CN104974218A - Separation method of low-abundance differential protein and application of separation method - Google Patents
Separation method of low-abundance differential protein and application of separation method Download PDFInfo
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Abstract
The invention provides a separation method of a low-abundance differential protein. The method comprises: obtaining a healthy control group protein sample, a patient control group protein sample, and a to-be-tested group protein sample; taking the healthy control group protein sample as an antigen for immunization to prepare a high-abundance protein polyclonal antibody, and taking the high-abundance protein polyclonal antibody as an affinity ligand to prepare a high-abundance affinity chromatography column; enabling the patient control group protein sample to pass through the high-abundance affinity chromatography column, and collecting a pass-through component; concentrating the pass-through component, and taking the concentrated pass-through component as an antigen for secondary immunization to prepare a low-abundance protein polyclonal antibody; and taking the low-abundance protein polyclonal antibody as an affinity ligand to prepare a low-abundance affinity chromatography column; and enabling the to-be-tested group protein sample to pass through the low-abundance affinity chromatography column, and collecting an eluted component. By adopting the separation method, an up-regulation protein, the content of which is relatively high in normal plasma, can be directly separated in one time, and complicatedness of the separation process is reduced. The method can directly aims to the low-abundance protein of differential expression, and has relatively high singularity and pertinency.
Description
Technical field
The invention belongs to protein detection technology field, be specifically related to a kind of separation method and application thereof of low abundance difference albumen.
Background technology
Through all diseases are hidden and the research of pathogenic process verified, potential disease or preclinical physiological status all have found well relevant with physiological status biomarker, and these biomarkers are the albumen that abundance is lower mostly.These low-abundance proteins, in blood plasma, all only have very low concentration, and often by high-abundance proteins such as albumin, can be difficult to be detected, be greatly delayed the discovery time of disease in causing detect the early stage of hiding in disease.So, the plasma proteins under the ratio compared with normal of otherness and improper physiological status, and find out corresponding biomarker, thus aided disease more early find that there is important medical significance.
But based on above-mentioned purpose, the plasma proteins under the ratio compared with normal of otherness and improper physiological status, and find out corresponding biomarker, adopt the conventional sense means of current proteomics, there is larger difficulty.Reason is to find in the process of biomarker protein, will first eliminate high-abundance proteins covering whole system, the albumen of very low concentrations can be detected.Current usual method is presented on 2D running gel by all albumen, then contrasts the differential protein between different sample; And wherein, first 2D electrophoresis can not demonstrate the albumen of extreme acidic or alkalescence, and the lipoprotein that hydrophobicity is larger; If protein is too much, the identification of protein spots also also exists larger difficulty; Secondly the high-abundance proteins in blood plasma can cover the trace of albumin really with very important biomolecule active function, although had the various affine method of report such as CB dye media, antibody medium etc. to remove high-abundance proteins, but the albumen that can remove is still very limited, most of house-keeping albumen and normal function albumen can not be removed; 3rd, the biomarker protein relevant with improper physiological status is not limited to low-abundance protein, and the albumen that some abundance is higher also occurs in disease report, therefore result also can be caused inaccurate so only use high abundance to remove.
Based on above-mentioned deficiency, the contriver of this case early proposed prior to 2010 and a kind ofly carries out enrichment to the differential protein between testing protein and reference protein in CN102430176A patent, thus promoted the method for testing protein or peptide concentration.The process that its technology realizes adopts carries out immunity using standard control albumen as antigen, prepare the immunoglobulin (Ig) that is combined of at least one albumen in this control sample or polypeptide (to be exactly the anti-antibody produced using standard control albumen as antigen in fact, belong to polyclonal antibody), then the chromatography column of affinity media is made with this immunoglobulin (Ig), be used for adsorbing a large amount of high-abundance proteins in testing sample, thus stream is worn component and is low abundance difference albumen in the testing protein that enrichment concentrates.
But in this previous way, normal control albumen is adopted to prepare antibody by immunity, what reference protein itself adopted is normal protein, the low-abundance protein that institute's isolation and identification obtains is the new potential marker protein occurring or raise just, and directly cannot determine that the expression of these albumen belongs to the upregulated protein that in down-regulation protein in improper blood plasma or normal plasma, content is more further, need further to carry out a large amount of comparison and detection in employing contrast, accuracy is reduced after increasing complicated step, improve workload, Shortcomings during aforesaid method is implemented.
Summary of the invention
Object of the invention process is the above-mentioned deficiency overcoming prior art, provide a kind of can the separation method of low abundance difference albumen of low abundance down-regulation protein directly in the improper blood plasma of separation and concentration and application thereof.
In order to realize foregoing invention object, the technical scheme of the embodiment of the present invention is as follows:
A separation method for low abundance difference albumen, comprises the steps:
Obtain normal healthy controls histone sample and patient's control group protein sample and histone sample to be measured;
Normal healthy controls histone sample is carried out immunity as antigen, and preparation high-abundance proteins resists more; And resist for affinity ligand and solid-phase matrix prepare high abundance affinity column with described high-abundance proteins more;
Patient's control group protein sample was carried out post in described high abundance affinity column, and collects stream and wear component;
Described stream is worn after component concentrates and carry out immunity as antigen, prepare low-abundance protein and resist more; And prepare low abundance affinity column as affinity ligand is fixing with solid-phase matrix so that how anti-described low-abundance protein is;
Histone sample to be measured is carried out post in described low abundance affinity column, and collects elution fraction.
The present invention, on the basis of the separation method of above-mentioned low abundance difference albumen, also proposes a kind of application of separation method on low abundance difference expressing protein detects of above-mentioned low abundance difference albumen.
The separation method of low abundance difference albumen of the present invention and application, on the basis of original recipe, first carry out primary immune response with the albumen of healthy group, preparation can be used for the high abundance of filtering a large amount of background high abundance differential protein and resist more, then remove the background high-abundance proteins in improper patient variation's expressing protein sample with this high-abundance proteins how anti-preparation high-abundance proteins chromatography column, the stream of collection wears the low-abundance protein that component is improper patient variation expression; Then this stream is worn after component concentrates and go to carry out second time immunity as antigen, obtain resisting of low abundance difference expression more, afterwards again with the how anti-chromatography column preparing low abundance difference albumen as part further that this low abundance difference is expressed, just can directly belong to the down-regulation protein in improper blood plasma or belong to the more upregulated protein of content in normal plasma in flash liberation histone to be measured sample.Overall process once directly can be separated and obtain the more upregulated protein of content in normal plasma, reduce the complicated property of sepn process, improve separation and detection efficiency, and last elution fraction is directly for the low-abundance protein of differential expression, has higher specificity and specific aim.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing:
Fig. 1 is that the embodiment of the present invention adopts human normal plasma and another liver cancer patient blood plasma to detect according to above-mentioned steps the picture that the elutriant obtained carries out SDS-PAGE Gel Electrophoresis Silver dye respectively as testing protein respectively.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Example of the present invention proposes a kind of separation method of low abundance difference albumen, comprises step summary as follows:
S10, obtains normal healthy controls histone sample, patient's control group protein sample and histone sample to be measured;
S20, carries out first time immunity using normal healthy controls histone sample as antigen, and preparation high-abundance proteins resists more;
S30, how anti-the high-abundance proteins obtained by step S20 is as affinity ligand, is fixed on the solid phase carrier of chromatography column, prepares high abundance affinity column;
S40, carried out post by high abundance affinity column prepared by patient's control group protein sample step S30, collected stream and wore liquid;
S50, the stream that step S40 collects is worn liquid carry out concentrated after, carry out immunity again as antigen, prepare low-abundance protein and resist more;
S60, how anti-the low-abundance protein obtained by step S50 is as affinity ligand, is fixed on the solid phase carrier of chromatography column, prepares low abundance affinity column;
S70, carried out post by histone sample to be measured in the described low abundance affinity column prepared by step S60, and collected elution fraction.
The separation method of low abundance difference albumen of the present invention, on the basis of original recipe, first carry out primary immune response with the albumen of healthy group, preparation can be used for the high abundance of filtering a large amount of background high abundance differential protein and resist more, then remove the background high-abundance proteins in improper patient variation's expressing protein sample with this high-abundance proteins how anti-preparation high-abundance proteins chromatography column, the stream of collection wears the low-abundance protein that component is improper patient variation expression; Then this stream is worn after component concentrates and go to carry out second time immunity as antigen, obtain resisting of low abundance difference expressing protein more, afterwards with the how anti-chromatography column preparing low abundance difference albumen as part further of this low abundance difference expressing protein, just can directly belong to the down-regulation protein in improper blood plasma or belong to the more upregulated protein of content in normal plasma in flash liberation histone to be measured sample.Thus overall employing aforesaid operations process of the present invention is carried out, and reduce the complicated property of sepn process, improve separation and detection efficiency, and last elution fraction is directly for the low-abundance protein of differential expression, has higher specificity and specific aim.
Wherein, said process of the present invention, both the albumen adopting the albumen that obtains from healthy normal people and ill patient to obtain in step slo is respectively as the source of high-abundance proteins and low abundance difference albumen, in the albumen that this patient obtains, the kind of low abundance difference albumen is obviously be different from arm's length standard with amount, is more easy to the expression directly reflecting low abundance difference albumen.Directly prepare for the affinity ligand to low-abundance protein in testing sample as sample with these two kinds contrasts after acquisition, reach the object being once directly separated and obtaining the more upregulated protein of content in normal plasma.Specifically, process is afterwards as follows:
In order to promote the specificity and specific aim that are separated low-abundance protein, the interference of the high abundance background proteins after avoiding in chromatographic separation, need the competitive inhibition reducing high-abundance proteins further, so in step S20 the present invention using normal healthy controls histone sample as antigen immune rabbit (also can be rat etc. other immunizator), prepared the polyclonal antibody of immunity by immunity; Most of albumen in immunologic process (be a large amount of high-abundance proteins substantially) produce immune due to; Only there is small portion albumen (substantially for low-abundance protein) because the reason of this body structure or the too low reason of protein content, immune response can not occur; So the polyclonal antibody being directed to high-abundance proteins prepared, remove background high-abundance proteins by this high-abundance proteins polyclonal antibody.
Further, how anti-the high-abundance proteins that step S20 prepares by step S30 is as affinity ligand, is fixed to the chromatography column that solid phase carrier prepares specific adsorption high-abundance proteins; Solid phase carrier, as the carrier of chromatography, can select the types such as dextrane gel to carry out; The mode of affinity ligand and solid phase carrier secure bond can adopt the usual modes such as covalent cross-linking to realize.
Step S40 is after preparing high abundance affinity column; with this high abundance chromatography column, post process was carried out to improper patient's protein sample; cross in the process of post because the affinity media of chromatography is the system rejecting high-abundance proteins for polyclonal antibody; so this time a large amount of high-abundance proteins crossed in post protein sample can be attracted on chromatography column, the stream obtained wears the low-abundance protein that composition in component and most improper patient variation express.Certainly, to wear amount in liquid at stream in order to eliminate as far as possible or reduce high abundance in this course, stream can be worn fluid component and again carry out post, to promote the removal to background proteins as far as possible.Simultaneously; in the process implemented; in order to reduce or reduce the non-specific adsorption of albumen in high abundance chromatography column and sample; void column of improper patient's protein control sample only containing solid-phase matrix not containing part is first crossed post process; a lot of impurity in improper like this patient's protein control sample or the factor of non-specific adsorption by preliminary filtering, can promote the specificity that high-abundance proteins chromatography column is affine.
Then; the step S50 stream collected by step S40 mainly containing the low abundance difference albumen of improper patient is worn liquid and is concentrated; then concentrated albumen is carried out second time immunity as antigen again, prepare the polyclonal antibody being directed to low abundance difference albumen.Wherein, it is pointed out that the amount due to low abundance difference protein expression is less, so the amount in order to it can be made to reach immunity; the amount of collected low abundance difference albumen the amount of improper patient's protein control sample can be strengthened, until can meet service requirements of the present invention in enforcement.Meanwhile, need to adopt the immunizator different from step S20 to carry out with the second time immunity that low-abundance protein carries out in this step S50, the first time immunity in such as step S30 adopts rabbit to carry out, and the immunity again of this step S40 just can adopt rat to carry out.
Specificity resisting for low abundance difference expressing protein can be obtained by the immunity of step S50 more, step S60 and S70 is by the affinity of this how anti-low abundance difference albumen had and specificity afterwards, just can the most at last in testing protein sample similar differential protein be separated.The process implemented remains the method for similar above-mentioned high abundance chromatography column, step S50 is carried out the immune low abundance difference albumen obtained of second time how anti-as affinity ligand, on covalency or non-covalent mode fixed value solid phase carrier, prepare the affinity column for low-abundance protein; Then with this low abundance affinity column process testing protein sample, low abundance difference albumen so in testing protein sample just can be adsorbed by this chromatography column, carries out wash-out just directly can obtain testing sample and belong to the more upregulated protein of content in normal plasma after absorption with elutriant.
Certainly, in the implementation process of step S70, because in testing protein sample own, the content of high-abundance proteins accounts for absolute predominance, stronger competitive advantage may can be produced in absorption, so in force in order to promote low-abundance specificity further, and reduce the competitive inferior position of low-abundance protein, therefore can adopt, before crossing post, histone sample to be measured first be carried out preliminary treatment by the mode of gradient centrifugation, just can tentatively remove number molecular weight by the mode of gradient centrifugation or measure large high-abundance proteins, thus be more beneficial to the specificity promoting the affine absorption of low-abundance protein.
Certainly, carried out in the process of post at histone sample to be measured, equally also be in order to avoid non-specific adsorption, also can adopt histone sample to be measured with not carrying out post containing the blank chromatography column that how anti-low-abundance protein is, this non-coupling is adopted to have the blank pillar of polyclonal antibody only containing solid-phase matrix to carry out post, the object of the invention is to it can be used as background, the factor of non-specific binding can be caused, and small-particle fragment etc. is delayed at this containing on blank chromatography column of base for post matter, thus realize tentatively removing background interference, further promote the effect of how anti-chromatography column for the specific adsorption of testing sample.
Simultaneously in above-mentioned steps S70, after testing sample carried out post process, can wash affinity column with damping fluid further, not have to combine and the albumen that is trapped in interstitial space to remove, reduce background and disturb.Damping fluid for washing can adopt any damping fluid of Ag-Ab association reaction that do not disturb can.
Finally the elutriants such as imidazoles are adopted to carry out wash-out again after absorption, i.e. the separable low-abundance protein obtained in testing sample.Then elutriant is concentrated, SDS-PAGE Gel Electrophoresis Silver dye, Western-blot analyze, just low abundance difference albumen can be shown soon after mass spectrum.The process of above-mentioned entirety is all fewer with amount based on the low-abundance protein kind of differential expression own, therefore final according to above-mentioned implemented column purification after, carry out the elution fraction that wash-out obtains afterwards again, after analyzing with LTQ-MASS, only need the difference function protein of more different physiological status from remaining tens protein haply; This considerably reduce the workload of protein differential disply, skipped complicated 2D electrophoresis, have larger potentiality in the early detection of disease and the discovery detection field of biomarker.The process simultaneously crossing post absorption and wash-out does not need repeatedly to repeat, and consuming time and workload shortens greatly.
The process high-abundance proteins that contriver adopts previously is prepared the many anti-chromatography collection stream that carries out of specificity and is worn liquid, and stream is worn in liquid containing a large amount of albumen much existed in normal plasma, as Apo E, Hemopexin, Sex hormone-binding globulin, Apo B-100 and Haptoglobin-related protein etc.; Why these albumen can be worn in solution at stream occurs, is that the concentration of these albumen in blood plasma increases greatly, has exceeded the ratio of polyclonal antibody on affine adsorption medium, makes to be adsorbed completely because under various different improper physiological status; Therefore final stream wears liquid also to be needed to carry out abstraction and purification further and just can compare and mass spectrum.And in the process of adsorbing in the present invention be all specific adsorption, the elutriant finally collected, be all the appointment albumen of specific adsorption substantially, be substantially devoid of above-mentioned high-abundance proteins, can directly compare and mass spectrum.
Above patient can select according to the type of different research various disease, can Selection radio as the easy lymphatic cancer, melanoma, cancer of the stomach, the rectum cancer, bacteriological infection, dysemia, mammary cancer, cervical cancer, colorectal carcinoma, esophagus cancer, lymphoma, leukemia, liver cancer, lung cancer, non-small cell lymphoma, mesothelioma, multiple sclerosis, nervous system infection etc. missing early diagnosis.Contrast patient protein sample adopt from be diagnosed as suffer from above-mentioned disease blood sample of patient obtain, gathered blood plasma after blood sample after centrifugal treating washed corpuscles as sample.
In process with affinity chromatography column purification of the present invention, the amount of preparative chromatography post matrix used must be adjusted to a little more than its saturated level, keeps the volume with polyclonal antibody matrix, and non specific background is reduced as far as possible.And polyclonal antibody is relevant with flow velocity to the quantity of the catch of the antigen of correspondence, when flow velocity is slower, column capacity is more effective, because the antibody of coupling just has the more time to be combined with antigen.Meanwhile, mechanical compression during loading should be little as far as possible, and avoid excessively contacting with air.
Based in above-mentioned implementation process, preferably adopt SepharoseCL 4B as the solid-phase matrix of chromatography column in chromatography media the present invention in chromatography column, in use of the present invention, Sepharose CL 4B compares the matrix of other kinds, there is reasonable porousness, the capacity of absorption can be increased, avoid because the competitiveness of the high-abundance proteins in testing sample causes the situation of target protein deficiency.
The separation method of above-mentioned low abundance difference albumen of the present invention, the 2D electrophoresis otherness comparing common proteomics is more still difficult to carry out and the method for antibody absorption of high-abundance proteins immunity, the present invention carries out the rear affine absorption of immunity using low-abundance protein as direct target, and in all steps and process, progressively weaken and reduce competitiveness and the background interference of high-abundance proteins, and then promote low-abundance protein and be separated and the ability of enrichment, finally obtain specificity comparatively by force and kind low abundance difference expressing protein comparatively completely; For the discovery of biomarker and relevant monitoring provide more excellent biological means.
So based on the object of relevant monitoring and detection, the present invention proposes the application of separation method in low abundance difference Protein Detection of above-mentioned low abundance difference albumen further, low abundance difference albumen directly can be enriched to by the method for above-mentioned separation, follow-up CCD can be directly used in and detect analysis, mass spectrum etc.The process be separated once can complete according to aforesaid method of the present invention, and overall accuracy is better.
The understanding of those skilled in the art can be easier to for the ins and outs and process approach that make above-mentioned enforcement of the present invention and implement reference, highlight the progressive that the inventive method compares the antibody absorption of existing 2D electrophoresis and high-abundance proteins immunity simultaneously, be illustrated below by way of specific embodiment and actual analysis data.
Embodiment 1
For the purpose of the biomarker of the low abundance difference albumen of being correlated with by liver cancer of analyzing and researching in this embodiment 1, therefore adopt the protein sample of acquisition to obtain from the liver cancer patient made a definite diagnosis and normal people's blood sample respectively, concrete steps implementation step is with reference to carrying out as follows:
S10 ', obtain respectively human normal plasma protein sample, the plasma proteins sample of liver cancer patient and testing protein plasma sample (above blood plasma be the blood sample of acquisition is added antithrombotics and after carry out centrifugal treating, discard the yellow clear liquid in the upper strata after cell precipitation).
S21 ', get human normal plasma sample 2mL in step S10 ', mix with 2ml Freund's complete adjuvant, ultrasonic emulsification is complete; Subcutaneous multi-point injection is carried out to 3 rabbit, and preserves in rabbit basal part of the ear venous blood sampling sample;
S22 ', booster immunization: equally again produce the protein solution obtained in 2mL step S21 ' after two weeks, mix with 2ml Freund's incomplete adjuvant, ultrasonic emulsification is complete; Subcutaneous multi-point injection is carried out to 3 rabbit, and preserves in rabbit basal part of the ear venous blood sampling sample;
S23 ', the polyclonal antibody rabbit blood sample ELISA measuring of getting in step S22 ' generated is to the specific adsorption situation of the protein solution obtained in S22 '; When the rabbit drop of blood degree of ELISA testing inspection immunity reaches 10
4~ 10
5time, put to death rabbit and get blood.If ELISA testing inspection result does not reach standard, so continue repeating step S22 ' and enhancing immunity is carried out to rabbit, until when reaching conformance with standard, put to death rabbit and get blood.
S24 ', separated plasma in the rabbit blood sample obtained from step S23 ', a general rabbit can bloodletting 100 ~ 120mL; The blood obtained is put into the container that antithrombotics is housed in advance, fully after mixing, 1000rpm4 DEG C of centrifugal 5min, takes out supernatant and is blood plasma;
S25 ', takes out 10mL from the rabbit plasma of step S24 ', extracts high-abundance proteins polyclonal antibody;
With the PBS damping fluid of pH7.0 dilute 5 times stand-by; Take out Protein G post (1ml), first use the PBS wash buffer of pH7.0, until the data of protein nucleic acid detector display reach baseline; Take out the rabbit plasma solution of the PBS damping fluid dilution of pH7.0, with Protein G post on the flow velocity of 0.5ml/min, use the PBS wash buffer of pH 7.0 to baseline after completion of the sample, collect stream and wear liquid; Certainly, the stream collected in this step is worn in liquid and also may also be contained not yet by the polyclonal antibody adsorbed, so this stream wears liquid repeatedly can cross post, to reduce the waste of polyclonal antibody;
With pH2.5Gly-HCl wash buffer medium, receive eluted protein solution, be the polyclonal antibody for high-abundance proteins, then adjust ph is extremely neutral immediately; Measure A280nm and A260nm, estimation protein concentration; SDS-PAGE analysis is carried out to eluted protein solution.
S30 ', prepares polyclonal antibody affinity column:
S31 ', obtains Sepharose CL 4B medium (purchased from the biological CAS No.61970-08-9 of source, Shanghai leaf);
S32 ', then by the Sepharose CL 4B medium of step S31 ', cleans with deionized water 500mL, drains into wet pie (settling volume is about 45mL), transfers in 200mL beaker; Gel suspension, in 100mL deionized water, stirs gently with magnetic stirring bar;
S33 ', gets 4.2g CNBr and is dissolved in 50ml HPLC level acetonitrile, join in gel suspension, puts into pH probe, and the NaOH with 20% maintains the pH of reaction mixture 11.0, and whole system is in ice bath, and temperature maintains 0 DEG C; During reaction 10 ~ 15min, check whether CNBr all dissolves.Now, NaOH spending rate will reduce; After pH is stabilized in 11.0, in the core glass funnel of reaction mixture impouring precooling, the deionized water ice-cold with 1L and 500mL ice-cold coupling buffering tuck in (pH8.5,0.1MNaHCO
3) detergent gel rapidly, due to the unstable of activated carrier, gel must coupling protein matter or aglucon immediately;
Filtrate 500mL0.1M FeSO
4the CNBr that neutralization is not reacted, pours waste liquid cylinder into;
S34 ', with the 0.1M NaHCO of pH 8.5
3the eluted protein solution (high abundance be namely separated immune step is how anti-) that dialysis prepares from step S25 ', and mix with medium, Homogeneous phase mixing 35h under 4 DEG C of environment; Add thanomin 0.5ml, vibrate under 4 DEG C of environment 5h; After the PBS buffer solution for cleaning medium of pH 7.0,0.01M, dress post.
S40 ' loading crosses post:
The patient of step S10 ' is contrasted plasma sample and gets 20 μ L by S41 ', adds 980 μ l 10mM PBS; Use formula: C (mg/ml)=1.45xA
280nm-0.74xA
260nmthe protein concentration of estimation sample; Get about for subsequent use containing 1mg testing sample according to the concentration of estimation;
S42 ' gets Sepharose CL 4B medium and prepares a blank chromatography column, after the blank chromatography column that on the testing sample prepared by step S41 ', the PBS of 10mM is equilibrated, and cleans 3 volume collection streams with PBS and wears liquid;
The stream collected after tentatively filtering with blank chromatography column in step S42 ' is worn liquid by S43 ', chromatography column prepared by the equilibrated step S34 ' of the PBS of upper 10mM; And with PBS flushing medium 3 ~ 4 column volumes, receive each stream and wear solution (this stream wears liquid can carry out post again, and repeated 2 ~ 3 times).
Whole for the plasma sample of whole improper patients stream crossed after post is worn liquid by S51 ', after all collecting (if amount can strengthen the amount of the plasma sample of improper patient not further), concentrates with dialysis tubing;
S52 ', then with reference to the process of step S21 ' ~ S25 ', using the low abundance difference expressing protein in concentrated for the step S51 ' patient plasma obtained as antigen, is that immunizator carries out immunity again with rat, the same last polyclonal antibody extracting low-abundance protein;
S60 ', the polyclonal antibody of low-abundance protein prepared by step S52 ', the low abundance affinity column that all processes preparation with reference to step S30 ' is affinity ligand with this low-abundance protein polyclonal antibody;
S71 ', for subsequent use after the mode of test plasma protein sample gradient centrifugation sedimentation is tentatively simply removed some high-abundance proteins;
S72 ', will tentatively eliminate the test plasma protein sample of some high-abundance proteins in step S71 ', prepare a blank chromatography column cross post with Sepharose CL 4B medium, removes background interference, collects stream and wears liquid;
S73 ', the stream after step S72 ' empty is crossed post wears liquid, low abundance affinity column prepared by the equilibrated step S60 of the PBS of upper 10mM, and with PBS flushing medium 3 ~ 4 column volumes;
S74 ', carries out wash-out by the low abundance affinity column imidazole elution that step S73 ' crosses after post, collects elution fraction.
Substantially step S74 ' collects among elutriant and namely there is low abundance difference expressing protein to be prepared.In order to verify this result, in the present invention, proceed following process:
The elutriant obtained in step S74 ' is then added the 50%TCA solution of 1/4 times of volume by S80 ', and after 4 DEG C of refrigerator overnight, at 4 DEG C, the centrifugal 30min of 12000rpm, gets precipitation;
Above-mentioned deposit sample adds 1mL ,-20 DEG C of pre-cold acetone cleanings, and 4 DEG C, 12000rpm, centrifugal 10min, after abandoning supernatant (repeating 3 times); Vacuum-drying, is stored in 4 DEG C of refrigerators stand-by.
In order to the situation that outstanding contrast liver cancer patient and normal people's protein diversity are expressed, choose respectively simultaneously and be diagnosed as liver cancer patient blood plasma and normal health human plasma respectively as sample to be tested 1 and sample to be tested 2, process, finally collect elutriant.Then carry out SDS-PAGE Gel Electrophoresis Silver dye, rear Western-blot, LTQ-MASS analyze, and the result of comparison is see shown in accompanying drawing.Wherein accompanying drawing 1 is adopt human normal plasma and another liver cancer patient blood plasma to detect according to above-mentioned steps the film that the elutriant obtained carries out SDS-PAGE Gel Electrophoresis Silver dye respectively as testing protein respectively; Band 1 ~ 3 in Fig. 1 is the eluted protein of human normal plasma after the affine absorption of polyclonal antibody, and band 4 ~ 6 is the eluted protein of liver cancer patient blood plasma after the affine absorption of polyclonal antibody; As can be seen from the band 1 ~ 6 of electrophoresis film obviously, the protein quantity of normal people's band is obviously less than the protein content of anon-normal ordinary person band.And in adhesive tape 4 ~ 6, the obvious protein quantity of dyeing of albumen is 2, adhesive tape 1 ~ 3 only has 1, and the traction phenomenon of adhesive tape 4 ~ 6 is obvious, is to express cause because there is more protein diversity.
Carry out the result of LTQ-MASS analysis further as following table:
The eluted protein of table 1 human normal plasma after the affine absorption of polyclonal antibody
The eluted protein of table 2 liver cancer patient blood plasma after the affine absorption of polyclonal antibody
From the comparison result of above-mentioned differential protein, the albumen that normal people and liver cancer patient are expressed there are differences, concrete relevant functionally possibility is accurately uncertain, await further following the tracks of and analyzing, but acetonitrile detects that there are differences is to determine.And it can also be seen that from above-mentioned the low abundance difference expressing protein that method of the present invention obtains, these adopt the proteomics means such as conventional 2D electrophoresis to obtain; And can direct disposable kind and the quantity that just can be separated and prepare low abundance difference expressing protein, the stream carrying out chromatography is further worn the result that liquid carries out LTQ-MASS analysis and can be found out, kind and the quantity of the normal plasma protein detected and normal cell functional protein decrease, illustrate the better effects if that the inventive method is removed for the background interference of high-abundance proteins, to low abundance difference expressing protein, to there is higher specificity and specific aim.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (7)
1. a separation method for low abundance difference albumen, is characterized in that, comprise the steps:
Obtain normal healthy controls histone sample, patient's control group protein sample and histone sample to be measured;
Normal healthy controls histone sample is carried out immunity as antigen, and preparation high-abundance proteins resists more; And resist for affinity ligand and solid-phase matrix prepare high abundance affinity column with described high-abundance proteins more;
Patient's control group protein sample was carried out post in described high abundance affinity column, and collects stream and wear component;
Described stream is worn after component concentrates and carry out immunity again as antigen, prepare low-abundance protein and resist more; And prepare low abundance affinity column as affinity ligand is fixing with solid-phase matrix so that how anti-described low-abundance protein is;
Histone sample to be measured is carried out post in described low abundance affinity column, and collects elution fraction.
2. the separation method of low abundance difference albumen as claimed in claim 1, is characterized in that, patient's control group protein sample was carried out post in described high abundance affinity column, and collects before stream wears component step, also comprises:
By described patient's control group protein sample with not resisting the blank chromatography column for affinity ligand to carry out post containing high-abundance proteins more.
3. the separation method of low abundance difference albumen as claimed in claim 1 or 2, is characterized in that, histone sample to be measured was carried out post in described low abundance affinity column, and before collecting elution fraction step, also comprises:
By described histone sample to be measured with not resisting the blank chromatography column for affinity ligand to carry out post containing low-abundance protein more.
4. the separation method of low abundance difference albumen as claimed in claim 1 or 2, is characterized in that, described solid-phase matrix is Sepharose CL 4B.
5. the separation method of low abundance difference albumen as claimed in claim 1 or 2, is characterized in that, patient's control group protein sample was carried out post in described high abundance affinity column, and collects before stream wears component step, also comprises:
Described patient's control group protein sample is carried out gradient sedimentation process.
6. the separation method of low abundance difference albumen as claimed in claim 1 or 2, is characterized in that, histone sample to be measured was carried out post in described low abundance affinity column, and after collecting elution fraction step, also comprises:
Described elution fraction is carried out concentrate, electrophoresis, Western-blot analyze and mass spectrum process.
7. the application of separation method on low abundance difference expressing protein detects of the low abundance difference albumen described in any one of claim 1 to 6.
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| PCT/CN2015/088825 WO2017008380A1 (en) | 2015-07-10 | 2015-09-02 | Isolation method for low-abundance difference protein and applications thereof |
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| CN114593979A (en) * | 2022-04-01 | 2022-06-07 | 清华大学 | Method for detecting low-abundance protein in body fluid sample based on mass spectrum |
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