CN108359704A - The processing method of goat dairy lactalbumin for proteomics research - Google Patents
The processing method of goat dairy lactalbumin for proteomics research Download PDFInfo
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- CN108359704A CN108359704A CN201810163765.5A CN201810163765A CN108359704A CN 108359704 A CN108359704 A CN 108359704A CN 201810163765 A CN201810163765 A CN 201810163765A CN 108359704 A CN108359704 A CN 108359704A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
Abstract
The processing method of the goat dairy lactalbumin for proteomics research of the present invention belongs to the technical field of lactalbumin extraction and processing method.Goat dairy sample high speed centrifugation is taken to obtain the whey of crude separation, then ultracentrifugation removes casein again, is purified with pre- cold acetone precipitation, obtains lactalbumin quality sample；Dithiothreitol (DTT), 3 acetic acid of indoles progress reductive alkylation are added into lactalbumin quality sample, trypsin solution is added and carries out enzymolysis incubation in super filter tube, much peptidase hydrolyzed liquor；To polypeptide solution desalting processing, peptide fragment sample is re-dissolved through centrifugal concentrating drying, directly upper machine analysis.The method of the invention is easy to operate, recovery rate is high, stability is strong and flux is high, especially for low abundance proteins, is more easy to realize the high-throughput detection of proteomics.There is preferable popularizing application prospect in terms of goat dairy whey protein group research.
Description
Technical field
The present invention relates to a kind of using proteome analysis as the extraction of purpose lactalbumin and processing method, especially
A kind of sample-pretreating method for the research of goat dairy lactalbumin group.
Background technology
The technology platform important as rear era gene, proteomics are to study a new field of protein group,
It substantially refers to the characteristic that protein is studied in extensive level, including protein expression level, amino acid sequence, turns over
Translate post-processing and protein interaction, understand on protein level the various functions of cell, various physiological and biochemical procedures with
And the pathologic process etc. of disease.The all protein expressed by a set of gene, one can be studied by proteomic techniques
The secondary kinds of protein number identified can up to thousands of kinds, and the pre-treatment of sample is a step of wherein most critical, directly determines
The success or failure of proteomics experiment.
Main component is lactoprotein, lactose and butterfat in breast, and wherein lactoprotein is most important constituent in breast.Breast
Kinds of protein is various, and the influence due to hereditary variation and posttranslational modification to lactoprotein makes the composition of lactoprotein become
It is more complicated, huge.In recent years, it is found by proteomics research, body defenses protein is as the maximum albumen of content
Matter type is present in lactalbumin, and body defenses protein includes the higher immunoglobulin of content and the lower benefit of content
Body systematic protein etc..Although people were to proteomics research more than 50 years, the composition, structure in relation to lactoprotein and modification
It is fully clarified yet that not yet etc. a series of problem.The research of Goats Milk is concentrated mainly on to lactoprotein functionality both at home and abroad
Low abundance proteins (protein abundance i.e. certain protein Proteomics content height, usually by concentration ng/mL~
The protein definition of pg/mL be low abundance proteins) identification and compare, but due to limitation of sample pretreating method etc. because
Element, still there are many there is the active albumen of critical function to have no identification and report.
One of the main problem that goat dairy protein science research at present faces is exactly that the extraction difficulty of protein groups is larger.It is main
There is following reason：1. kinds of protein is various in breast, the milk sample in different genera source has differences at being grouped as, physics
The step of sex differernce is bigger, and the milk sample of separate sources can influence sample preparation processing and parameter, method used at present are difficult to
Realize the high efficiency extraction to all albumen；2. the abundance difference of Goat milk albumen is larger, some protein contents are considerably less, mesh
Preceding method, which can usually leak, carries low abundance proteins；3. traditional procedure is all relatively cumbersome, it be easy to cause influence liquid
The uncertain factor of matter result.
Invention content
Due to the particularity of proteomics research purpose, other are for the purpose of dairy products manufacture, conventional physical and chemical analysis etc.
The preparation processing step of goat dairy whey protein be not fully suitable for sample for the purpose of proteomics research
Preparation is handled；The milk sample in different genera source because it is also differed at the different requirement to processing step and parameter being grouped as,
The technical problem to be solved by the present invention is to overcome protein in domestic existing goat dairy whey protein group research to be not easy
Extraction, and the problem of leakage carries is easy for the less low abundance proteins of content, provide it is a kind of it is easy to operate, recovery rate is high,
The sample preparation processing method of the strong and high-throughput goat dairy whey protein group research of stability.
To achieve the above object, the present invention provides the following technical solutions.
The implementation of the present invention mainly has the following steps：The selection of goat dairy sample, sample pretreatment, pre- cold acetone precipitation,
Reductive alkylation, protein digestion, the extraction of peptide fragment and purifying, peptide fragment desalting processing and drying.
1) selection of goat dairy sample：The fresh sheep breast of healthy goat is selected, usual sample number is in 5-50, to eliminate individual
Difference this method chooses the sheep breast of 30 goats as whole milk samples, and the general number that samples is not easy very little, the very few easy influence of sample number
Final test result, sample number, which is more than the kinds of protein number detected with increases of sampling number after 50, to be increased, but increase
Amplitude is little；Every goat takes the goat dairy of 20-80mL, selects to take 50mL goat dairies in this method.The fresh sheep taken from pasture
Breast must transport laboratory treatment back with ice chest or -20 DEG C as early as possible, and -60 DEG C of the following conditions guarantors need to be positioned over by being such as unable to timely processing
It deposits, 2-6 DEG C of refrigerator defrosting is placed it in before use, this method must use (- 60 DEG C or less) preservations of fresh or condition of ultralow temperature
Sheep breast, 2-6 DEG C and -20 DEG C are only applicable to transport in short-term, it is impossible to be used in the storage of sheep milk sample product is placed, and albumen otherwise can occur
Degradation influence protein science measurement result.
2) sample pretreatment：The sheep breast of step 1) (is such as stored in -60 DEG C of following temperature, sheep breast need to be placed in 4 DEG C of refrigerators
Thaw), 15~30min 5000~15000g, is centrifuged under the conditions of 4 DEG C, it is careful to remove upper layer butter oil layer and lower layer's casein
Particle obtains thick lactoalbumin soln；Take thick lactoalbumin soln centrifuge 0.5 under the conditions of 50000~150000g, 4 DEG C~
2h removes the casein in whey, collects upper layer whey liquid.Centrifuging temperature should not increase in this method, and increasing temperature can cause
In the subsequent operation that room temperature carries out, (operation for scraping off fat deposit at ambient temperature needs to consume the regular hour, especially exists
When needing largely to handle sample, this section operation time in can cause centrifugation after milk sample heating) when fat deposit occurs
Back dissolving.Currently preferred centrifugal condition twice is respectively：The condition of centrifugation is 8000g, when centrifuging under the conditions of 4 DEG C for the first time
Between 20min；The condition of second centrifugation is 100000g, centrifugation time is 1h under the conditions of 4 DEG C.
3) pre- cold acetone precipitation：- 20 DEG C of pre- cold acetones of 2~6 times of volumes are added into the whey liquid obtained in step 2),
Rapid mixing (this process should be carried out in ice bath)；(8~15 hours) are stood overnight in -18 DEG C of following temperature, are contained in whey at this time
Some part lipid is dissolved in acetone, and high speed centrifugation abandons supernatant, is then washed twice with the 500 pre- cold acetones of μ L, small after high speed centrifugation
The heart removes supernatant, uncaps and volatilizes, and air-dries precipitation, and air-dry time is 5~10min, should not be over-drying, if air-dry time mistake
Long, the lactalbumin of precipitation is likely to occur undissolved situation when redissolving.
4) reductive alkylation：The albumen precipitated in step 3) is redissolved with ultra-pure water, the albumen for measuring above-mentioned protein solution is dense
After degree, take after the 200 μ L dilution of Tris-Hcl buffer solutions of 100~300 μ g albumen ureas containing 8M loading to 10K super filter tubes, on
Sample amount should not be too large, and try not more than 350 μ g, and applied sample amount is bigger, and ultrafiltration is slower, the dithiothreitol (DTT) of the 1M of 1 μ L of addition, and 37
15~30min is reacted under the conditions of DEG C；Then it waits for that protein sample temperature is cooled to room temperature the indole-3-acetic acid for the 1M that 5 μ L are added, mixes
After even 15~30min is placed at the place of being protected from light.
5) protein digestion：The ammonium bicarbonate soln that 50mM is added into the super filter tube of step 4) is replaced 2~3 times, in room
Centrifugation discards efflux under the conditions of temperature；It uses the ammonium bicarbonate soln of 50mM to be resuspended again, trypsase is added and is digested, tryptose
The mass ratio of enzyme and protein is about 1:30~120, preferable ratio of choosing is 1:80, by the super filter tube after addition trypsase
It is positioned in 37 DEG C of insulating boxs and digests 3-7 hours；It is added again after the completion of enzymolysis for the first time and digests same amount of pancreas for the first time
Protease is completed second and is digested, obtains the enzymolysis polypeptide solution of lactalbumin for (8~15 hours) overnight under the conditions of 37 DEG C；
6) extraction and purifying of peptide fragment：Polypeptide solution obtained by middle to step 5), 7000~12000g centrifugations 10~
20min, collects efflux, then with twice of the ammonium bicarbonate soln diafiltration of 50mM, merging efflux；Then it is acidified with trifluoroacetic acid
It terminates and reacts to final concentration 0.3%~0.6%；
7) peptide fragment desalting processing：Desalination is carried out to peptide fragment using Pierce C18Tips (ThermoFisher companies), is taken off
Salt flow is：C18 desalting columns first are activated with 100~200 μ L methanol, room temperature centrifuges 5~15min, abandons efflux；With 100~200
80% acetonitriles of the μ L containing 0.2% trifluoroacetic acid soaks desalting column, and centrifugation is abandoned efflux, is repeated once；With 100~200 μ L
0.2% trifluoroacetic acid balances desalting column, is repeated once；Enzymolysis sample is injected into desalting column；With 100~200 μ L's 0.2%
Trifluoroacetic acid elutes salt plug, washes 2 times；It is eluted, is washed 2 times with 80% acetonitriles of 100~200 μ L containing 0.2% trifluoroacetic acid again, received
Collection merges eluate twice；
8) eluate is after room temperature is spin-dried in centrifuge concentrator, directly upper Nano1200, QE after being dissolved with buffer solution
(thermoFisher companies) carries out proteome analysis.
According to above-mentioned technical solution and latter embodiments, technical scheme of the present invention is summarized as follows.
A kind of preparation processing method of goat dairy lactalbumin for proteomics research, there is the choosing of goat dairy sample
It takes, at sample pretreatment, pre- cold acetone precipitation, reductive alkylation, protein digestion, the extraction of peptide fragment and purifying, peptide fragment desalination
Reason and dry processing step；
The selection of the goat dairy sample selects the sheep breast of healthy goat as whole milk samples；
The sample pretreatment is that whole milk samples are centrifuged to 15~30min under the conditions of 5000~15000g, 4 DEG C, removes
Upper layer butter oil layer and lower layer's casein particle are removed, thick lactoalbumin soln is obtained；Take thick lactoalbumin soln 50000~
150000g, 0.5~2h is centrifuged under the conditions of 4 DEG C, remove the casein in whey, collect upper layer whey liquid；
The pre- cold acetone precipitation is that pre- cold acetone mixing is added into whey liquid, and pre- cold acetone addition is whey
2~6 times of liquid product；It is placed 8~15 hours in -18 DEG C of following temperature, then centrifuges and abandon supernatant, washed 2 times with pre- cold acetone；Centrifugation
Supernatant is carefully removed afterwards, is uncapped and is volatilized, the air-dried albumen precipitated；
The reductive alkylation after the albumen concentration for measuring protein solution, is taken with the albumen of ultra-pure water redissolution precipitation
Loading is to super filter tube after redissolving the Tris-Hcl buffer solutions dilution of albumen urea containing 8M；The dithiothreitol (DTT) of addition 1M, 37 DEG C
Under the conditions of react 15~30min；Be cooled to room temperature the indole-3-acetic acid that 1M is added, after mixing the place of being protected from light placement 15~
30min；Wherein, the mass volume ratio (g/L) of albumen and Tris-Hcl buffer solutions is 100~300:200, albumen and two sulphur are revived
The mass volume ratio (g/L) of sugar alcohol is 100~300:1, the mass volume ratio (g/L) of albumen and indole-3-acetic acid is 100~
300:5；
The protein digestion is that the ammonium bicarbonate soln of from the addition 50mM to super filter tube is replaced 2~3 times, and centrifugation is abandoned
Remove efflux；It uses the ammonium bicarbonate soln of 50mM to be resuspended again, trypsase is added and is digested, the quality of trypsase and albumen
Than being 1:30~120, the super filter tube after addition trypsase is positioned in 37 DEG C of insulating boxs and is digested 3~7 hours；First time enzyme
Solution is added and digests same amount of trypsase for the first time again after the completion, is digested 8~15 hours for second under the conditions of 37 DEG C,
Obtain the enzymolysis polypeptide solution of lactalbumin；
The extraction and purifying of the peptide fragment are to centrifuge 10~20min to 7000~12000g of polypeptide solution, collect outflow
Liquid, then with twice of the ammonium bicarbonate soln diafiltration of 50mM, merge efflux；Then it is acidified to final concentration 0.3% with trifluoroacetic acid
~0.6% terminates reaction, the peptide fragment purified；
The peptide fragment desalting processing is using Pierce C18Tips (ThermoFisher companies) to the peptide fragment of purifying
Desalination is carried out, eluate is collected；
The drying is dissolved with Tris-HCl buffer solutions by eluate after room temperature is spin-dried in centrifuge concentrator
Directly upper Nano1200, QE (thermoFisher companies) carry out proteome analysis afterwards.
In the selecting step of goat dairy sample, the whole milk samples are fresh goat dairies, or -60 DEG C or less
The goat dairy placed is stored, or stores 3 hours goat dairies below at -20~6 DEG C.
In sample pretreatment step, centrifugal condition is respectively twice：Centrifugation for the first time is 8000g, is centrifuged under the conditions of 4 DEG C
Time 20min；Second centrifugation is 100000g, centrifugation time is 1h under the conditions of 4 DEG C.
In pre- cold acetone precipitation step, the pre- cold acetone is the pre- acetone for being cooled to -20 DEG C；The whey liquid
It is middle that pre- cold acetone mixing is added, it is carried out under ice bath environment.
In pre- cold acetone precipitation step, the air-dried precipitation, air-dry time 5-10min.
It is to utilize Pierce C18Tips (ThermoFisher companies) to the peptide of purifying in peptide fragment desalting processing step
Duan Jinhang desalinations, specific desalination flow are：C18 desalting columns first are activated with 100~200 μ L methanol, room temperature centrifuges 5~15min,
Abandon efflux；Desalting column is soaked with 80% acetonitriles of 100~200 μ L containing 0.2% trifluoroacetic acid, efflux is abandoned in centrifugation, is repeated
Once；Desalting column is balanced with the trifluoroacetic acid of 100~200 μ L 0.2%, is repeated once；The peptide fragment of purifying is injected into desalting column；
Salt plug is eluted with the trifluoroacetic acid of 100~200 μ L 0.2%, is washed 2 times；Contain 0.2% trifluoroacetic acid with 100~200 μ L again
80% acetonitrile elution, washes 2 times, collects the eluate merged twice.
Compared with prior art, the beneficial effects of the invention are as follows：1) using the extraction side of " high speed centrifugation-high speed centrifugation "
Method, operation is simpler, and the stability of protein is stronger, the salt used in traditional isoelectric point precipitation when extracting lactalbumin
Acid can influence the stability of protein, and then influence the result of proteomics identification to a certain extent；2) protein is adopted
With being digested in super filter tube, the step before upper machine testing carries out all in super filter tube, and avoiding needs repeatedly to turn in traditional treatment method
The operation of centrifuge tube is moved, multistep transfer operation easily leads to the loss of protein loss, especially low abundance proteins.The present invention's
Method makes the extraction efficiency higher of whey protein, especially for low abundance proteins, is more easy to realize the height of proteomics
Flux detects.
Description of the drawings
Fig. 1 is the process flow figure of the goat dairy lactalbumin of the present invention.
Fig. 2 is the comparison of the detected lactalbumin type number difference after embodiment and comparative example handle milk sample product
Figure.
Specific implementation mode
With reference to specific embodiment, present invention is further described in detail, to enable those skilled in the art with reference to explanation
Book word can be implemented according to this, but not as a limitation of the invention.
Embodiment 1
1) selection of goat dairy sample：The fresh sheep breast for selecting healthy Guan zhong dairy goat, to eliminate individual difference this method
The sheep breast of 30 goats is chosen as whole milk samples, every goat takes the sheep breast of 50ml.
2) sample pretreatment：50ml is stored in -80 DEG C of sheep breast and is placed in 4 DEG C of refrigerator defrostings (or using fresh sheep breast),
8000g, 20min is centrifuged under the conditions of 4 DEG C, it is careful to remove upper layer butter oil layer and lower layer's casein particle, obtain thick whey egg
White solution；It takes thick lactoalbumin soln to centrifuge 1h under the conditions of 100000g, 4 DEG C, removes the casein in whey, collect upper layer
Whey liquid.
3) pre- cold acetone precipitation：- 20 DEG C of pre- cold acetones of 5 times of volumes are added into the whey liquid obtained in step 2), it is fast
Fast mixing (this process should be carried out in ice bath)；It is placed overnight at -20 DEG C, high speed centrifugation abandons supernatant, then with 500 μ L precoolings third
Ketone is washed twice, and supernatant is carefully sucked after high speed centrifugation, is uncapped and is volatilized, and precipitation is air-dried, and air-dry time 5-10min is sunk
The albumen in shallow lake.
4) reductive alkylation：The albumen that precipitation is redissolved with ultra-pure water, with the egg for the protein solution that BCA kit measurements precipitate
White concentration, the 1M of 2 μ L is added to 10K super filter tubes in loading after taking the Tris-Hcl buffer solutions of 200 μ g albumen ureas containing 8M to dilute
Dithiothreitol (DTT), react 2h under the conditions of 37 DEG C；Then wait for that protein sample is cooled to room temperature the indoles -3- second for the 1M that 5 μ L are added
Acid places 30min after mixing at the place of being protected from light；The dithiothreitol (DTT) solution of the 1M of 2 μ L is added into mixed solution again, placement is kept away
25min at light obtains the protein solution after reductive alkylation.
5) protein digestion：The ammonium bicarbonate soln that 50mM is added into super filter tube is replaced twice, and room temperature centrifugation discards stream
Go out liquid；It is resuspended with the ammonium bicarbonate soln of 50mM, trypsase is added and is digested, the mass ratio of trypsase and protein is about
It is 1:80,37 DEG C digest 4 hours, obtain first time enzymolysis polypeptide solution, pancreas egg is added into first time enzymolysis polypeptide solution
White enzyme (about 1:80, w:W), it digests overnight under the conditions of 37 DEG C, obtains the enzymolysis polypeptide solution of second of lactalbumin.
6) extraction and purifying of peptide fragment：To the polypeptide solution of gained in step 5), 10000g centrifuges 15min, collects outflow
Liquid, then with twice of the ammonium bicarbonate soln diafiltration of 50mM, merge efflux；Then it is acidified to final concentration 0.4% with trifluoroacetic acid
Terminate reaction, the peptide fragment purified；
7) peptide fragment desalting processing：Desalination is carried out to peptide fragment using Pierce C18 desalinations pillars, desalination flow is：First use
200 μ L methanol activate C18 desalting columns, and room temperature centrifuges 10min, abandons efflux；With 80% of 200 μ L containing 0.2% trifluoroacetic acid
Acetonitrile soaks desalting column, and centrifugation is abandoned efflux, is repeated once；Desalting column is balanced with the trifluoroacetic acid of 200 μ L 0.2%, repeats one
It is secondary；Enzymolysis sample (peptide fragment of purifying) is injected into desalting column；Salt plug is eluted with the trifluoroacetic acid of 200 μ L 0.2%, is washed 2 times；Again
It is eluted, is washed 2 times with 80% acetonitriles of the 200 μ L containing 0.2% trifluoroacetic acid, collect the eluate merged twice；
8) eluate is after room temperature is spin-dried in centrifuge concentrator, direct Shang nanoliter efficient liquid phase-after being dissolved with buffer solution
Mass spectrometry (thermoFisher companies) carries out liquid quality detection and then uses proteome analysis.
The method and the method digested in super filter tube, mirror of " high speed centrifugation-high speed centrifugation " have been selected in the present embodiment simultaneously
The protein classes number made is most, detects 368 kinds of albumen (see Fig. 2).
Embodiment 2
Compared with Example 1, second of centrifugal condition is 50000g, centrifuges 2h at 4 DEG C in step 2) sample pretreatment,
Remaining process and condition are same as Example 1.The protein classes number identified is 357 kinds (see Fig. 2).
Embodiment 3
Compared with Example 1, second of centrifugal condition is 150000g, is centrifuged at 4 DEG C in step 2) sample pretreatment
0.5h, remaining process and condition are same as Example 1.The protein classes number identified is 360 kinds (see Fig. 2).
Following embodiment is used for check experiment.
The processing method that comparative example 1 is digested using prior art isoelectric point precipitation and centrifuge tube
1) selection of goat dairy sample：The fresh sheep breast for selecting healthy Guan zhong dairy goat, to eliminate individual difference this method
The sheep breast of 30 goats is chosen as whole milk samples, every goat takes the sheep breast of 50ml.
2) sample pretreatment：50ml is stored in -80 DEG C of sheep breast and is placed in 4 DEG C of refrigerator defrostings (or using fresh sheep breast),
1500g, 15min is centrifuged under the conditions of 4 DEG C, it is careful to remove upper layer butter oil layer and lower layer's casein particle, obtain thick whey egg
White solution；PH value is adjusted to 5.0 with the hydrochloric acid of 1M and 0.1M by the thick lactoalbumin soln of acquisition, is stood under room temperature
30min centrifuges 10min in 5000g, with filtered through gauze, collects whey supernatant；Then use the hydrochloric acid of 1M and 0.1M by pH again
Value is adjusted to 4.6, is stored at room temperature 30min, abandons precipitation in 5000g centrifugation 10min filterings, finally obtains upper layer whey solution.
3) pre- cold acetone precipitation：- 20 DEG C of pre- cold acetones of 5 times of volumes are added in the whey liquid obtained into step (2), it is fast
Fast mixing (this process should be carried out in ice bath)；At -20 DEG C overnight, high speed centrifugation abandons supernatant, is then washed with the 500 pre- cold acetones of μ L
Twice, supernatant is carefully sucked after high speed centrifugation, uncaps and volatilizes, air-drying precipitation, (air-dry time is 5~10min, not done excessively
Dry, if air-dry time is long, the lactalbumin of precipitation is likely to occur undissolved situation when redissolving).
4) reductive alkylation：It after measuring albumen concentration, takes the albumen of 200 μ g in centrifuge tube, two sulphur of the 1M of 2 μ L is added
Threose alcoholic solution shakes mixing, 37 DEG C of water-bath 2h on ice；Wait for that protein sample is cooled to room temperature the indoles second for the 1M for adding 5 μ L
Acid places 30min after mixing at the place of being protected from light, and the dithiothreitol (DTT) solution of the 1M of 2 μ L is added again, and placement is protected from light place 25min, obtains
Protein solution after to reductive alkylation.
5) protein digestion：The lactoalbumin soln obtained in step (4) is diluted to the ammonium bicarbonate soln of 50mM
600 μ L are added trypsase and are digested, and the mass ratio of trypsase and protein is about 1:80,37 DEG C digest 4 hours, obtain
To first time enzymolysis polypeptide solution, trypsase (about 1 is added into first time enzymolysis polypeptide solution:80, w:W), 37 DEG C of conditions
Lower enzymolysis overnight, obtains the enzymolysis polypeptide solution of second of lactalbumin, is then acidified to final concentration 0.4% eventually with trifluoroacetic acid
Only react.
6) peptide fragment desalting processing：Desalination is carried out to peptide fragment using Pierce C18 desalinations pillars, desalination flow is：First use
200 μ L methanol activate C18 desalting columns, and room temperature centrifuges 10min, abandons efflux；With 80% of 200 μ L containing 0.2% trifluoroacetic acid
Acetonitrile soaks desalting column, and centrifugation is abandoned efflux, is repeated once；Desalting column is balanced with the trifluoroacetic acid of 200 μ L 0.2%, repeats one
It is secondary；Enzymolysis sample is injected into desalting column；Salt plug is eluted with the trifluoroacetic acid of 200 μ L 0.2%, is washed 2 times；Contained again with 200 μ L
80% acetonitrile of 0.2% trifluoroacetic acid elutes, and washes 2 times, collects the eluate merged twice；
7) eluate is after room temperature is spin-dried in centrifuge concentrator, direct Shang nanoliter efficient liquid phase-after being dissolved with buffer solution
Mass spectrometry (thermoFisher companies) carries out liquid quality detection and then uses proteome analysis.The protein classes number identified
It is 132 kinds (referring to Fig. 2).
Traditional " isoelectric point precipitation " and centrifuge tube enzyme solution are selected in this comparative example, the experimental results showed that, it is used for
When proteome analysis, the kinds of protein number detected is far less than the albumen prime number that can be detected out in embodiment 1
Amount, this is different from the result in a kind of methods of extraction Goat milk of patent CN201610806041, and the analysis of causes is as follows：1.
Test objective is different, and the present invention is the processing method for the purpose of proteome analysis.Proteomics original meaning refers to a kind of " base
Because of a full set of protein expressed by group ", i.e., including all protein expressed by a kind of cell or even a kind of biology, it is with group
Knit, even the difference of ambient condition and change.Refer to a full set of protein in detection sheep breast in the present invention.Traditional " waits electricity
So that casein precipitate is precipitated according to the different addition hydrochloric acid of lactalbumin and casein isoelectric point in the point precipitation method ", Jin Erda
To the purpose of extraction lactalbumin, and the addition of hydrochloric acid can make partially protein be denaturalized to a certain extent, to make
A full set of protein composition in sheep breast changes, and influences last proteomics testing result.2. the selection of centrifugal rotational speed
It is different.The selection of centrifugal speed is very crucial in " high speed centrifugation-high speed centrifugation " step in the present invention.
Comparative example 2
" high speed centrifugation-high speed centrifugation " method same as Example 1 is selected in this comparative example when step 2) sample pretreatment,
Second of centrifugal condition 100000g, 1h is centrifuged under the conditions of 4 DEG C, remaining step is identical as comparative example 1, that is, selects centrifuge tube enzymolysis
Method.Far fewer than the method (referring to Fig. 2, protein classes number is less than 300 kinds) of the present invention, explanation makes the protein classes number identified
The super filter tube that the present invention is worse than with centrifuge tube enzymolysis detection result digests detection result.
Comparative example 3
" isoelectric point precipitation " identical with comparative example 1 is selected when step 2) sample pretreatment in this comparative example, remaining step
It is rapid same as Example 1, that is, select super filter tube centrifugal method.Method (ginseng of the protein classes number identified far fewer than the present invention
See Fig. 2, protein classes number is close to 200 kinds), as long as illustrating that adding hydrochloric acid makes casein precipitate be precipitated, detection result is with regard to poor
In the method for the present invention.
Summarize the conclusion (of pressure testing) that the above specific implementation mode can obtain：Pass through a nanoliter efficient liquid phase-mass spectrometry (nano
HPLC-MS/MS method detection), be used in combination the proteomic analytical methods obtained goat dairy lactalbumin of analysis embodiment and
The goat dairy lactalbumin that comparative example obtains, in this method the combination of operating procedure and the selection of parameter be more suitable for albumen
The processing of goat dairy lactalbumin for the purpose of the research of matter group.The results show that in the goat dairy lactalbumin that embodiment 1 obtains
Detect the protein classes number and reality that 368 species specificity albumen, lactalbumin property are stablized, and embodiment 2 and 3 detects in testing
It applies example 1 to be not much different, respectively 357 kinds and 360 kinds；And the use of hydrochloric acid and the transfer of multiple centrifuge tube are to sheep in comparative example
Protein groups are affected in breast, and lactalbumin quantity that comparative example testing inspection goes out is far below the albumen number that embodiment detects
Amount, wherein it is minimum with the protein quantity that comparative example 1 detects, it is only capable of detecting 132 kinds of lactalbumins, illustrates prepared by embodiment
Goat dairy lactalbumin effectively prevents the loss of some low abundance proteins, and extraction effect is obviously improved, and reality is more easy to
The now high-throughput detection of the lactalbumin for the purpose of proteome analysis.
Claims (6)
1. a kind of preparation processing method of goat dairy lactalbumin for proteomics research, there is the choosing of goat dairy sample
It takes, at sample pretreatment, pre- cold acetone precipitation, reductive alkylation, protein digestion, the extraction of peptide fragment and purifying, peptide fragment desalination
Reason and dry processing step；
The selection of the goat dairy sample selects the sheep breast of healthy goat as whole milk samples；
The sample pretreatment is that whole milk samples are centrifuged to 15~30min under the conditions of 5000~15000g, 4 DEG C, in removing
Layer butter oil layer and lower layer's casein particle, obtain thick lactoalbumin soln；Take thick lactoalbumin soln 50000~
150000g, 0.5~2h is centrifuged under the conditions of 4 DEG C, remove the casein in whey, collect upper layer whey liquid；
The pre- cold acetone precipitation is that pre- cold acetone mixing is added into whey liquid, and pre- cold acetone addition is whey liqs
Long-pending 2~6 times；It is placed 8~15 hours in -18 DEG C of following temperature, then centrifuges and abandon supernatant, washed 2 times with pre- cold acetone；It is removed after centrifugation
Supernatant is removed, uncaps and volatilizes, the air-dried albumen precipitated；
The reductive alkylation is after the albumen concentration for measuring protein solution, to take redissolution with the albumen of ultra-pure water redissolution precipitation
Loading is to super filter tube after the Tris-Hcl buffer solutions dilution of albumen urea containing 8M；The dithiothreitol (DTT) of 1M, 37 DEG C of conditions are added
15~30min of lower reaction；It is cooled to room temperature the indole-3-acetic acid that 1M is added, in 15~30min of the place of being protected from light placement after mixing；Its
In, the mass volume ratio of albumen and Tris-Hcl buffer solutions is 100~300:200, the mass body of albumen and dithiothreitol (DTT)
Product is than being 100~300:1, the mass volume ratio of albumen and indole-3-acetic acid is 100~300:5；
The protein digestion is that the ammonium bicarbonate soln of from the addition 50mM to super filter tube is replaced 2~3 times, and centrifugation discards stream
Go out liquid；It uses the ammonium bicarbonate soln of 50mM to be resuspended again, trypsase is added and is digested, the mass ratio of trypsase and albumen is
1:30~120, the super filter tube after addition trypsase is positioned in 37 DEG C of insulating boxs and is digested 3~7 hours；It has digested for the first time
It is added again after and digests same amount of trypsase for the first time, digested 8~15 hours for second, obtain under the conditions of 37 DEG C
The enzymolysis polypeptide solution of lactalbumin；
The extraction and purifying of the peptide fragment are to centrifuge 10~20min to 7000~12000g of polypeptide solution, collect efflux,
Again with twice of the ammonium bicarbonate soln diafiltration of 50mM, merge efflux；Then with trifluoroacetic acid be acidified to final concentration 0.3%~
0.6% terminates reaction, the peptide fragment purified；
The peptide fragment desalting processing is to carry out desalination to the peptide fragment of purifying using Pierce C18 Tips, collects eluate；
The drying is by eluate after room temperature is spin-dried in centrifuge concentrator, straight after being dissolved with Tris-HCl buffer solutions
It connects Nano1200, QE and carries out proteome analysis.
2. the preparation processing method of the goat dairy lactalbumin according to claim 1 for proteomics research,
It is characterized in that, in the selecting step of goat dairy sample, the whole milk samples are fresh goat dairies, or -60 DEG C or less
The goat dairy placed is stored, or stores 3 hours goat dairies below at -20~6 DEG C.
3. the preparation processing method of the goat dairy lactalbumin according to claim 1 for proteomics research,
It is characterized in that, in sample pretreatment step, centrifugal condition is respectively twice：Centrifugation for the first time is 8000g, under the conditions of 4 DEG C from
Heart time 20min；Second centrifugation is 100000g, centrifugation time is 1h under the conditions of 4 DEG C.
4. the preparation processing method of the goat dairy lactalbumin according to claim 1 for proteomics research,
It is characterized in that, in pre- cold acetone precipitation step, the pre- cold acetone is the pre- acetone for being cooled to -20 DEG C；The whey liquid
It is middle that pre- cold acetone mixing is added, it is carried out under ice bath environment.
5. the preparation processing method of the goat dairy lactalbumin according to claim 1 for proteomics research,
It is characterized in that, in pre- cold acetone precipitation step, the air-dried precipitation, air-dry time is 5~10min.
6. at the preparation for the goat dairy lactalbumin of proteomics research according to claim 1,2,3,4 or 5
Reason method, which is characterized in that in peptide fragment desalting processing step, carried out to the peptide fragment of purifying using Pierce C18 Tips
Desalination, specific desalination flow are：C18 desalting columns first are activated with 100~200 μ L methanol, room temperature centrifuges 5~15min, and abandoned stream goes out
Liquid；Desalting column is soaked with 80% acetonitriles of 100~200 μ L containing 0.2% trifluoroacetic acid, centrifugation is abandoned efflux, is repeated once；With
The trifluoroacetic acid of 100~200 μ L 0.2% balances desalting column, is repeated once；The peptide fragment of purifying is injected into desalting column；With 100~
The trifluoroacetic acid of 200 μ L 0.2% elutes salt plug, washes 2 times；Contain 80% acetonitrile of 0.2% trifluoroacetic acid with 100~200 μ L again
Elution, is washed 2 times, collects the eluate merged twice.
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| CN115197895A (en) * | 2022-07-28 | 2022-10-18 | 中国人民解放军军事科学院军事医学研究院 | Based on TiO 2 Exosomes omics series extraction technology and application thereof |
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