pulmonary nodule patient
Total protein, trypsin digestion, iTRAQ labels, two-dimensional liquid chromatography and series connection are extracted in the blood plasma of group and normal healthy controls person's group
Mass Spectrometer Method, relative quantitative assay obtain lung cancer/pulmonary nodule GAP-associated protein GAP characteristic spectrum；In lung cancer associated proteins characteristic spectrum, lung
In cancer patient group and normal healthy controls person's group the albumen of differential expression be upregulated protein (SAA1, SERPINA1, CRP, IGHG4 and
CFH)；In pulmonary nodule GAP-associated protein GAP characteristic spectrum, the albumen of differential expression is in pulmonary nodule patient group and normal healthy controls person's group
Upregulated protein (SERPINA1 and CRP) and down-regulation protein (SAA1, IGHG4 and CFH).The invention helps to study lung cancer and lung
Tubercle biology essence, it is significant to lung cancer and pulmonary nodule specification parting.
Currently, the application about iTRAQ protein sciences during D-ALPHA-Hydroxypropionic acid strain fermentation, does not there is patent of invention also.Cause
This, the present invention is using born of the same parents caused by optimal technological condition for fermentation in the protein groups technology analysis L.delbrueckii based on iTRAQ
Interior protein groups variation, identifies the albumen of significant difference expression during D-ALPHA-Hydroxypropionic acid high-efficiency fermenting, to instruct subsequent strain to be promoted
With advanced optimizing for technological condition for fermentation.
Invention content
The object of the present invention is to provide a kind of using based on significance difference in iTRAQ protein sciences identification D-ALPHA-Hydroxypropionic acid fermentation process
The method of different expression albumen；It is as follows：
1), D-ALPHA-Hydroxypropionic acid production is carried out using L.delbrueckii under optimal technological condition for fermentation；
2), extract and quantify L.delbrueckii logarithmic phase intracellular total proteins under optimal technological condition for fermentation；
3), to extract and quantitative intracellular total protein carry out enzymolysis and isotopic tag mark；
4) LC-MS/MS detections are carried out to the peptide fragment for digesting and marking；
5) database retrieval, is carried out to the mass spectrometric data of detection, carries out the qualitative and quantitative analysis of albumen；
6) differentially expressed protein analysis, the egg of identification significant difference expression, are carried out according to abundance to qualitative and Quantitative Western
In vain.
The step 2) extracts L.delbrueckii in optimal technological condition for fermentation using total bacterial protein extracts kit
Lower logarithmic phase intracellular total protein, and extraction albumen is quantified using Braford methods；
The step 3) is tried to extracting and quantitative intracellular total protein is digested using trypsase, and using iTRAQ
Agent box carries out isotopic tag label to peptide hydrolysis.
The peptide fragment that the step 4) is digested and marked using liquid chromatograph mass spectrography centering is detected analysis.
The step 5) is to carry out database retrieval to the mass spectrometric data of detection, carries out the qualitative and quantitative analysis of albumen,
Searching database is SWISS-PROT online databases.
The step 6) combines capital of a country gene and genome encyclopedia routing database, to the difference identified in step 5)
It expresses albumen and carries out feature path analysis, determine the metabolic pathway belonging to differentially expressed protein.
Qualitative and Quantitative Western expresses level difference multiple >=2 according to abundance and carries out differentially expressed protein in the step 5)
Analysis；Identify the albumen of significant difference expression, including upregulated protein >=2 times and down-regulation protein≤0.5 times.
The present invention relates to absolute and relative quantitative (the Isobaric tags for relative using isotope labelling
And absolute quantitation, abbreviation iTRAQ) technology identification D-ALPHA-Hydroxypropionic acid fermentation process in differentially expressed protein side
Method.We are analyzed using iTRAQ protein groups technologies, pass through intracellular protein Extraction and determination, proteolysis label, label peptide fragment liquid matter
Detection, protein quantification is qualitative, differential protein identifies and the experiment flows such as feature path analysis determine Lactobacillus delbrueckii
(Lactobacillus delbrueckii, Laboratories Accession are bought in China General Microbiological culture presevation administrative center, bacterium
Kind of number CGMCC1.2624) intracellular protein group profile under optimal technological condition for fermentation.ITRAQ proteome analysis common recognition is other
With quantified 934 kinds of albumen, wherein 139 kinds of albumen have compared with the expression of control group under optimal technological condition for fermentation it is apparent poor
Different (97 kinds of up-regulations, 42 kinds of downwards).Path function analysis is carried out to this 139 species diversity albumen and finds what 9 significant differences were expressed
Function module, including：Central carbon metabolism, energetic supersession, amino acid metabolism, polypeptide hydrolysis, transdermal delivery, hereditary information processing,
Nucleic acid metabolism, pressure response and unknown function albumen.The present invention is analyzed for the first time by systems biology means
L.delbrueckii is under different fermentations process conditions, the intracellular protein group profile of high-efficiency fermenting D-ALPHA-Hydroxypropionic acid process, is referred to reaching
Lead the purpose of follow-up strain promotion and further optimization of fermentation condition.
Specific implementation mode
As the preferred embodiment of the present invention, one kind is provided in L.delbrueckii using based on iTRAQ protein science skills
The method that art identifies significant difference expression albumen during D-ALPHA-Hydroxypropionic acid high-efficiency fermenting.
It is as follows：
1), protein extraction and quantitative：Use total bacterial protein extracts kit (work is given birth in C600596, Shanghai) extraction
L.delbrueckii (purchase is in China General Microbiological culture presevation administrative center, bacterium numbering CGMCC1.2624) is optimal
Logarithmic phase intracellular total protein under technological condition for fermentation, and extraction albumen is quantified using Braford methods；
2) it, digests and marks：1) extraction and quantitative intracellular total protein in are digested using trypsase, and adopted
Isotopic tag label is carried out to peptide hydrolysis with iTRAQ kits；
3), liquid matter is analyzed：Using liquid chromatograph mass spectrography (LC-MS/MS) technology pair 2) in enzymolysis and label peptide fragment
It is detected analysis；
4), data retrieval：Database retrieval is carried out to 3) the middle mass spectrometric data detected, the qualitative of albumen is carried out and quantitatively divides
Analysis, searching database are SWISS-PROT online databases (http://www.uniprot.org)；
5) differentially expressed protein, is carried out according to abundance (expression fold differences >=2) to qualitative in 4) and Quantitative Western
Analysis, the albumen of identification significant difference expression, including upregulated protein (>=2 times) and down-regulation protein (≤0.5 times)；
6), in conjunction with capital of a country gene and genome encyclopedia (Kyoto Encyclopedia of Genes and
Genomes, abbreviation KEGG) routing database, feature path analysis is carried out to 5) the middle differentially expressed protein identified, determines difference
Express the metabolic pathway belonging to albumen.
Embodiment
1, L.delbrueckii carries out D-ALPHA-Hydroxypropionic acid production under optimal technological condition for fermentation
Lactobacillus delbrueckii (buy in China General Microbiological by Lactobacillus delbrueckii, Laboratories Accession
Strain
Preservation administrative center, bacterium numbering CGMCC1.2624)
First, the ripe L.delbrueckii shake-flask seeds of culture are inoculated into the ratio of 10% (v/v) equipped with 4.5L
(Tween 80 1g/L, peptone 22.5g/L, yeast extract 7.5g/L, hydrogen citrate two in the 7.5L fermentation tanks of fermentation medium
Ammonium 2g/L, dipotassium hydrogen phosphate 2g/L, anhydrous sodium acetate 3g/L, anhydrous magnesium sulfate 0.2g/L, manganese sulfate 0.1g/L, glucose 80g/
L, pH=5.9).42 DEG C, 150rpm fermentations 48h.Lead to nitrogen 0.5h after inoculation, fermentation 18h starts stream plus glucose, flows rate of acceleration
4.6g/ (Lh) flows 12h between the added-time, stream plus glucose total amount 55g/L.Using calcium hydroxide as neutralizer, whole-process control fermentation
PH=5.9.
2, intracellular protein extracts
(1) it takes respectively under control group and optimal technological condition for fermentation, the sample 25mL to ferment under 18h time points is in precooling
In 50mL centrifuge tubes, 4 DEG C are immediately placed in refrigerated centrifuge, 5000rpm centrifuges 10min, and brine is used after abandoning supernatant
Three times, -80 DEG C save backup thalline.Each technological condition for fermentation is repeated 2 times experiment.
(2) thalline in 50mL centrifuge tubes is transferred in the mortar of precooling, 1mL lysates are added in the thalline into mortar
L3SDS is drawn to liquid-transfering gun in 1.5mL EP pipes after thalline is completely dissolved, and carries out ultrasonication processing later, is broken
Fragmentation nucleus acid, supersound process condition are：Power 80W is opened 1 second, is closed 1 second, continuous switch 10 times, be placed on cooled on ice；To reach
To good crushing effect, it is at least repeated eight times the above ultrasonic flow.After ultrasound, it is immediately placed in high speed freezing centrifuge
4 DEG C, 10000rpm centrifuges 15min, collects supernatant.
(3) by supernatant average mark in 4 1.5mLEP pipes, every EP pipe fills about 250 μ L supernatants, later toward every
1mL acetone is respectively added in branch EP pipes, is placed in -20 DEG C of refrigerators and precipitates 12h.After precipitation, in 4 DEG C, 10000rpm is centrifuged
15min abandons supernatant, opens the lid of EP pipes, is buckled on clean paper handkerchief, blots residual liquid in pipe as far as possible, then exist
It spontaneously dries at room temperature, after thorough drying, you can obtain protein agglomerate, saved backup in -80 DEG C of ultra low temperature freezers.
(4) 100 μ L lysates L3 is taken to be added in the protein agglomerate being completely dried, with pipette tips by protein agglomerate stab at
Small bulk, and suction piping and druming repeatedly, until protein is thoroughly dispersed in lysate.Later albumen is helped by the way of ultrasound
Quickly dissolving, be ultrasonically treated in condition and step (2).In on 4 DEG C, 10000rpm centrifugation 15min, absorption after ultrasonic dissolution assisting
In clear liquid to another clean 1.5mL EP pipes.It is the intracellular total protein extracted that the supernatant obtained is handled through the above process.
3, intracellular total protein is quantitative
The measurement of intracellular total protein concentration is carried out using Bradford methods, specific experiment flow is as follows：Take 5 respectively, 10,
15, the BSA of 20,25,30,35 μ g carries out the making of standard curve.1mL Bradford are drawn respectively is added to every 1.5mL
In EP pipes, protein staining is carried out, vortex oscillation 30s mixings measure its light absorption value using ultraviolet specrophotometer.Toward cuvette
It is as possible slowly and uniform when the middle BSA solution being added after dyeing, to avoid the generation of bubble.It is quantified using " two-step method ",
After the concentration of each sample quantitatively tentatively first is calculated, all samples concentration is then adjusted to concentration relatively, then into
Row second metering.Difference when due to protein quantification on instrument state and operation technique can cause experimental error, every time
Quantification of protein is both needed to draw standard curve, to ensure the accuracy of quantitative result.
4, proteolytic cleavage
(1) protein solution after each sample takes 200 μ g quantitative respectively, is added in 1.5mL EP pipes；
(2) 4 μ L TCEP Reducing Reagent (iTRAQ kits are included) is taken to be added to equipped with protein solution respectively
1.5mL EP pipes in, in 60 DEG C react 60min；
(3) after reaction, 2 μ l MMTS Cysteine-Blocking Reagent (iTRAQ kits are continuously added
It is included), 30min is placed at ambient temperature, completes the reductive alkylation of protein；
(4) above-mentioned protein solution is transferred in 10K super filter tubes, 10000rpm centrifuges 15min, collecting pipe bottom solution；
(5) 100 μ l urea liquids (8M, pH=8.5) are taken to be added in the solution of back collection, 10000rpm centrifugations
15min, collecting pipe bottom solution, is repeated 2 times；
(6) 100 μ l TEAB solution (0.25M, pH=8.5) are taken to be added in the solution that back is collected, 10000rpm centrifugations
15min, collecting pipe bottom solution, is repeated 3 times；
(7) a new collecting pipe separately is taken, it is 50 μ l that TEAB solution (0.5M) to final volume is added in super filter tube, is added
(trypsase is 1 with albumen quality ratio to a certain amount of trypsase:50), 12h is reacted in 37 DEG C of water-baths；
(8) after reaction, continuously adding a certain amount of trypsase, (trypsase is 1 with albumen quality ratio:100),
4h is reacted in 37 DEG C of water-baths, 10000rpm centrifuges 15min after reaction, and it is enzyme to collect the solution that bottom of the tube centrifuges
Solve postdigestive peptide fragment；
(9) 50 μ l TEAB solution (0.5M) are taken to be added in super filter tube, 10000rpm centrifuges 15min again, (8) and (9)
The solution that two steps are collected merges, and the sample after 100 μ l enzymolysis is obtained.
5, iTRAQ reagents mark
(1) iTRAQ kits are generally preserved in -20 DEG C, need to take out and equilibrate to room temperature in advance before use, later with centrifugation
The machine slow-speed of revolution centrifuges several seconds, and iTRAQ reagents is made to be entirely located in preservation bottom of the tube；
(2) 150 μ l isopropanols is taken to be added in every pipe iTRAQ reagents respectively, vortex oscillation mixing, 5000rpm centrifugations
30s；
(3) protein sample after 50 μ l enzymolysis is taken, is added in clean 1.5mL EP pipes；
(4) iTRAQ reagents being added in above-mentioned enzymolysis protein sample, vortex oscillation mixing, 5000rpm centrifuges 30s, in
2h is reacted at room temperature, and iTRAQ reagents is made to be attached on peptide fragment；
(5) it takes 100 μ l distilled water to be added in reaction system, terminates reaction；
(6) sample after marking iTRAQ reagents mixes, and vortex oscillation mixing, and 5000rpm centrifuges 30s；
(7) processing is dried in mixing sample by the way of vacuum refrigeration；
(8) after sample is completely dried, it is for use to be placed in preservation in -20 DEG C of refrigerators.
Explanation：In this chapter researchs, two biology of control group are marked respectively with iTRAQ 8-plex 113 and 115 labels
Repeating sample marks two biology under optimal technological condition for fermentation to repeat respectively with iTRAQ 8-plex 117 and 121 labels
Peptide fragment in sample after digestion.
6, the LC-MS/MS analyses of label peptide fragment
(1) the first high pH-RP liquid phase separations of dimension
1) with 95% A phases (water phase, with ammonium hydroxide adjust pH=10) 5%B phases (and 80% acetonitrile, with ammonium hydroxide adjust pH=10)
Pillar 30min is balanced, the sample after iTRAQ reagents are marked is redissolved with the A phases of 200 μ l；
2) chromatographic condition：Sample size 100 μ l, flow velocity 0.2ml/min, Detection wavelength 214nm.In chromatographic separation process, B phases
Ratio is gradually increased to 37% from 5%, and the duration of the process is 75min, later carries B Phase Proportions within the time of 5min
Up to 95%, and 5min is kept, entire chromatographic isolation total duration amounts to 85min；
3) 24 components are collected according to peak type since linear gradient, every 1 pipe connects 1 time, gradient 85min for every 50 seconds,
Iterative cycles connect sample；
4) 24 components of collection are dried in a manner of vacuum refrigeration, carry out second dimension reversed-phase liquid chromatography-matter
Spectrum combination (LC-MS) detection and analysis.
(2) second dimension LC-MS
1) 24 component peptide fragments after vacuum freeze drying are dissolved with sample lysate (0.1% formic acid, 5% acetonitrile), are filled
Divide oscillation mixing, 4 DEG C, 12000rpm, centrifuges 15min, supernatant is taken to be added in loading pipe, carry out liquid phase chromatogram-mass spectrometry combination
With detection；
2) chromatographic condition：Mobile phase A is 0.1% formic acid, and Mobile phase B is 0.1% formic acid and 80% acetonitrile, flow velocity are
300nL/min.Using gradient elution, i.e. B phases rise to 35% in 45min from 5%, are promoted to 90% and are protected in 50min
5min is held, be finally reduced to 5% and keeps 10min, each component bulk analysis time is 65min；
3) peptide fragment after detaching is directly entered mass spectrograph Thermo Scientific Q Exactive and carries out on-line checking.
7, mass spectrometric data is retrieved
Original document under mass spectrum machine needs to be converted into MASCOT formats using software Pro teome Discoverer 1.4
The file of (.mgf) could carry out the analysis of next step.After obtaining the file of MASCOT formats (.mgf), software is utilized
Protein Pilot 5.0, setting false discovery rate (FDR) are no more than 1%, carry out protein groups quantitative analysis, identify thick polypeptide.
The thick polypeptide data of identification are retrieved according to SWISS-PROT online databases.
8, differentially expressed protein identifies
According to protein abundance level, when fold differences reach 2 times or more or are less than 0.5 times, and its p value is small through statistical check
It is considered as differential protein in 0.05.Wherein it is considered as upregulated protein more than or equal to 2 times, is considered as down-regulation protein less than or equal to 0.5 times.
139 significant difference expression albumen are identified altogether, wherein 97 are notable upregulated protein, 42 are notable down-regulation protein.
9, differential protein feature path is analyzed
Differentially expressed protein is carried out according to functional role of the differential protein in cell in conjunction with KEGG routing databases
Feature path is analyzed, it is found that these albumen belong to 9 function modules (metabolic pathway), including：Central carbon metabolism, energetic supersession,
Amino acid metabolism, polypeptide hydrolysis, transdermal delivery, hereditary information processing, nucleic acid metabolism, pressure response and unknown function albumen.
10, seed culture condition
The bacterial strain for taking 2mL glycerol tube preservations is inoculated in 200mL seed culture mediums ((Tween 80 1g/L, peptone 10g/
L, yeast extract 5g/L, diammonium hydrogen citrate 2g/L, dipotassium hydrogen phosphate 2g/L, anhydrous sodium acetate 3g/L, anhydrous magnesium sulfate 0.2g/
L, manganese sulfate 0.1g/L, glucose 20g/L, pH=5.9), 42 DEG C of stationary cultures to exponential phase, generally 11~13h, bacterium
Body just starts to precipitate.Layering between thalline and supernatant is not obvious, and when microscopy, most of thalline were rod-short, also there is part
Elongated rod shape in splitting status.At this time between thalline OD600=1.5~2.1.
11, the measurement of D-ALPHA-Hydroxypropionic acid yield and chiral purity
(1) D-ALPHA-Hydroxypropionic acid yield is detected using high performance liquid chromatography (Agilent 1200, USA).Concrete operations are as follows：
1) 1mL zymotic fluids, 10000rpm is taken to centrifuge 2min.
2) 100 μ L of supernatant are taken, are added in 900 μ L 0.1M dilution heat of sulfuric acid, 10000rpm centrifuges 5min after mixing.
3) the water system filter membrane that supernatant crosses 0.22 μm is taken, efficient liquid phase chromatographic analysis is used for：A：Chromatographic column HPX-87H
(250mm × 4.6mm, BioRad, America), mobile phase：2.5mM sulfuric acid solutions, flow velocity 0.4ml/min, 65 DEG C of column temperature, profit
It is detected with parallax monitor, 20 μ L of sample size；B：Chromatographic column ZOBAX SB-C18 (250mm × 4.6mm, Agilent,
America), mobile phase：5mM sulfuric acid solutions, flow velocity 0.5mL/min, 30 DEG C of column temperature, ultraviolet detection wavelength 210nm, sample size 20
μL。
(2) D-ALPHA-Hydroxypropionic acid chiral purity carries out separation determination using high performance liquid chromatography.Concrete operations are as follows：
1) 1mL zymotic fluids, 10000rpm is taken to centrifuge 2min.
2) 100 μ L of supernatant are taken, are added in 900 μ L water, 10000rpm centrifuges 5min after mixing.
3) the water system filter membrane that supernatant crosses 0.22 μm is taken, efficient liquid phase chromatographic analysis is used for：Chromatographic column Chiral
Separation columns CRS10W (50mm × 4.6mm, MCI GEL, Japan), mobile phase：2mM copper-baths, stream
Fast 0.5ml/min, 25 DEG C of column temperature, ultraviolet detection wavelength 254nm, 20 μ L of sample size.
The utilization that the present invention is disclosed and proposed is based on significance difference during iTRAQ protein sciences identification D-ALPHA-Hydroxypropionic acid high-efficiency fermenting
The method of different expression albumen, those skilled in the art can be by using for reference present disclosure, and the appropriate links such as condition route that change are realized,
Although the methods and techniques of the present invention are described by preferred embodiment, related technical personnel can obviously not depart from
Methods and techniques described herein route is modified or is reconfigured in the content of present invention, spirit and scope, to realize most
Whole technology of preparing.In particular, it should be pointed out that all similar replacements and change are aobvious for a person skilled in the art
And be clear to, they are considered as being included in spirit of that invention, range and content.
Claims (8)
1. a kind of method identifying significant difference expression albumen during D-ALPHA-Hydroxypropionic acid high-efficiency fermenting based on iTRAQ protein sciences；Tool
Steps are as follows for body：
1), D-ALPHA-Hydroxypropionic acid production is carried out using L.delbrueckii under optimal technological condition for fermentation；
2), extract and quantify L.delbrueckii logarithmic phase intracellular total proteins under optimal technological condition for fermentation；
3), to extract and quantitative intracellular total protein carry out enzymolysis and isotopic tag mark；
4) LC-MS/MS detections are carried out to the peptide fragment for digesting and marking；
5) database retrieval, is carried out to the mass spectrometric data of detection, carries out the qualitative and quantitative analysis of albumen；
6) differentially expressed protein analysis, the albumen of identification significant difference expression, are carried out according to abundance to qualitative and Quantitative Western.
2. the method as described in claim 1, it is characterized in that step 2) is extracted using total bacterial protein extracts kit
L.delbrueckii logarithmic phase intracellular total proteins under optimal technological condition for fermentation, and using Braford methods to extraction albumen into
Row is quantitative.
3. the method as described in claim 1, it is characterized in that step 3) is to extracting and quantitative intracellular total protein uses tryptose
Enzyme is digested, and carries out isotopic tag label to peptide hydrolysis using iTRAQ kits.
4. the method as described in claim 1, it is characterized in that step 4) is digested and marked using liquid chromatograph mass spectrography centering
Peptide fragment be detected analysis.
5. the method as described in claim 1, it is characterized in that step 5) is to carry out database retrieval to the mass spectrometric data of detection, into
The qualitative and quantitative analysis of row albumen, searching database are SWISS-PROT online databases.
6. the method as described in claim 1, it is characterized in that step 6) combines capital of a country gene and genome encyclopedia number of path
According to library, feature path analysis is carried out to the differentially expressed protein identified in step 5), determines the metabolism belonging to differentially expressed protein
Path.
7. the method as described in claim 1, it is characterized in that qualitative in step 5) and Quantitative Western expresses level error according to abundance
Different multiple >=2 carry out differentially expressed protein analysis.
8. the method as described in claim 1, it is characterized in that the albumen that identification significant difference is expressed in step 5), including up-regulation egg
In vain >=2 times and down-regulation protein≤0.5 times.
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| CN110018256A (en) * | 2019-04-24 | 2019-07-16 | 湖北省农业科学院粮食作物研究所 | A method of identification rice quality differential protein |
| 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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| CN110018256A (en) * | 2019-04-24 | 2019-07-16 | 湖北省农业科学院粮食作物研究所 | A method of identification rice quality differential protein |
| 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 |
| CN111830148A (en) * | 2020-06-15 | 2020-10-27 | 南宁牟合蛋白科技有限公司 | Quantitative analysis method for obtaining high-flux macroproteins based on itraq technology |
| CN112505328A (en) * | 2020-11-02 | 2021-03-16 | 武汉金开瑞生物工程有限公司 | Isotope labeling kit and labeling method |
| CN112505328B (en) * | 2020-11-02 | 2024-02-13 | 武汉金开瑞生物工程有限公司 | Isotope labeling kit and labeling method |
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