CN104977417A - Glycopeptide chip and preparation method thereof - Google Patents

Glycopeptide chip and preparation method thereof Download PDF

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Publication number
CN104977417A
CN104977417A CN201510248845.7A CN201510248845A CN104977417A CN 104977417 A CN104977417 A CN 104977417A CN 201510248845 A CN201510248845 A CN 201510248845A CN 104977417 A CN104977417 A CN 104977417A
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China
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glycopeptide
chip
preparation
centrifugal
bath
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Inventor
李铮
钟耀刚
马恬然
杜昊骐
贾丽苑
秦鑫敏
张志伟
于汉杰
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Northwest University
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Northwest University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Abstract

The invention provides a glycopeptide chip and a preparation method thereof, and relates to a glycopeptide chip for identifying diseases. A purpose of the present invention is to provide a glycopeptide chip and a preparation method thereof, wherein the preparation cost of the glycopeptide chip is low, the glycopeptide chip can well reflect the natural interaction state of the saccharide chain and the binding protein and reduces non-specific recognition, and the problems that the immobilization efficiency is low and the binding specificity is unreal can be solved with the glycopeptide chip. According to the present invention, the glycopeptide is separated from the natural source sample by using an agglutinin filtration membrane assisted method, and is covalently coupled to an epoxidized derivatized solid-phase carrier in a N-C bond manner by using the N-terminal of the peptide or the amino on the amino acid residue; and the preparation cost of the preparation method is low compared with the conventional technology, the prepared glycopeptide chip can well reflect the natural interaction state of the saccharide chain and the binding protein and reduces the non-specific recognition, and the problems that the immobilization efficiency is low and the binding specificity is unreal can be solved with the glycopeptide chip.

Description

A kind of glycopeptide chip and preparation method thereof
Technical field
The present invention relates to a kind of glycopeptide chip of identifying disease.
Background technology
Carbohydrate chip is that research sugar group is learned and carbohydrate-binding protein (glycan-binding protein, GBP) with sugar chain the most effective interactional high throughput analysis instrument, its potential range of application widely, such as, comprises the aspects such as screening GBP, Antibody specificity analyses, the adhesion of bacterium and virus and enzyme viability analysis.Carbohydrate chip also can help researcher to develop the medicine of new diagnostic method and monitor disease states and development disease therapy.The research method that carbohydrate chip is becoming a kind of standard is for Large-scale Screening sugar and illustrate sugar institute's role in biosystem.Such as carbohydrate chip technology has been used for the research of bird flu aspect, for the outburst of fast monitored current H5N1 avian influenza subtypes and assessment virus variation to the potential menace of the mankind.
At present, the preparation method of the carbohydrate chip reported has three kinds, and the first is fixed on solid phase carrier with the method for physisorption by sugar chain; Another kind is to the further chemical modification of sugar chain, and then modified sugar chain covalent coupling on the such as solid phase carrier such as nitrocellulose filter or slide.There is following shortcoming in these two kinds of methods: if prepare carbohydrate chip by the method for physisorption, then the sugar chain be fixed on solid phase carrier easily comes off in experimentation, affects the accuracy of result; To the further chemical modification of sugar chain (amination or sulfhydrylation) and purifying, and then sugared covalent coupling on such as surface aldehydes or the solid phase carrier such as carboxylated nitrocellulose filter or slide, the method process is complicated, and cost is high, poor practicability.The third method preparing carbohydrate chip is the reduction end utilizing sugar chain, and with oximido covalent coupling, it arrives-O-NH 2on the slide of derivatization or with the form valency coupling of glycosidic bond its on the slide of hydroxyl derivatization, due to sugar chain source (isolated or synthesized) difficulty, these carbohydrate chip preparation costs are higher.
Raw material mainly sugar chain prepared by current carbohydrate chip, technology relates to the immobilization of sugar chain, the high flux of carbohydrate-binding protein is expressed and the part such as the examination and analysb of carbohydrate chip.For mammal, the carbohydrate in its body is primarily of about 10 kinds of common monosaccharide units compositions, and have linear and branched structure simultaneously, and comprise α and β two kinds of spatial configurations, this makes sugar chain have high potential complicacy.According to calculating, sugar chain type possible in mammal can reach about 1012 kinds, the sugar chain that the N found in reality connects is more than 2000 kinds, and this numeral is at present also in continuous increase. because sugar chain will be fixed on carrier also as specific " probe " in detection by carbohydrate chip, the development of carbohydrate chip depends on sugar chain to a great extent, and obtaining a large amount of applicable sugar chains preparing chip needs is primary problem.
The separation of carbohydrate for the sequence of sugar chain in analyzing sugared group and physiological function extremely important, but the carbohydrate of natural source is very limited, when the sugar particularly needing a large amount of purity very high.Development can in a large number, efficiently extremely important for setting up of sugared library from the method for the higher glycan of nature separation purity.On the other hand, polysaccharide or oligosaccharides can be undertaken by multi-step biological synthesis combination chemistry and Biocatalysis method, and this deficiency for natural source carbohydrate can play supplementary function.Research for carbohydrate synthesis continue for century more than one, and through a large amount of research work, present many oligosaccharides can Prof. Du Yucang.But the difference of complicacy due to carbohydrate structure, some oligosaccharides is synthesized in the laboratory of specialty may need even time several years several months.
Summary of the invention
The object of the present invention is to provide a kind of preparation cost low, the state of nature of sugar chain and its associated proteins effect can better be reacted, reduce the non-specific of identification, solve fixed efficiency low, false a kind of glycopeptide chip of binding specificity and preparation method thereof.
The invention provides a kind of glycopeptide chip preparation method, from the sample of natural source, glycopeptide is isolated with agglutinin filter membrane auxiliary law, utilize peptide on glycopeptide N-end or amino acid residue on amino, with N-C key form covalent coupling on the solid phase carrier of epoxidation derivatization, specifically: first albumen activation trypsase trypsin enzymolysis is obtained protein enzymatic hydrolyzate; Add agglutinin to protein enzymatic hydrolyzate again, the glycoprotein in cleaning, wash-out Selective Separation protein enzymatic hydrolyzate, obtains the glycopeptide of specific isolation; Utilize the glycopeptide of described specific isolation to make sampling liquid, sampling liquid is added in 384 orifice plates, then use point sample instrument at the array in epoxidation sheet Ji Shangdianzhi 4 district, obtain glycopeptide chip.
Describedly comprise proteolysis specifically: get protein sample 1 ~ 1.5mg, add in centrifuge tube, carry out centrifugal concentrating, add alkalescent cleaning fluid again, again centrifugal, final volume 100 μ L, centrifugal product 500 μ L final concentration 8M urea dissolve, room temperature leaves standstill at least 30min, concentrated centrifugal to 100 μ L, adding the DTT(dithiothreitol (DTT) of 200 μ L 10mmol/L again) solution carries out water-bath, 100 μ L 20mmol/L IAM(iodoacetamides are added after water-bath) solution, lucifuge leaves standstill, water-bath is carried out after adding the DTT solution of 100 μ L 10mmol/L again, the activation trypsase trypsin of 0.125mg/mL is added after water-bath terminates, enzymolysis spends the night, high-temperature heating after enzymolysis, collected by centrifugation centrifugate, obtain protein enzymatic hydrolyzate.
Glycoprotein in described Selective Separation protein enzymatic hydrolyzate is specifically: get 300 μ g agglutinins and join in protein enzymatic hydrolyzate, room temperature reaction 3h in air concussion bath, be transferred in 30KD centrifugal column, by the cleaning repeatedly with binding buffer liquid, 600 μ L/ time, wash away the protein of non-specific adsorption, then (elution buffer of Con A is the damping fluid containing 0.5mol/L Alpha-Methyl mannoside to add 300 μ L elution buffers, the elution buffer of LCA is the damping fluid containing 0.2mol/L fucose), wash-out 1h in air concussion bath, collect eluent, namely the glycopeptide of specific isolation from sample is obtained.
The concrete steps of the point of described glycopeptide chip are
1) glycopeptide of purifying is mixed with sampling liquid, the effective constituent of this sampling liquid is the glycopeptide with a class sugar chain structure;
2) sampling liquid of preparation is added in 384 orifice plates, then uses point sample instrument at the array (point sample) in epoxidation sheet Ji Shangdianzhi 4 district;
3) chip made by point is overnight incubation in the environment of 40% ~ 60% in humidity; The chip of hatching is vacuumized in the vacuum dryer of 37 DEG C in the vacuum dryer of 3 hours or 60 DEG C and vacuumize 1 hour, make glycopeptide be fixed on chip;
4) the glycopeptide chip fixed is placed in exsiccator, keeps in Dark Place; Weighing 0.02g/mLBSA dissolves in the phosphate buffer-polysorbas20 of pH7.4, dissolves the membrane filtration with 0.2 μm after mixing, the glycopeptide chip prepared is placed in confining liquid 1 hour;
5) the glycopeptide chip phosphate buffer-polysorbas20 after closing and phosphate buffer are cleaned 1 time respectively successively, each 5 minutes, more for subsequent use after drying;
6) the glycopeptide chip after drying is added to the incubation buffer of 600 ~ 700 μ L, as the reaction environment with sample to be tested;
Described phosphate buffer pH7.4 is with 1L total amount, and component is: NaCl 8.0g, KCl 0.2g, KH 2pO 40.2g, Na 2hPO 412H 2o 2.9g;
Described phosphate buffer-polysorbas20 is the described phosphate buffer also containing massfraction 0.05 polysorbas20.
In described proteolysis process, alkalescent cleaning fluid adopts 100mmol/L NH 4hCO 3.
All centrifugal all employings 10KD centrifugal columns in described proteolysis process, column temperature 4 DEG C, 14,000g is centrifugal, centrifugation time 15min.
Add the also again centrifugal process of alkalescent cleaning fluid in described proteolysis process to repeat twice.
Bath temperature 37 DEG C, time 1h is in all water bath processing in described proteolysis process.
Described proteolysis process IAM solution keeps in Dark Place, and lucifuge room temperature leaves standstill 1h.
Activate the mass ratio proteinase of trypsase trypsin and albumen in described proteolysis process: albumen=1:100, and use 25mmol/L NH 4hCO 3dilution.
The present invention has following beneficial effect:
Technical solution of the present invention based on agglutinin to specific sugar chain identification binding specificity, from the sample of natural source, glycopeptide is isolated with agglutinin filter membrane auxiliary law, utilize peptide on glycopeptide N-end or amino acid residue on amino, with N-C key form covalent coupling on the solid phase carrier of epoxidation derivatization, prepare glycopeptide chip, solve the problem that sugar chain source is difficult and expensive, the carbohydrate chip that cost and technology are all more traditional is lower; The state of nature of sugar chain and its associated proteins effect can better be reacted, the non-specific of identification can be reduced, solve fixed efficiency low, the technical matterss such as binding specificity is untrue.
Accompanying drawing explanation
Fig. 1 is schematic flow sheet prepared by glycopeptide chip of the present invention;
Fig. 2 is lectin chip detection figure, and in figure, A is standard protein ribonuclease B holoprotein, and B is the glycopeptide obtained after enzymolysis, and C is the glycopeptide after ConA enrichment;
Fig. 3 is Quality Control figure, A is after point sample, and B is after point sample cleaning, and C is after closing, and D is the negative Quality Control figure of BSA-Cy3, and E is MAL-II-Cy3(specific recognition Sia2-3Gal β 1-4Glc (NAc) structure);
Fig. 4 is glycopeptide chip sample application array figure, and in figure, blueness represents negative reference mark, and green represents positobe focus (Marker), and grey represents the glycopeptide of separate sources;
Fig. 5 is that glycopeptide chip hatches result figure;
Fig. 6 is difference sample liquid pH check result figure, left figure be the glycopeptide chip detection result figure that pH7.4 time point system is removed, right figure is the glycopeptide chip detection result figure that pH9.0-9.5 time point system is removed;
Fig. 7 is the Tris of the 0.1M of pH9.3, PBS, NaCO 3vacuumize 3 hours with NaAc tetra-kinds of solution testing result figure, left figure in the vacuum dryer of 37 DEG C, right figure vacuumizes 1 hour in the vacuum dryer of 60 DEG C;
Fig. 8 is different point sample humidity chip scans figure, and left figure is humidity 60%, and right figure is humidity 45%;
Fig. 9 hatches result figure under different drying condition;
Figure 10 is the confining liquid of different damping fluid, and left figure is the confining liquid of 1% BSA phosphate buffer, and right figure is the confining liquid of the phosphate buffer of 2%BSA, 0.5mol/L glycocoll;
Figure 11 is the incubation buffer of heterogeneity, the left side is 1%BSA(v/v), 0.58%NaCl phosphate buffered solution (containing 0.05%Tween-20) as incubation buffer, the right is that the phosphate buffered solution (containing 0.05%Tween-20) of 2%BSA, 0.5mol/L glycocoll is as incubation buffer;
Figure 12 is that the glycopeptide chip of normal healthy controls old women and the diabetes B patient old women saliva mixing sample adopting LCA to extract breeds result figure;
Figure 13 is that the glycopeptide chip of normal healthy controls old women and the diabetes B patient old women serum mixing sample adopting ConA to extract breeds result figure.
Embodiment
Below by the specific embodiment provided, the present invention will be further described, but not as a limitation of the invention.
As shown in Figure 1, be glycopeptide chip preparation flow schematic diagram of the present invention, specifically: first albumen activation trypsase trypsin enzymolysis is obtained protein enzymatic hydrolyzate; Add agglutinin to protein enzymatic hydrolyzate again, the glycoprotein in cleaning, wash-out Selective Separation protein enzymatic hydrolyzate, obtains the glycopeptide of specific isolation; Utilize the glycopeptide of described specific isolation to make sampling liquid, sampling liquid is added in 384 orifice plates, then use point sample instrument at the array in epoxidation sheet Ji Shangdianzhi 4 district, obtain glycopeptide chip.Concrete steps are as follows:
(1) proteolysis
Get protein sample (1 ~ 1.5mg) and be added on 10KD centrifugal column, column temperature 4 DEG C, the centrifugal 15min of 14,000g, add the 100 mmol/L NH of 450 μ L 4hCO 3to add in post 4 DEG C, 14, the centrifugal 15min of 000g, repeat twice, final volume 100 μ L, centrifugal product is that 8M urea dissolves with 500 μ L final concentrations, be placed in room temperature at least 30 min, concentrated centrifugal to 100 μ L, the DTT solution adding 200 μ L 10 mmol/L again carries out water-bath, water bath condition is temperature bath 1h in 37 DEG C, add the IAM solution that 100 μ L 20mmol/L keep in Dark Place again, room temperature lucifuge reaction 1h, the DTT solution adding 100 μ L 10 mmol/L after reaction terminates again carries out water-bath, water bath condition is temperature bath 1h in 37 DEG C, the trypsase trypsin adding the activation of 0.125 mg/mL (uses 25mmol/L NH 4hCO 3dilution, and proteinase: albumen=1:100), 37 DEG C of enzymolysis spend the night, high-temperature heating sex change trypsase after enzymolysis spends the night, collected by centrifugation centrifugate,
(2) agglutinin Selective Separation glycoprotein
The agglutinin (molten with binding buffer solution) getting 300 μ g joins in the centrifugate of collection, is placed in air concussion bath room temperature reaction 3h.Transfer in 30KD centrifugal column, by the cleaning repeatedly with binding buffer liquid, 600 μ L/ time, wash away the protein of non-specific adsorption, and then add elution buffer (elution buffer of Con A is the damping fluid containing 0.5mol/L Alpha-Methyl mannoside, and the elution buffer of LCA is the damping fluid containing 0.2mol/L fucose) wash-out 1h in air concussion bath of 300 μ L, collect the eluent i.e. glycopeptide of special separation from sample;
(3) the some system of glycopeptide chip
1) glycopeptide of purifying is mixed with sampling liquid, the effective constituent of this sampling liquid is the glycopeptide with a class sugar chain structure;
2) sampling liquid of preparation is added in 384 orifice plates, then uses point sample instrument at the array (point sample) in epoxidation sheet Ji Shangdianzhi 4 district;
3) chip made by point is overnight incubation in the environment of 40% ~ 60% in humidity; The chip of hatching is vacuumized in the vacuum dryer of 37 DEG C in the vacuum dryer of 3 hours or 60 DEG C and vacuumize 1 hour, make glycopeptide be fixed on chip;
4) the glycopeptide chip fixed is placed in exsiccator, keeps in Dark Place; Weighing 0.02g/mLBSA dissolves in the phosphate buffer-polysorbas20 of pH7.4, dissolves the membrane filtration with 0.2 μm after mixing, the glycopeptide chip prepared is placed in confining liquid 1 hour;
5) the glycopeptide chip phosphate buffer-polysorbas20 after closing and phosphate buffer are cleaned 1 time respectively successively, each 5 minutes, more for subsequent use after drying;
6) the glycopeptide chip after drying is added to the incubation buffer of 600-700 μ L, as the reaction environment with sample to be tested;
Described phosphate buffer pH7.4 is with 1L total amount, and component is: NaCl 8.0g, KCl 0.2g, KH 2pO 40.2g, Na 2hPO 412H 2o 2.9g;
Described phosphate buffer-polysorbas20 is the described phosphate buffer also containing massfraction 0.05 polysorbas20.
Fig. 2 is lectin chip detection figure in proteolysis of the present invention and agglutinin selection course, and in figure, part A is standard protein ribonuclease B holoprotein, and part B is the glycopeptide obtained after enzymolysis, and C part is the glycopeptide after ConA enrichment.
Fig. 3 is glycopeptide chip point different phase Quality Control figure, in figure, part A is after point sample, and part B is after point sample after cleaning, and C part is after closing, D part is the negative Quality Control figure of BSA-Cy3, and E is MAL-II-Cy3(specific recognition Sia2-3Gal β 1-4Glc (NAc) structure).
Embodiment 2
Glycopeptide chip of the present invention is determined the foundation of glycopeptide chip detection carbohydrate-binding protein method and condition thereof
During method for building up, select protein surface without sugar chain structure, the BSA that can not be combined with glycopeptide and sampling liquid are as negative Quality Control, the BSA of Cy3 mark is as Marker, form the matrix (as shown in Figure 4) of 9 × 7 with the glycopeptide of LCA, ConA enrichment, then hatch with the fluorescently-labeled ribonuclease B of Cy3 (RNaseB), result of determination (as shown in Figure 5) is carried out with the presence or absence of the fluorescence signal value of the glycopeptide of LCA, ConA enrichment, power.Determine sampling liquid pH and classification, point sample humidity, drying time, sealing condition, the best incubation buffer of glycopeptide chip respectively, and demonstrate the specificity of ConA, GNA and RNaseB combination.
Be specially:
1. sampling liquid pH and classification are determined
Consider that the kind of salt ion in the pH of sampling liquid and sampling liquid can affect to the combination of glycopeptide and epoxidation sheet base, first the sampling liquid determination point sample pH of the same race of about pH7.0 and pH9.0-9.5 two pH scopes is chosen, found that, pH be 7.4 time points make glycopeptide chip detection time be negative signal, after first time cleaning, sampling point is washed off, illustrate that sampling point is not well combined with sheet base, and during pH9.0 ~ 9.5, detection signal is positive, is more conducive to the combination (as shown in Figure 6) of glycopeptide and epoxidation sheet base when pH9.0 ~ 9.5 are described.On this basis, the kind of sampling liquid is tested, choose the Tris of the 0.1M of pH9.3 respectively, PBS, NaCO 3test with NaAc tetra-kinds of solution, test findings shows: the detection signal of Tris sampling liquid display is the most weak, and PBS, NaCO 3then show stronger detection signal with NaAc, the PBS of pH9.3 is described, NaCO 3the sampling liquid (as shown in Figure 7) of glycopeptide chip is all can be used as with NaAc.
2. the determination of point sample humidity
Be the environment mid point system of 40%-60% in humidity by glycopeptide chip, choosing humidity in this experiment is 60% and 45% for experiment condition, point sample is complete, when found that humidity is 60%, there are diffusion phenomena in point of sample, and during humidity 45%, can be good at the form of support level sampling point, compared with the maintenance (as shown in Figure 8) of convenience point form when illustrating that point sample humidity is 45%.
3. the determination of drying time
Being hatch 12 hours in the environment of 45% in humidity by putting the chip that makes, the glycopeptide chip of hatching being vacuumized in the vacuum dryer of 37 DEG C in the vacuum dryer of 3 hours or 60 DEG C and not vacuumizing 1-3 hour not etc., making glycopeptide be fixed on chip; With again with Cy3 fluorescence labeling after RNaseB hatch.Found that, 3 hours are vacuumized in the vacuum dryer of 60 DEG C, there is point reduction phenomenon and detection signal is lower, constantly little 1, substantially can the original state of support level sampling point and detection signal obviously increase, 3 hours are vacuumized in the vacuum dryer of 37 DEG C, can the original state of support level sampling point and the vacuum drying of signal compared with 60 DEG C wants high, say in the vacuum dryer of 37 DEG C in the vacuum dryer vacuumizing 3 hours or 60 DEG C vacuumize 1 hour can the shape of support level sampling point, detection signal is better (as shown in Figure 9).
4. the determination of sealing condition
Will other be closed without protein example region by carrier after glycopeptide is fixing, combine with it to prevent protein example to be measured, form false positive and chip background is raised. for this reason, selected containing 1%BSA(v/v) or 2%BSA, 0.5mol/L glycocoll phosphate buffered solution (containing 0.05%Tween-20) in respectively as confining liquid, chip is closed, then to hatch with the RNaseB after Cy3 fluorescence labeling.Found that, it is all lower that both close rear backdrop, the positive assay signal that the chip closed with 1% BSA phosphate buffer obtains and the phosphate buffer (as shown in Figure 10) be greater than containing 2%BSA, 0.5mol/L glycocoll, illustrate that the sealing effect of 1% BSA phosphate buffer is better than the phosphate buffer of 2%BSA, 0.5mol/L glycocoll.
5. the determination of best incubation buffer
Containing 1%BSA(v/v), respectively as Incubating Solution, chip is hatched in the phosphate buffered solution (containing 0.05%Tween-20) of 0.58%NaCl or 2%BSA, 0.5mol/L glycocoll, compare the impact that these 2 kinds of damping fluids are combined with the RNaseB that Cy3 marks glycopeptide chip.Found that containing 1%BSA(v/v), 0.58%NaCl phosphate buffered solution (containing 0.05%Tween-20) is as incubation buffer, detect and all can obtain desirable fluorescence signal, phosphate buffered solution containing 2%BSA, 0.5mol/L glycocoll is (containing 0.05%Tween-20 as incubation buffer, fail to occur fluorescence signal, this illustrates that glycocoll can affect the specific binding (Figure 11) of the two.
Embodiment 3
As shown in Figure 12 and Figure 13, from glycopeptide chip scans figure, obtain the fluorescence signal value that glycopeptide that each separate sources LCA and ConA extract respectively is combined with carbohydrate-binding protein, will the value of 2 times of background standard deviations be greater than as effective value.In each district on a slice, thin piece, the glycopeptide of the non-homology that LCA and ConA extracts has 3 to repeat a little, chooses the intermediate value of the fluorescence signal value of often kind of glycopeptide, tries to achieve mean value and standard deviation value.The relatively saliva of type-II diabetes patient and normal healthy controls, the differential expression situation of carbohydrate-binding protein in serum.Calculate the ratio R atio of type-II diabetes patient saliva, serum sample fluorescence signal and normal healthy controls saliva sample fluorescence signal.General situation, statistical theory using the value of Ratio value between 0.66 to 1.5 as the value without significant difference, and Ratio value be greater than 1.5 and be less than 0.66 as the value that there are differences.
Be the different corresponding test result of glycopeptide of source property that LCA with ConA extracts with following table one, table two.
Table one is the testing result of normal healthy controls old women of the present invention and diabetes B patient old women saliva mixing sample.
Table one
Note: FH: healthy geriatric female control sample, FD:2 diabetes mellitus type old women sample, wherein * P < 0.05; * P < 0.01; * * P <0.001.
Table two is the detection knot of normal healthy controls old women of the present invention and diabetes B patient old women serum mixing sample
Really.
Table two
Note: FH: healthy geriatric female control sample, FD:2 diabetes mellitus type old women sample, wherein * P < 0.05; * P < 0.01; * * P < 0.001.
Above content is the further description done the present invention in conjunction with concrete embodiment, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.

Claims (10)

1. a glycopeptide chip preparation method, it is characterized in that, from the sample of natural source, glycopeptide is isolated with agglutinin filter membrane auxiliary law, utilize peptide on glycopeptide N-end or amino acid residue on amino, with N-C key form covalent coupling on the solid phase carrier of epoxidation derivatization, specifically: first albumen activation trypsase trypsin enzymolysis is obtained protein enzymatic hydrolyzate; Add agglutinin to protein enzymatic hydrolyzate again, the glycoprotein in cleaning, wash-out Selective Separation protein enzymatic hydrolyzate, obtains the glycopeptide of specific isolation; Utilize the glycopeptide of described specific isolation to make sampling liquid, sampling liquid is added in 384 orifice plates, then use point sample instrument at the array in epoxidation sheet Ji Shangdianzhi 4 district, obtain glycopeptide chip.
2. glycopeptide chip preparation method according to claim 1, it is characterized in that, describedly comprise proteolysis specifically: get protein sample 1 ~ 1.5mg, add in centrifuge tube, carry out centrifugal concentrating, add alkalescent cleaning fluid again, again centrifugal, final volume 100 μ L, centrifugal product 500 μ L final concentration 8M urea dissolve, room temperature leaves standstill at least 30min, concentrated centrifugal to 100 μ L, the DTT solution adding 200 μ L 10mmol/L again carries out water-bath, 100 μ L 20mmol/L IAM solution are added after water-bath, lucifuge leaves standstill, water-bath is carried out after adding the DTT solution of 100 μ L 10mmol/L again, the activation trypsase trypsin of 0.125mg/mL is added after water-bath terminates, enzymolysis spends the night, high-temperature heating after enzymolysis, collected by centrifugation centrifugate, obtain protein enzymatic hydrolyzate.
3. glycopeptide chip preparation method according to claim 1, it is characterized in that, glycoprotein in described Selective Separation protein enzymatic hydrolyzate is specifically: get 300 μ g agglutinins and join in protein enzymatic hydrolyzate, room temperature reaction 3h in air concussion bath, be transferred in 30KD centrifugal column, by the cleaning repeatedly with binding buffer liquid, 600 μ L/ time, wash away the protein of non-specific adsorption, then 300 μ L elution buffers are added, the elution buffer of Con A is the damping fluid containing 0.5mol/L Alpha-Methyl mannoside, the elution buffer of LCA is the damping fluid containing 0.2mol/L fucose, wash-out 1h in air concussion bath, collect eluent, namely the glycopeptide of specific isolation from sample is obtained.
4. glycopeptide chip preparation method according to claim 1, it is characterized in that, the concrete steps of the point of described glycopeptide chip are:
1) glycopeptide of purifying is mixed with sampling liquid, the effective constituent of this sampling liquid is the glycopeptide with a class sugar chain structure;
2) sampling liquid of preparation is added in 384 orifice plates, then uses point sample instrument at the array in epoxidation sheet Ji Shangdianzhi 4 district;
3) chip made by point is overnight incubation in the environment of 40% ~ 60% in humidity; The chip of hatching is vacuumized in the vacuum dryer of 37 DEG C in the vacuum dryer of 3 hours or 60 DEG C and vacuumize 1 hour, make glycopeptide be fixed on chip;
4) the glycopeptide chip fixed is placed in exsiccator, keeps in Dark Place; Weighing 0.02g/mLBSA dissolves in the phosphate buffer-polysorbas20 of pH7.4, dissolves the membrane filtration with 0.2 μm after mixing, the glycopeptide chip prepared is placed in confining liquid 1 hour;
5) the glycopeptide chip phosphate buffer-polysorbas20 after closing and phosphate buffer are cleaned 1 time respectively successively, each 5 minutes, more for subsequent use after drying;
6) the glycopeptide chip after drying is added to the incubation buffer of 600 ~ 700 μ L, as the reaction environment with sample to be tested;
Described phosphate buffer pH7.4 is with 1L total amount, and component is: NaCl 8.0g, KCl 0.2g, KH 2pO 40.2g, Na 2hPO 412H 2o 2.9g;
Described phosphate buffer-polysorbas20 is the described phosphate buffer also containing massfraction 0.05 polysorbas20.
5. glycopeptide chip preparation method according to claim 2, is characterized in that: in described proteolysis process, alkalescent cleaning fluid adopts 100mmol/L NH 4hCO 3.
6. glycopeptide chip preparation method according to claim 3, is characterized in that: all centrifugal all employings 10KD centrifugal columns in described proteolysis process, column temperature 4 DEG C, 14,000g is centrifugal, centrifugation time 15min.
7. glycopeptide chip preparation method according to claim 5, is characterized in that: add the also again centrifugal process of alkalescent cleaning fluid in described proteolysis process and repeat twice.
8. glycopeptide chip preparation method according to claim 2, is characterized in that: be bath temperature 37 DEG C, time 1h in all water bath processing in described proteolysis process.
9. glycopeptide chip preparation method according to claim 4, is characterized in that: described proteolysis process IAM solution keeps in Dark Place, and lucifuge room temperature leaves standstill 1h.
10. the glycopeptide chip preparation method according to any one of claim 1 ~ 9, is characterized in that: the mass ratio proteinase activating trypsase trypsin and albumen in described proteolysis process: albumen=1:100, and uses 25mmol/L NH 4hCO 3dilution.
CN201510248845.7A 2015-05-15 2015-05-15 Glycopeptide chip and preparation method thereof Pending CN104977417A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105424625A (en) * 2015-11-16 2016-03-23 西北大学 Method for quantitatively detecting sugar chain structures in glycosphingolipid
CN106645743A (en) * 2016-10-17 2017-05-10 天津科技大学 Technology for quickly constructing carbohydrate chip through neoglycoprotein

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105424625A (en) * 2015-11-16 2016-03-23 西北大学 Method for quantitatively detecting sugar chain structures in glycosphingolipid
CN106645743A (en) * 2016-10-17 2017-05-10 天津科技大学 Technology for quickly constructing carbohydrate chip through neoglycoprotein

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