CN106770872A - A kind of authentication method for serum of broilers protein group - Google Patents
A kind of authentication method for serum of broilers protein group Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/02—Column chromatography
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Abstract
A kind of authentication method for serum of broilers protein group, comprises the following steps：(1) collection of blood serum sample；(2) SDS PAGE electrophoresis；(3) protein gel film dosim；(4) analyses of the nano HPLC MS/MS to protein, the data obtained is identified by Protein Pilot software retrieval Protein Data Banks.The value volume and range of product of serum of broilers protein expression can quickly, be with high throughput searched out using the method for the present invention, method and thinking are provided further to carry out broiler chicken nutrition, the research of broiler chicken immunologic function.
Description
Technical field
The present invention relates to analytical chemistry and life science, and in particular to a kind of mirror for serum of broilers protein group
Determine method.
Background technology
There is serum normal viscosity, acid-base value, osmotic pressure for maintaining blood etc. to act on, and the protein source in serum is almost
It is relevant with all cells, tissue, organ, it is medical diagnosis on disease and life such that it is able to the pathology and physiological status of directly reflection body
The important sample that thing mark finds.Compared with histone and cell protein, haemocyanin species is various, distinct, also
There is substantial amounts of intein body, posttranslational modification, degradation fragment.In addition, huge (the albumen of the abundance difference of serum proteins
Concentration range crosses over 10 orders of magnitude), high-abundance proteins occupy more than the 99% of Tot Prot, severe jamming low abundance proteinses
Detection and identification.Therefore in protein science research is carried out, it is necessary to the high-abundance proteins in serum are first removed, to reduce height
Cover effect of the abundance protein to low abundance proteinses, improves sensitivity, helps preferably to identify other albumen, promotes low rich
Spend the detection of protein.
Conventional technology has precipitation of protein, affine technolog etc. at present.Precipitation of protein uses acetonitrile, ethanol, third
Ketone etc. to settle the high-abundance proteins in serum to some extent, but the specificity of the method is not high；Affine technolog utilizes egg
Interaction between white matter and its part or antibody carrys out the high abundance target protein such as specific bond albumin, IgG, and specificity is high, but
It is the cost increase of the method, and it is worth noting that, for source of species behaviour and the blood of mouse more than existing commercially produced product
Develop clearly, make its application limited.
And the relevant report of serum of broilers protein group authentication method is there is no at present.
The content of the invention
The purpose of the present invention is to solve the shortcomings of the prior art, there is provided a kind of identification for serum of broilers protein group
Method, using film dosim combination mass-spectrometric technique in the method for high throughput identification serum of broilers albumen, can effectively reduce serum
Interference when high-abundance proteins are to mass spectral analysis, increases the value volume and range of product of detection albumen, solves broiler chicken species removal high abundance
The method limitation problem of albumen, method is provided further to carry out the research immune with broiler chicken nutrition, broiler chicken of serum of broilers albumen
Foundation.
To achieve these goals, the technical solution adopted by the present invention is as follows：It is a kind of for serum of broilers protein group
Authentication method, classification separation is carried out using the method serum of broilers albumen of film dosim, is divided by nano-HPLC-MS/MS
Analysis, and obtains protein expression profiles, then analyzed by GO the function that obtains albumen and.
Implement step as follows：
(1) blood serum sample collection：The n blood of normal broiler chicken is collected, is placed in the negative tube without heparin, negative tube exists
Aggegation 15-20min on ice, temperature be 4-8 DEG C, centrifugal force be 1600-1800g under the conditions of take after centrifugation time 15-20min
Clear liquid, n as required blood serum sample, the blood serum sample of acquisition carries out protein quantification；
(2) SDS-PAGE electrophoresis：1. taking 10~30 μ g albumen carries out the SDS-PAGE electrophoresis that mass fraction is 10~15%,
Deposition condition：60~90V, 20~30min, 90~110V electrophoresis can terminate electrophoresis when bromophenol blue reaches gel leading edge；
2. the gel that 1. step obtains is dyeed with coomassie brilliant blue staining method：Gel is placed in mass fraction for 0.1%R-
Dyeing 1~2h, destainer decolouring 30min~1h, ultra-pure water washing, per 30- are vibrated in 250 dyeing liquors, on 25-40 DEG C of shaking table
40min changes water once, to gel background is purified, band is clear；The dyeing liquor is ethanol, acetic acid and water composition, wherein
Ethanol and acetic acid volume ratio are 1:1~1:4, containing the R-250 that mass fraction is 0.1~0.125%, destainer is ethanol, acetic acid
With water composition, wherein ethanol and acetic acid volume ratio is 1:1~1:4；
(3) protein gel film dosim：1. 40~70kDa of high-abundance proteins, 20~25kDa are cut into 1 according to stripe size
~2mm3The blob of viscose of size, remaining protein band is cut into 1~2mm respectively according to the distribution of molecular weight3The blob of viscose of size；2. step
1. gel 100-200 μ l destainers decolouring 2-3 times in, 15-20min is repeated and decolourized, until Coomassie brilliant blue color is de-
Untill to the greatest extent, supernatant is abandoned, the lyophilised gel in freeze dryer is concentrated in vacuo；The destainer is that volume ratio is 50:50 acetonitrile and 25-
100mM NH4HCO3Solution composition；3. 10-15mM, volume 100-200 μ l DTT solution is added to be incubated in 37-56 DEG C of insulating box
45min-1h, abandons waste liquid；4. the 50-100mM iodoacetamide solutions of certain volume are added so that the amount 5 of the material of iodoacetamide
Times step 3. in added DTT material amount, room temperature dark reaction 30-45min abandons waste liquid；5. 100-200 μ l destainers are added
Cleaning 2-3 times, each 10-15min, the lyophilised gel in freeze dryer is concentrated in vacuo；6. 10-15 μ l trypsin solutions are added, in 4 DEG C
Or 20-30min is placed on mixture of ice and water, treat gel swelling；7. 15-20 μ l, 25-100mM NH are added4HCO3Solution submergence is solidifying
Glue, 37 DEG C of -40 DEG C of overnight incubations；8. 50-100 μ l extract solutions are added, the extract solution is 1-5% formic acid solutions or trifluoroacetic acid
Solution, 37 DEG C of -40 DEG C of incubation 30min-1h, takes supernatant and is transferred in clean centrifuge tube；9. 50-100 μ are added in blob of viscose again
L extract solutions, the extract solution is that volume ratio is 50:50 acetonitrile and 1-5% formic acid solutions or trifluoroacetic acid solution；10. step is 8.
9. extract solution twice merges, concentrated freeze-dried in freeze dryer is concentrated in vacuo, and obtains peptide fragment sample；
(4) sample mass spectral analysis：The peptide fragment sample that step (3) is obtained, adds 10-15 μ l, 0.1-0.5% formic acid molten
Liquid, carries out mass spectral analysis, obtains the mass-to-charge ratio of peptide fragment and its fragment ion.
The sample mass spectral analysis is as follows：
Liquid phase chromatogram condition：Using liquid chromatographic system, liquid chromatographic system manager containing binary solvent, the auto injection
Device；Chromatographic column be C18 posts, specification be 0.075 × 150mm, 5 μm,Sample size：1-5μL；Loading mobile phase：3-5% second
The aqueous formic acid of nitrile+0.1%；Separate mobile phase：A is 3-5% acetonitrile+5%DMSO+0.1% aqueous formic acids, and B is 90-95%
Acetonitrile+5%DMSO+0.1% formic acid solutions；Peptide fragment mixture is separated with condition of gradient elution, after post efflux without
Shunting is introduced directly into mass spectrometer system detection；
Mass Spectrometry Conditions：Detected using time-of-flight mass spectrometry system, condition is electron spray nanoESI ion guns,
Scan mode：Cation scan pattern, sets dry gas stream speed and removes cluster voltage, MS sweep limits m/z 350~1250, scanning
Speed is 4-5spectrum/s, MS/MS sweep limits m/z 100~1500, and sweep speed is 20-30spectrum/s, wherein
Parent ion dynamic function sets the exclusion time for 20s 20-30s, under data dependence pattern, selection abundance highest 40-50
Parent ion is collided, wherein abundance absolute threshold：150-200cps, valence state meets 2+~4+, and collision energy exists with mass number
Adjust automatically in the range of desired value ± 10V.
The data retrieval is as follows：Retrieved using search engine, database source is albumen database, kind selection
Gallus gallus, sample type is Identification, cysteine modified selection Iodoacetamide, pancreatin enzyme
Cut, instrument source is Tripple TOF 6600, and protein level FDR (false discovery rate) is set to 1%.
The Gene Ontology are analyzed as follows：Albumen to identifying carries out Gene Ontology analyses, using biology
Bioinformatic tool AmiGO v1.8 carry out enrichment analysis, obtain the cellular component of serum protein group, molecular function and are participated in
Biological process.
The n is 5 integral multiple.
Condition of gradient elution in the liquid phase chromatogram condition：0~0.5min is changed to 92%A phases, 0.5- for 95%
To 70%A phases, 40-48min linear changes to 10%A phases simultaneously keep 6min to 40min linear changes, then switch 95%A phases and protect
Hold 6min；Flow velocity is 0.3 μ l/min.
In the Mass Spectrometry Conditions, dry gas stream speed 3psi removes cluster voltage 80V.
The present invention is carried out during Proteomic analysis to n serum sample, and n sample is carried out to wait mass mixing, with
Individual difference is eliminated, and carries out film dosim twice and LC-MS analysis, to ensure to obtain reliable data.
The blood serum sample that the present invention is obtained may be from after 21 ages in days to the broiler chicken sample butchered in preceding all growth periods.
The present invention has the effect that：Film dosim procedure of the present invention is simple, low cost, while using
Receive stream high performance liquid chromatography-proteomics research platform associated with time-of-flight mass spectrometry, it is possible to achieve to complex sample
High-throughout separation, and highly sensitive detection, the protein groups identified find in immune response, oxidative stress after analysis
With in cell surface receptor signal pathway be enriched with, this for further development serum of broilers protein group research work provide according to
According to..
Brief description of the drawings
Fig. 1 realizes flow chart for the inventive method；
Fig. 2 is serum of broilers SDS-PAGE in the present invention；
Fig. 3 is the TIC of LC-MS analysis after film dosim in the present invention；
Fig. 4 is the Identification of Fusion Protein result Wei Entu for repeating in the present invention to obtain twice；
Fig. 5 is the GO analysis results of serum of broilers protein group in the present invention.
Specific embodiment
As shown in figure 1, the inventive method to implement process as follows：
(1) blood serum sample collection：5 blood of normal broiler chicken are collected, is placed in the negative tube without heparin, negative tube exists
Aggegation 15-20min on ice, temperature be 4-8 DEG C, centrifugal force be 1600-1800g under the conditions of take after centrifugation time 15-20min
Clear liquid, the blood serum sample of acquisition carries out protein quantification, and waits mass mixing.
(2) SDS-PAGE electrophoresis：1. taking 20 μ g albumen carries out the SDS-PAGE electrophoresis that mass fraction is 12%, electrophoresis strip
Part：After 80V electrophoresis 30min, 110V electrophoresis can terminate electrophoresis when bromophenol blue reaches gel leading edge；2. Coomassie brilliant blue is used
Colouring method is dyeed to the gel that 1. step obtains：Gel is placed in mass fraction in 0.1%R-250 dyeing liquors, 25-
Dyeing 2h, destainer decolouring 1h, ultra-pure water washing about 4h are vibrated on 40 DEG C of shaking tables, water is changed once per 30min, it is de- to gel background
Only untill, band is clear；The dyeing liquor is that volume ratio is 25:10:65 ethanol, acetic acid and water composition, be containing mass fraction
0.1% R-250, destainer is that volume ratio is 45:10:45 ethanol, acetic acid and water composition；Fig. 2 shows serum of broilers
It is high that SDS-PAGE, seralbumin (about 70kDa) and apolipoprotein (about 52kDa) account for the Total albumen content, from figure
In it can be seen that 55~72kDa band colour developing most substantially, illustrate the protein concentration highest of the part, abundance is high, mass spectral analysis
Result is also accredited as seralbumin and apolipoprotein.
(3) protein gel film dosim：1. high-abundance proteins are cut into about 1mm according to stripe size3The blob of viscose of size, remaining
Protein band is cut into about 1mm respectively according to the distribution of molecular weight3The blob of viscose of size；2. step 1. in gel decolourized with 100 μ l
Liquid is decolourized twice, and each 15min is repeated and decolourized, and untill Coomassie brilliant blue color takes off, supernatant is abandoned, in LabconcoMiddle lyophilised gel；The destainer is that volume ratio is 50:50 acetonitrile and 50mM NH4HCO3Solution composition；③
100 μ l 10mM DTT solution are added, 45min is incubated in 56 DEG C of insulating boxs, abandon waste liquid；4. 100 μ l 50mM iodoacetamides are added
Solution, room temperature dark reaction 30min, abandons waste liquid；5. 100 μ l destainers are added to clean twice, each 10min, in LabconcoMiddle lyophilised gel；6. 15 μ l trypsin solutions (10ng/ μ l) are added, 30min is placed in 4 DEG C, treat gel swelling；⑦
Add 15 μ l 50mM NH4HCO3Solution submerges gel, 37 DEG C of overnight incubations；8. the 50 μ l extract solutions, the extract solution are added to be
5% formic acid solution, 37 DEG C of incubation 1h, takes supernatant and is transferred in clean centrifuge tube；9. 50 μ l extract solutions are added in blob of viscose again,
The extract solution is that volume ratio is 50:50 acetonitrile and 5% formic acid solution；10. step extract solution twice 8. and 9. merges, in
LabconcoIn it is concentrated freeze-dried.
(4) sample mass spectral analysis：
The sample that step (2) is obtained, adds the formic acid solutions of 10 μ l 0.1%, treats mass spectral analysis.
Liquid phase chromatogram condition：Using AB Sciex companies ekspert nano LC liquid chromatographic systems, the system contains binary
Solvent manager, automatic sampler；Chromatographic column be C18 posts, specification be 0.075 × 150mm, 5 μm,Sample size：1-5μ
L；Loading mobile phase：The aqueous formic acid of 5% acetonitrile+0.1%；Separate mobile phase：A is 5% acetonitrile+5%DMSO+0.1% formic acid
The aqueous solution, B is 90% acetonitrile+5%DMSO+0.1% formic acid solutions；Condition of gradient elution：0~0.5min is changed to for 95%
92%A phases, 0.5-40min linear changes to 70%A phases, 40-48min linear changes to 10%A phases simultaneously keep 6min, then switch
95%A phases simultaneously keep 6min；Flow velocity is 0.3 μ l/min, and efflux is introduced directly into mass spectrometer system and detects without shunting after post；
Mass Spectrometry Conditions：Detected using the time-of-flight mass spectrometry systems of AB Sciex Tripple TOF 6600, its
Condition is electron spray nanoESI ion guns, scan mode：Cation scan pattern, dry gas stream speed 3psi, removes cluster voltage 80V,
MS sweep limits m/z 350~1250, sweep speed is 4spectrum/s, MS/MS sweep limits m/z100~1500, scanning
Speed is 20spectrum/s, and wherein parent ion dynamic function sets the exclusion time for 20s, under data dependence pattern, selection
40 parent ions of abundance highest are collided (abundance absolute threshold：150cps, valence state meets 2+~4+), collision energy is with matter
Amount number adjust automatically in the range of desired value ± 10V.Fig. 3 illustrates blood serum sample through 1 cut sample introduction after film dosim point
The TIC obtained after analysis, lateral coordinates represent appearance time, and longitudinal coordinate represents the height at peak.
(5) data retrieval：
Retrieved using Protein Pilot search engines, database source is Swissprot albumen databases
(http://www.uniprot.org/downloads), kind selection Gallus gallus, sample type is
Identification, cysteine modified selection Iodoacetamide, pancreatin digestion, instrument source TrippleTOF
6600, protein level FDR (false discovery rate) are set to 1%.Fig. 4 displayings repeat down the albumen for identifying twice
Information, it can be seen that the albumen being accredited in film dosim twice and LC-MS analysis has 551, each
In there are 84 and 258 albumen uniquely to come across once identification, identification albumen number 893 is amounted to.
(6) Gene Ontology analyses：Albumen to identifying carries out Gene Ontology analyses, using bioinformatics
Instrument AmiGO v1.8 carry out enrichment analysis, and the cellular component of serum protein group, molecular function and the biology for being participated in are discussed
Process.Serum is the important biomolecule sample for reflecting fuselage state, and the research object of serum protein group is the complete of appearance in serum
Portion's protein, including：Tissue secretion albumen, immunoglobulin, long-range part and receptor protein, local part and receptor protein,
Transient albumen, tissue spill albumen, abnormal secretion albumen and extrinsic protein etc..Fig. 5 shows serum protein group thin
The GO annotations of born of the same parents' component, molecular function and the aspect of biological process three, abscissa is albumen number, and ordinate is all kinds of annotations.
As shown in Figure 5, serum protein group mainly constitutes the plasma membrane of cell, film inner structure, cytoplasmic matrix, mitochondria and small point of blood
Son, has played molecule binding function, including with nucleotides, albumen, kinases, carbohydrate, extracellular matrix binding function,
And the regulatory function of enzyme, participate in albumen, fat and carbohydrate metabolic process in, in immune response, oxidative stress
With cell surface receptor signal pathway, also there is albumen to be accredited in these important biological processes, this is further development
The research work of serum of broilers protein group provides foundation.
Description of the invention is not elaborated and partly belongs to techniques well known.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (7)
1. a kind of authentication method for serum of broilers protein group, it is characterised in that realize that step is as follows：
(1) blood serum sample collection：The n blood of normal broiler chicken is collected, is placed in the negative tube without heparin, negative tube is on ice
Aggegation 15-20min, temperature be 4-8 DEG C, centrifugal force be 1600-1800g under the conditions of take supernatant after centrifugation time 15-20min
Liquid, n as required blood serum sample, the blood serum sample of acquisition carries out protein quantification；
(2) SDS-PAGE electrophoresis：1. taking 10~30 μ g albumen carries out the SDS-PAGE electrophoresis that mass fraction is 10~15%, electrophoresis
Condition：60~90V, 20~30min, 90~110V electrophoresis can terminate electrophoresis when bromophenol blue reaches gel leading edge；2. use
Coomassie brilliant blue staining method is dyeed to the gel that 1. step obtains：Gel is placed in mass fraction for 0.1%R-250 contaminates
1~2h of dyeing is vibrated in color liquid, on 25-40 DEG C of shaking table, destainer decolouring 30min~1h, ultra-pure water washing is changed per 30-40min
Water once, to gel background is purified, band is clear；The dyeing liquor is ethanol, acetic acid and water composition, wherein ethanol and second
Sour volume ratio is 1:1~1:4, containing the R-250 that mass fraction is 0.1~0.125%, destainer is ethanol, acetic acid and water composition,
Wherein ethanol and acetic acid volume ratio is 1:1~1:4；
(3) protein gel film dosim：1. 40~70kDa of high-abundance proteins, 20~25kDa are cut into 1~2mm according to stripe size3
The blob of viscose of size, remaining protein band is cut into 1~2mm respectively according to the distribution of molecular weight3The blob of viscose of size；2. step 1. in
Gel 100-200 μ l destainers decolouring 2-3 times, 15-20min, repeat decolourize, until Coomassie brilliant blue color take off for
Only, supernatant is abandoned, the lyophilised gel in freeze dryer is concentrated in vacuo；The destainer is that volume ratio is 50:50 acetonitrile and 25-
100mM NH4HCO3Solution composition；3. 10-15mM, volume 100-200 μ l DTT solution is added to be incubated in 37-56 DEG C of insulating box
45min-1h, abandons waste liquid；4. the 50-100mM iodoacetamide solutions of certain volume are added so that the amount 5 of the material of iodoacetamide
Times step 3. in added DTT material amount, room temperature dark reaction 30-45min abandons waste liquid；5. 100-200 μ l destainers are added
Cleaning 2-3 times, each 10-15min, the lyophilised gel in freeze dryer is concentrated in vacuo；6. 10-15 μ l trypsin solutions are added, in 4 DEG C
Or 20-30min is placed on mixture of ice and water, treat gel swelling；7. 15-20 μ l, 25-100mM NH are added4HCO3Solution submergence is solidifying
Glue, 37 DEG C of -40 DEG C of overnight incubations；8. 50-100 μ l extract solutions are added, the extract solution is 1-5% formic acid solutions or trifluoroacetic acid
Solution, 37 DEG C of -40 DEG C of incubation 30min-1h, takes supernatant and is transferred in clean centrifuge tube；9. 50-100 μ are added in blob of viscose again
L extract solutions, the extract solution is that volume ratio is 50:50 acetonitrile and 1-5% formic acid solutions or trifluoroacetic acid solution；10. step is 8.
9. extract solution twice merges, concentrated freeze-dried in freeze dryer is concentrated in vacuo, and obtains peptide fragment sample；
(4) sample mass spectral analysis：The peptide fragment sample that step (3) is obtained, adds 10-15 μ l, 0.1-0.5% formic acid solutions,
Mass spectral analysis is carried out, the mass-to-charge ratio of peptide fragment and its fragment ion is obtained.
2. the authentication method for serum of broilers protein group according to claim 1, it is characterised in that：The sample matter
Analysis of spectrum is as follows：
Liquid phase chromatogram condition：Using liquid chromatographic system, liquid chromatographic system manager containing binary solvent, the automatic sampler；
Chromatographic column be C18 posts, specification be 0.075 × 150mm, 5 μm,Sample size：1-5μL；Loading mobile phase：3-5% acetonitriles+
0.1% aqueous formic acid；Separate mobile phase：A is 3-5% acetonitrile+5%DMSO+0.1% aqueous formic acids, and B is 90-95% second
Nitrile+5%DMSO+0.1% formic acid solutions；Peptide fragment mixture is separated with condition of gradient elution, after post efflux without point
Stream is introduced directly into mass spectrometer system detection；
Mass Spectrometry Conditions：Detected using time-of-flight mass spectrometry system, condition is electron spray nanoESI ion guns, scanning
Mode：Cation scan pattern, sets dry gas stream speed and removes cluster voltage, MS sweep limits m/z 350~1250, sweep speed
Be 4-5spectrum/s, MS/MS sweep limits m/z 100~1500, sweep speed is 20-30spectrum/s, wherein it is female from
It is 20s 20-30s that sub- dynamic function sets the exclusion time, under data dependence pattern, 40-50 mother of selection abundance highest from
Son is collided, wherein abundance absolute threshold：150-200cps, valence state meets 2+~4+, and collision energy is with mass number in target
Adjust automatically in the range of value ± 10V.
3. the authentication method for serum of broilers protein group according to claim 1, it is characterised in that：The data inspection
Rope is as follows：Retrieved using search engine, database source is albumen database, kind selection Gallus gallus, sample
Type is Identification, cysteine modified selection Iodoacetamide, and pancreatin digestion, instrument source is Tripple
TOF 6600, protein level FDR (false discovery rate) are set to 1%.
4. the authentication method for serum of broilers protein group according to claim 1, it is characterised in that：The Gene
Ontology is analyzed as follows：Albumen to identifying carries out Gene Ontology analyses, using bioinformatics tools AmiGO
V1.8 carries out enrichment analysis, obtains the cellular component of serum protein group, molecular function and the biological process for being participated in.
5. the authentication method for serum of broilers protein group according to claim 1, it is characterised in that：The n is 5
Integral multiple.
6. the authentication method for serum of broilers protein group according to claim 2, it is characterised in that：The liquid phase color
Condition of gradient elution in spectral condition：0~0.5min is changed to 92%A phases, 0.5-40min linear changes to 70%A for 95%
Phase, 40-48min linear changes to 10%A phases simultaneously keep 6min, then switch 95%A phases and keep 6min；Flow velocity is 0.3 μ l/
min。
7. the authentication method for serum of broilers protein group according to claim 2, it is characterised in that：The mass spectrum bar
In part, dry gas stream speed 3psi removes cluster voltage 80V.
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