CN109001363A - A kind of high pH reverse-phase chromatography high throughput Isolation method of whole protein group - Google Patents
A kind of high pH reverse-phase chromatography high throughput Isolation method of whole protein group Download PDFInfo
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- CN109001363A CN109001363A CN201810922316.4A CN201810922316A CN109001363A CN 109001363 A CN109001363 A CN 109001363A CN 201810922316 A CN201810922316 A CN 201810922316A CN 109001363 A CN109001363 A CN 109001363A
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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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Abstract
The present invention relates to a kind of high pH reverse-phase chromatography high throughput Isolation methods of whole protein group.The high pH reverse-phase chromatography high throughput Isolation method of whole protein group, it is characterized in that, protein mixture is added in SPE column, the protein mixture is mixed with the reverse-phase chromatography filler in SPE column, the eluent of opposed polarity is sequentially added in SPE column, the eluent discharge SPE column of opposed polarity is dissolved to the fraction of different albumen using syringe, centrifuge tube receives fraction.Compared with prior art, compared with prior art, the method for the present invention is easy to operate, quick, flux is high.
Description
Technical field
The present invention relates to protein pre-separation research fields, more particularly, to a kind of high pH reverse-phase chromatography of whole protein group
High-throughput Isolation method.
Background technique
The pre-separation technology of protein, has great importance in the research of proteomics.In proteomics
The dynamic range of protein analysis and the coverage rate of protein identification can be improved using the method for orthogonal separation.Yang F et al.
(Yang F,Shen Y,Camp D G,et al.High-pH reversed-phase chromatography with
fraction concatenation for 2D proteomic analysis[J].Expert Review of
Proteomics, 2012,9 (2): 129-34.) it establishes with high pH reverse-phase chromatography pre-separation, using low pH reverse-phase chromatography as most
It is one-dimensional afterwards that Separation Research carried out to protein sample, and by this clastotype and ion exchange-reverse phase chromatographic isolation mode into
Comparison is gone；As a result, it has been found that the separating effect of high-low pH reversed phase chromatography separation mode is better than ion exchange-reverse phase chromatographic isolation
Mould, the reverse-phase chromatography pre-separation under the conditions of high pH can effectively replace ion-exchange chromatography pre-separation.Wang Z et al. (Wang
Z,Ma H,Smith K,et al.Two-Dimensional Separation Using High pH and Low pH
Reversed Phase Liquid Chromatography for Top-down Proteomics[J].International
Journal of Mass Spectrometry, 2017,427.) be using specification (3.5 μm, 2.1mm × 250mm)
C4 chromatographic column, the offline separation method of two dimension associated with high pH and low pH efficient liquid phase reverse-phase chromatography, to whole Escherichia coli egg
White to have carried out pre-separation and Separation Research, the applied sample amount of this method is 50 μ g, while reverse phase of the research also to high pH and low pH
The orthogonality of chromatographic isolation is studied, and the identification quantity of protein obviously mentions after two methods of result of study display combination
It is high.
It is above-mentioned research shows that two dimensional separation method associated with high pH and low pH separation has good orthogonality.Two kinds points
From the identification quantity that the combination of method can significantly improve protein.Currently, for the reverse phase color of albumen whole under the conditions of high pH
Spectrum separation has certain research；But be all based on mostly business chromatographic column or homemade capillary chromatographic column etc. it is online or from
The separation method of line；The flux that these separation methods handle sample is low, and separating rate is relatively slow, when facing a large amount of sample
Than relatively time-consuming.For this purpose, the pre-separation of protein needs a kind of quick method of high throughput, to increase the flux of protein pre-separation
And separating rate.
105890960 A of patent CN uses size exclusion chromatography filler, reverse-phase chromatography filler or ion-exchange chromatography filler
As fixed phase stuffing, after fixed phase stuffing is mixed with protein mixture, the elution buffer night of varying strength is added, sufficiently
Simultaneously centrifugal treating is mixed, elution buffer is collected.It is collected separately based on protein of different nature in protein mixture
In the elution buffer of varying strength, the pre-separation of whole protein mixture is realized.The fast speed of this method, but the party
In method separation method, contains salt in obtained partially protein fraction, need offline desalination, will cause a large amount of eggs in desalting process
The loss of white matter, while the complicated operation such as needing to be centrifuged, since fraction and filler are all in the same centrifuge tube, this makes portion
It is easily pumped into filler when fractionation part residual or absorption fraction, and remaining fraction can be mixed into the component of next fraction, meeting
Pre-separation effect is caused to be deteriorated, and the filler being sucked out will increase the pretreated difficulty of fraction.
Summary of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of whole protein groups
High-throughput Isolation method.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of high pH reverse-phase chromatography high throughput Isolation method of whole protein group, is added protein mixture in SPE column,
The protein mixture is mixed with the reverse-phase chromatography filler in SPE column, and the eluent of opposed polarity is sequentially added in SPE column,
The eluent discharge SPE column of opposed polarity is dissolved to the fraction of different albumen using syringe, centrifuge tube receives fraction.
In the specific embodiment of the present invention, the high pH reverse-phase chromatography high throughput pre-separation of whole protein group
Method specifically includes the following steps:
(1) protein mixture is added in SPE column, so that protein sample is slowly passed through chromatograph packing material and is adsorbed, SPE
The liquid of outflow is accepted with centrifuge tube in the lower end of column；
(2) mobile phase of 8 kinds of opposed polarities is added in above-mentioned steps (1) treated reverse-phase chromatography filler, slowly pushes away
Syringe successively elutes, and the polarity of the mobile phase of addition is ascending, and the lower end of SPE column accepts the fraction of outflow with centrifuge tube；
(3) each fraction is concentrated and dried with vacuum concentration instrument.
In the specific embodiment of the present invention, the pH of the mobile phase of the step (2) is greater than 8.
In the specific embodiment of the present invention, the liquid for flowing out the lower end of SPE column in the step (1) is again
It is added in SPE column, repetitive operation 4-6 times.
In the specific embodiment of the present invention, the outlet of the syringe is connect by adapter with SPE column, institute
It states the usage mode of syringe: disconnecting the connection between SPE column and adapter, syringe pulls back, and it is mixed that albumen is added in SPE column
After closing liquid or eluent, couples SPE column and adapter, slowly inject emitter, flow out the liquid in SPE column.
In the specific embodiment of the present invention, the mobile phase of the opposed polarity is respectively by the buffering of different volumes
Liquid A and buffer solution B are formulated, the acetonitrile concentration of the ascending mobile phase of polarity is respectively 5.0%, 20.0%, 35%,
40%, 48%, 55%, 75%, 90%, the acetonitrile concentration is indicated with volume ratio.
In the specific embodiment of the present invention, the buffer solution A is the formic acid aqueous ammonium of 10mM；Described
The composition volume ratio of buffer solution B is the formic acid aqueous ammonium of 90% acetonitrile and 10%10mM.
In the specific embodiment of the present invention, two panels sieve plate, the reverse-phase chromatography filler are placed in the SPE column
Among two panels sieve plate.
In the specific embodiment of the present invention, the reverse-phase chromatography filler includes C4 reverse-phase chromatography filler.
The specific embodiment of the present invention is as follows:
(1) mobile phase A (the formic acid aqueous ammonium of 10mM) is prepared；Prepare Mobile phase B (90% acetonitrile/10% ammonium formate water
Solution).
(2) 8 the mobile phase C, each different mobile phase C being made of the mobile phase A and Mobile phase B of different proportion are designed
Volume be 300 μ L, each mobile phase is saved with independent centrifuge tube.
(3) connection between SPE column and adapter is first disconnected, C4 reverse-phase chromatography filler is added in SPE column.
(4) chromatograph packing material is balanced using mobile phase A (the formic acid aqueous ammonium of 10mM), is flowed out from SPE column lower end
Liquid, be centrifuged 5 minutes with supercentrifuge, to check whether filler leaks out, determine after filler will not leak out carry out it is other
Separating step.
(5) after completing step 4, whole Escherichia coli protein mixed liquor is added in SPE column, syringe post-tensioning, then
SPE column and adapter are connected together, the lower end of SPE column accepts the liquid of outflow with centrifuge tube, slowly inject emitter, make
Protein mixed liquor slowly passes through chromatograph packing material and is adsorbed.
(6) liquid flowed out is taken out with liquid-transfering gun and is added in SPE column again, is repeated step 5 totally 6 times.
(7) using 8 different proportions mobile phase A (the formic acid aqueous ammonium of 10mM) and Mobile phase B (90% acetonitrile/
10% formic acid aqueous ammonium) composition mobile phase C, E. coli SampLes are eluted, the mobile phase of each different proportion is washed
De- protein example is a fraction, the operation of elution are as follows: disconnect the connection between SPE column and adapter, syringe to
Post-tensioning takes the mobile phase A of 300 μ L and the mixed liquor of Mobile phase B with liquid-transfering gun, is added in SPE column, couples SPE column and adapter,
Emitter slowly is injected, flows out mobile phase, the centrifuge tube of the different numbers of the fraction flowed out under different mobile phase ratios receives.
It is repeated 8 times elution action, is collected altogether to 8 different fractions.
(8) 8 fractions obtained are concentrated and dried with vacuum concentration instrument, are delayed with pure water or with the loading of liquid chromatogram
Fliud flushing dissolved to get pre-separation after 8 fraction samples.
(9) method for using PAGE gel electrophoresis carries out separating effect verifying to 8 different fraction samples.
Compared with prior art, the present invention uses the eluent of high pH, and the mode with syringe separation is easy to operate
Quickly, flux is high, can thoroughly extrude each fraction, make to interfere with each other between fraction, since what is utilized is to wave
Hair property alkali and salt, can remove volatile alkali by centrifugal concentrating, in addition fraction can realize online desalination；As long as increasing chromatography to fill out
The dosage of material, so that it may increase the flux of separation.
Detailed description of the invention
Fig. 1: the component part of pre-separate device used in the present invention；
Fig. 2: pre-separate device structural schematic diagram used in the present invention；
Gel electrophoresis figure of Fig. 3: the E.coli sample through high pH pre-separation fraction.
Band 1 is Marker, and band 2 to 9 is respectively that fraction 1 arrives fraction 8, and band 10 is E.coli protein mixed liquor.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
1, mobile phase A and B are prepared
Prepare mobile phase A (the formic acid aqueous ammonium of 10mM): (1) weigh the ammonium formate of 0.0859g, be placed in 15mL from
In heart pipe, add the ultrapure water of 10.00mL, sufficiently dissolve, measuring its volume is 10.1mL, obtains the ammonium formate that concentration is 0.135M
Solution；(2) ammonium formate solution for taking 1.1111mL 0.135M adds the ultrapure water of 18.8889mL, sufficiently in 15mL centrifuge tube
It mixes, obtains mobile phase A；(3) it takes the concentrated ammonia liquor of 50 μ L in the solution of step (2), a pH often plus is once measured, to 5 times
When be reduced to 20 μ L or less every time, finally with pH meter measure pH value, consume 335 μ L ammonium hydroxide altogether, obtain pH be 10 ammonium formates-ammonia delay
Solution is rushed, this solution is mobile phase A (10mM formic acid aqueous ammonium).
It prepares Mobile phase B (90% acetonitrile/10% formic acid aqueous ammonium): weighing the ammonium formate of 0.0095g, add 1.5mL's
Then plus the acetonitrile of 13.5mL ultrapure water dissolution obtains the ammonium formate solution of 10mM, adds the ammonium hydroxide of 335 μ L, this solution is flowing
Phase B (90% acetonitrile/10% formic acid aqueous ammonium).
2,8 kinds of mobile phase C being made of the mobile phase A and Mobile phase B of different proportion are designed:
The concentration for the corresponding acetonitrile of mobile phase C that number is 1 to 8 is respectively as follows: 5.0%, 20.0%, 35%, 40%,
48%, 55%, 75%, 90%, the volume of each different mobile phase C is 300 μ L, the independent centrifugation of each mobile phase
Pipe is saved, and specific configuration proportion is shown in Table 1.
The composition of 1 mobile phase C of table
3, using Fig. 2 device, the connection between SPE column and adapter is first disconnected, weighs 50mg's with electronic analytical balance
C4 reverse-phase chromatography filler is added in SPE column.
4, the mobile phase A of 500 μ L is taken with liquid-transfering gun, is added in SPE column, adapter and SPE is connected in syringe post-tensioning
A centrifuge tube is placed in the lower end of column, SPE column, slowly injects emitter, and the solution flowed out from SPE column lower end uses supercentrifuge
It is centrifuged 5 minutes under 12000rpm, checks whether filler leaks out, determined and carrying out other separation steps after filler will not leak out
Suddenly.
5, after completing step 4, the protein mixing that the salt-free E.coli of 500 μ L, 2.06 μ g/ μ L (1030 μ g) is broken is taken
Liquid is added in SPE column, syringe post-tensioning, then SPE column and adapter is connected together, and the lower end of SPE column is accepted with centrifuge tube
The solution of outflow, slowly inject emitter, so that protein mixed liquor is slowly passed through chromatograph packing material and is adsorbed.
6, the liquid flowed out is taken out with liquid-transfering gun and is added in SPE column again, repeats step 4 and step 5 totally 6 times, repeatedly on
Sample improves ratio of adsorption.
7, e. coli protein sample is eluted by 8 different mobile phase C that table 1 is prepared, by second in mobile phase C
The sequence of nitrile concentration from small to large.The protein example of each difference mobile phase C elution is a fraction, the operation of elution
Are as follows: the connection between SPE column and adapter is disconnected, syringe pulls back, and the mobile phase C of 300 μ L is taken with liquid-transfering gun, and SPE is added
In column, couple SPE column and adapter, slowly inject emitter, flow out mobile phase, the fraction flowed out under different mobile phase ratios is used
500 μ L centrifuge tubes of difference number receive.It is repeated 8 times elution action, collects 8 different fractions such as table 1 altogether.
8,8 fractions obtained are concentrated and dried with vacuum concentration instrument, are dissolved with the pure water of 50 μ L to get pre-
8 fractions after separation.
9, with the method for PAGE gel electrophoresis, separating effect verifying is carried out to 8 different fraction samples: specific behaviour
As:
(1) preparation and the encapsulating of 15% separation gel are prepared by table 2:
The preparation method of 2 separation gel of table
The separation gel of 6.5mL is added in gel slot, with water-stop, stands 30 minutes.
(2) 5% concentration glue and encapsulating are prepared by table 3
The preparation method of the concentration glue of table 3
Sealing water in gel slot is gone, blots water with dust-free paper；Prepared concentration glue is added to full, 10 holes of insertion
Comb, stand 30 minutes, remove comb.
(3) in 8 centrifuge tubes, it is separately added into 2 × electrophoretic buffer of 15 μ L and 1 to No. 8 fraction solution of 15 μ L, respectively
The applied sample amount of fraction is 15 μ L；E.coli original protein sample applied sample amount is 12 μ L.
(4) constant pressure 120V, electrophoresis 80 minutes, electrophoresis pressed the specification of coomassie brilliant blue staining kit after the completion, utilized
Coomassie brilliant blue staining kit is dyed and is decolourized to gel.Experimental result is shown in Fig. 3, E.coli to obtain original protein sample quilt
Separation is in fraction 1 to 8.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Claims (9)
1. a kind of high pH reverse-phase chromatography high throughput Isolation method of whole protein group, which is characterized in that protein mixing is added
Reverse-phase chromatography filler of the object in SPE column, the protein mixture and SPE column mixes, and sequentially adds opposed polarity in SPE column
Eluent, the eluent of opposed polarity discharge SPE column has been dissolved to the fraction of different albumen, centrifuge tube using syringe
Receive fraction.
2. the high pH reverse-phase chromatography high throughput Isolation method of whole protein group according to claim 1, which is characterized in that tool
Body the following steps are included:
(1) protein mixture is added in SPE column, protein sample is made slowly to pass through chromatograph packing material and be adsorbed, SPE column
The liquid of outflow is accepted with centrifuge tube in lower end；
(2) mobile phase of 8 kinds of opposed polarities is added in above-mentioned steps (1) treated reverse-phase chromatography filler, slowly injects and penetrates
Device successively elutes, and the polarity of the mobile phase of addition is ascending, and the lower end of SPE column accepts the fraction of outflow with centrifuge tube；
(3) each fraction is concentrated and dried with vacuum concentration instrument.
3. the high pH reverse-phase chromatography high throughput Isolation method of whole protein group according to claim 2, which is characterized in that institute
The pH for stating the mobile phase of step (2) is greater than 8.
4. the high pH reverse-phase chromatography high throughput Isolation method of whole protein group according to claim 2, which is characterized in that institute
It states in step (1) and rejoins the liquid that the lower end of SPE column is flowed out in SPE column, repetitive operation 4-6 times.
5. the high pH reverse-phase chromatography high throughput Isolation method of whole protein group according to claim 2, which is characterized in that institute
State the outlet of syringe to connect by adapter with SPE column, the usage mode of the syringe: disconnection SPE column and adapter it
Between connection, syringe pulls back, and after mixed liquid of protein or eluent is added in SPE column, couples SPE column and adapter, slowly
Emitter is injected on ground, flows out the liquid in SPE column.
6. the high pH reverse-phase chromatography high throughput Isolation method of whole protein group according to claim 2, which is characterized in that institute
The mobile phase for stating opposed polarity is formulated by the buffer solution A of different volumes and buffer solution B respectively, the ascending flowing of polarity
The acetonitrile concentration of phase is respectively 5.0%, 20.0%, 35%, 40%, 48%, 55%, 75%, 90%, the acetonitrile concentration body
Product is than indicating.
7. the high pH reverse-phase chromatography high throughput Isolation method of whole protein group according to claim 6, which is characterized in that institute
The buffer solution A stated is the formic acid aqueous ammonium of 10mM；The composition volume ratio of the buffer solution B is 90% acetonitrile and 10%10mM
Formic acid aqueous ammonium.
8. the high pH reverse-phase chromatography high throughput Isolation method of whole protein group according to claim 1, which is characterized in that institute
Placement two panels sieve plate in SPE column is stated, the reverse-phase chromatography filler is among two panels sieve plate.
9. the high pH reverse-phase chromatography high throughput Isolation method of whole protein group according to claim 1, which is characterized in that institute
Stating reverse-phase chromatography filler includes C4 reverse-phase chromatography filler.
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Application publication date: 20181214 |