CN107991277B - Serotonin-magnetic particle composite and method for enriching sialylated glycoprotein - Google Patents
Serotonin-magnetic particle composite and method for enriching sialylated glycoprotein Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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Abstract
The invention provides a serotonin-magnetic particle compound and a method for enriching sialylated glycoprotein, wherein the compound is a magnetic particle with serotonin fixed on the surface. According to the invention, epoxy modified magnetic particles are used as a carrier for fixing serotonin, and the serotonin is covalently coupled on the surface of the epoxy magnetic particles through the ring-opening reaction of amino and epoxy under a slightly alkaline condition. The sialic acid glycoprotein enrichment method which is simple and convenient to operate and good in stability is established through the directional response of the magnetic particles to a magnetic field and the specific recognition of the serotonin to sialic acid. The complex protein sample is enriched with the sialoglycoprotein, and shows better results.
Description
Technical Field
The invention relates to detection of sialylated glycoprotein, in particular to a serotonin-magnetic particle compound and a method for enriching sialylated glycoprotein by using the compound.
Background
Sialic acid is a generic term of a class of carboxyl-containing nine-carbon monosaccharide compounds and derivatives thereof, protein sialylation modification is one of important post-translational protein modifications, has very important influence on the structure and function of protein, and plays an important biological role in organisms, such as recognition between viruses and cells, signal conduction, immune response and interaction between cells, and in addition, abnormal sialylation in organisms is closely related to the occurrence and development of a plurality of tumors. Because the abundance of sialoglycoprotein in a complex sample is low and the microstructure is not uniform, especially the content of sialoglycopeptide after enzymolysis is lower, the research on sialoglycoprotein firstly needs to solve the enrichment problem of sialoglycoprotein/glycopeptide. Currently, the enrichment method for sialoglycoprotein/glycopeptide mainly comprises the steps of using special lectin for enrichment, hydrophilic chromatography, strong cation exchange chromatography, titanium dioxide chromatography, wood fiber chromatography, and using serotonin (5-HT for short) as a ligand for enrichment. However, these methods all have their own disadvantages, such as low specificity and high cost of the method using lectin enrichment, and hydrophilic chromatography, strong cation exchange chromatography, titanium dioxide chromatography, and lignocellulose chromatography are affected by phosphorylated proteins, and the enrichment process takes a long time and the reaction conditions are harsh. At present, serotonin is used as a ligand to enrich the sialoglycoprotein/glycopeptide, and the main problem is that the bound sialoglycoprotein/glycopeptide is difficult to be quickly separated after the ligand is identified and bound.
The magnetic particles are used as a carrier which has superparamagnetism and can be modified by functional groups, and are widely applied to the fields of biochemistry, cell biology, microbiology, clinical medicine and the like. The reason why the magnetic fine particles are widely used is that they have the following characteristics: the magnetic responsiveness is good, the coercive force is basically zero, namely possess the superparamagnetism; the grain diameter can reach the nanometer level; has good chemical stability; the production process is relatively simple and the price is reasonable.
At present, no report is found on the preparation of magnetic particles coupled with serotonin. The main research difficulty lies in the design of a modification structure for coupling serotonin on the surface of the magnetic particle, so that the capability of recognizing and combining the sialic acid glycoprotein/glycopeptide by the serotonin is fully exerted.
Disclosure of Invention
The invention aims to provide a serotonin-magnetic particle compound and a method for enriching sialylated glycoprotein.
In order to achieve the purpose, the invention adopts the following technical scheme:
a serotonin-magnetic particle compound is a magnetic particle with serotonin fixed on the surface.
The complex comprises binding to Fe3O4Silicon atoms on the surfaces of the magnetic particles and a functional group connected with the silicon atoms, wherein the structure of the functional group is shown as formula 1:
in the formula 1, n is 3-5, and R is
Namely R is the part remained after one hydrogen atom is removed from the amino group of the serotonin.
Preferably, the value of n is 3.
Said Fe3O4The magnetic particles are prepared by a chemical coprecipitation method.
The preparation method of the serotonin-magnetic particle compound comprises the following steps:
epoxy modified magnetic particles are used as a carrier for fixing serotonin, and under an alkaline condition, the serotonin is covalently coupled to the surface of the carrier through the ring-opening reaction of amino and epoxy.
The preparation method specifically comprises the following steps:
1) 0.4-1.2 g Fe uniformly dispersed3O4Adding 3-7 mL of an epoxidation reagent and 0.2-0.6 mL of glacial acetic acid into the magnetic particles, then reacting in a water bath at 20-30 ℃ for 2-4 h, cleaning with absolute ethyl alcohol after the reaction is finished, and then drying at 60-80 ℃ to obtain epoxidation modified magnetic particles;
2) mixing 1mg of epoxy modified magnetic particles with 1mL of a serotonin hydrochloride solution with the concentration of 10-50 mu M, and reacting for 2-8 h in a dark place, wherein the serotonin hydrochloride solution adopts a coupling buffer solution as a solvent and 0.1-0 mu M of the coupling buffer solution.3M NaHCO3And (3) reacting the magnetic particles with a mixed solution of 0.2-0.8M NaCl, washing the magnetic particles by using a coupling buffer solution, and drying at 50-80 ℃.
Preferably, the epoxidizing agent is selected from 3-glycidoxypropyl trimethoxysilane, 3-glycidoxybutylethyltrimethoxysilane or 3-glycidoxypentylethyltrimethoxysilane.
A method of enriching for sialylated glycoproteins comprising the steps of:
mixing the collected sample with a binding buffer solution and a serotonin-magnetic particle compound (the compound is sealed in advance), then oscillating for 2-4 h in a dark place at 20-25 ℃, then sequentially washing and oscillating for elution, and collecting the eluent; the serotonin-magnetic particle compound is a magnetic particle with serotonin fixed on the surface.
The conditions for shaking elution include: the elution temperature is 20-25 ℃, and the elution time is 20-60 min.
The sample is selected from milk (such as milk and the like) and a separated substance thereof of animals or human beings, blood and a separated substance thereof (such as pig serum) and a homogenate of plants (such as peanuts, soybeans and the like), during mixing, the dosage of the serotonin-magnetic particle compound is 1-5 mg, the total volume of the sample and the binding buffer solution is 0.5-1.5 mL, and the volume ratio of the sample to the binding buffer solution is 1: 0.25-8.
The cleaning method comprises the steps of adopting a combined buffer solution which is a 10-30 mM sodium phosphate solution, and adopting 0.05-0.15M NaHCO as an eluant for shaking elution3Mixed with 0.2-0.8M NaCl, or 10-30 mM Na3PO4And 0.2-0.8M NaCl.
The invention has the beneficial effects that:
according to the invention, epoxy modified magnetic particles are used as a carrier for fixing serotonin, and under a slightly alkaline condition, serotonin is covalently coupled to the surface of an epoxy magnetic particle through the ring-opening reaction of amino and epoxy. The sialic acid glycoprotein enrichment method which is simple and convenient to operate and good in stability is established through the directional response of the magnetic particles to a magnetic field and the specific recognition of the serotonin to sialic acid. Complex protein samples (such as milk) are enriched with the sialoglycoprotein, and the obtained glycoprotein is subjected to SDS-PAGE protein gel electrophoresis, color development and Lectin-blot verification to show a better sialoglycoprotein enrichment result.
Drawings
FIG. 1 is a schematic diagram of the synthesis scheme of three serotonin-magnetic particle complexes No. 1#, No. 2# and No. 3 #.
FIG. 2 is an infrared detection spectrum of a serotonin-magnetic particle complex; wherein, Fe3O4O represents an epoxidized modified magnetic fine particle, Fe3O4-O-5HT stands for serotonin-magnetic particle complex, Fe3O4Representing ferroferric oxide magnetic particles.
FIG. 3 is an infrared detection spectrum of coupling products of serotonin and epoxy magnetic particles with different concentrations.
FIG. 4 is an infrared detection spectrum of the coupling product of the epoxy magnetic particle and the serotonin under different reaction times.
FIG. 5 is a schematic diagram showing the determination of the optimal volume ratio (v/v) of milk (milk) to binding buffer.
FIG. 6 shows the results of the enrichment efficiency of different serotonin-magnetic particle complexes and different elution solutions; wherein, lane 1: protein marker; lane 2: diluting a sample with milk; lane 3: the magnetic particle complexes are not bound to the sample; lane 4: cleaning the sample by the magnetic particle compound; lane 5: 1# serotonin-magnetic particle Complex, 20mM Na3PO40.5M NaCl, pH 6.0; lane 6: 1# serotonin-magnetic particle Complex, 0.1M NaHCO30.5M NaCl, pH8.3 elution sample; lane 7: 2# serotonin-magnetic particle Complex, 0.1M NaHCO30.5M NaCl, pH8.3 elution sample; lane 8: 3# serotonin-magnetic particle Complex, 0.1M NaHCO3The sample was eluted with 0.5M NaCl, pH 8.3.
FIG. 7 shows the results of Lectin blot (Lectin blot) detection of extracted protein samples.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
First, experimental part
1. Reagent and apparatus
3-glycidoxypropylether trimethoxysilane abbreviated as GPTS (C3), 3-glycidylbutylethyltrimethoxysilane abbreviated as GPTS (C4), 3-glycidopentylether trimethoxysilane abbreviated as GPTS (C5), FeCl3·6H2O，FeCl2·4H2O, NaOH, glacial acetic acid, ethanolamine, potassium bromide (KBr) from Sigma-Aldrich, USA; acrylamide, N-methylene acrylamide from Amresco; sodium chloride, trisodium phosphate, serotonin amine hydrochloride (serotonin hydrochloride), sodium bicarbonate and other common reagents are domestic analytical pure reagents; a magnetic separator: magnetic nano-biotechnology, seikang limited; model zwhy-2101C constant temperature air oscillator: shanghai Ching Analyzer manufacturing, Inc.; infrared spectrometer (FT-IR) Nicolet 5700: thermo electron corporation, usa.
The solutions used, unless solvents are specified, are all water as solvent.
2. Procedure of experiment
2.1 preparation of epoxidized modified magnetic particles (i.e., epoxidized magnetic particles)
Firstly, preparing Fe by using a chemical coprecipitation method3O4Magnetic fine particles having an average diameter of 3.5 μm, prepared Fe3O40.8g of magnetic particles were placed in a 1L beaker, 200mL of absolute ethanol was added and the mixture was shaken thoroughly to induce Fe3O4The magnetic particles are suspended in absolute ethanol. Placing the beaker on a high-strength magnet to enable the beaker to be Fe3O4Separating magnetic particles, removing supernatant, repeatedly washing magnetic particles with anhydrous ethanol by shaking for 5 times, and removing Fe3O4Impurities in the magnetic particles. The cleaned Fe3O4The magnetic fine particles were transferred to a 1L three-necked flask, mounted on an iron support, and then 200mL of absolute ethanol was added thereto to prepare Fe3O4The magnetic particles are suspended in absolute ethanol and then in N2Stirring with a stirrer at high speed under protection. After 15-20 min, continuously introducing nitrogen, and dropwise adding 5mL of epoxidation reagent (with different carbon arm lengths) into the flaskEpoxidizing reagents GPTS (C3), GPTS (C4) or GPTS (C5)) with the concentration of 0.4mL of glacial acetic acid, and reacting in a water bath at 25 ℃ for 2h to obtain epoxidizing magnetic particles with 3 different carbon arm lengths, specifically Fe3O4The hydroxyl groups on the surface of the magnetic particles are covalently bonded with Si atoms in the epoxidizing agent to obtain the epoxidized magnetic particles with different carbon arm lengths. After the reaction is finished, transferring the reactant into a 1L beaker, repeatedly cleaning the reactant for 5 times by using 200mL of absolute ethyl alcohol, removing the residual epoxidation reagent and glacial acetic acid in the reaction system, finally, fixing the volume of the prepared epoxidized magnetic particles to 50mL by using the absolute ethyl alcohol, drying 2mL at 70 ℃, weighing the dried particles, and measuring the solid content of the magnetic particles in the volume fixing system by using a mass difference method (the purpose of volume fixing is that the error of the result can be reduced by weighing the particles after the volume fixing is finished because the cleaning volume is larger after the epoxidation modification is finished and the volume fixing is smaller).
2.2 preparation of serotonin-magnetic microparticle complexes
A certain amount of serotonin hydrochloride is weighed and dissolved in a coupling buffer solution (0.1M NaHCO)30.5M NaCl, pH8.3) to obtain a serotonin hydrochloride solution, wherein the final concentration of the serotonin hydrochloride in the solution is 1, 2, 4, 10 and 50 mu M, and the coupling buffer solution is used as a reference (the final concentration of the serotonin hydrochloride is 0 mu M). Respectively putting 1mg of epoxidized magnetic particles with different carbon arm lengths into a 2mL centrifuge tube, placing the centrifuge tube on a magnetic separation frame for magnetic separation, then washing the centrifuge tube for 3 times by using a coupling buffer solution, removing absolute ethyl alcohol, adding 1mL of serotonin hydrochloride solutions with different concentrations, shaking the shaker by 160-210 rpm (preferably 180rpm), reacting overnight at room temperature in a dark place, after the reaction is finished, performing magnetic separation, discarding supernatant, and washing the magnetic particles for 3 times by using the coupling buffer solution to prepare 3 types (with different carbon arm lengths) of serotonin-magnetic particle complexes (1 #, 2# and 3#, which are shown in figure 1). The active ingredients of the serotonin hydrochloride are the same as those of the serotonin, but the serotonin is easy to decompose, so that the more stable serotonin hydrochloride is adopted in the reaction.
2.3 coupling Effect of serotonin and epoxidized magnetic particles
And drying the product obtained after the coupling of the prepared serotonin and the epoxy magnetic particles at 50 ℃ overnight. Infrared spectrum of infraredConnecting the instrument to a computer, preheating for 2 hr, taking a small amount of oven-dried sample, grinding with about 200mg of potassium bromide (KBr) oven-dried overnight, mixing well, placing in a mold, and molding with 8 × 106And pressing the mixture into a transparent sheet on a tablet press under Pa pressure, and performing infrared determination by using an infrared spectrometer to detect the coupling effect of the serotonin and the epoxy magnetic particles.
2.4 enrichment of sialic acid glycoprotein in milk by serotonin-magnetic microparticle complexes
Centrifuging fresh milk at 5000rpm and 4 deg.C for 30min, removing upper layer oil layer, packaging lower layer liquid, storing at-80 deg.C, and thawing at 4 deg.C before use. Taking 3mg of serotonin-magnetic particle composite and 1-3% (preferably 2%) of ethanolamine aqueous solution with volume fraction, oscillating (160-210 rpm) for 0.5-1.5 hours (preferably 1 hour) under the conditions of room temperature (20-25 ℃) and light shielding, sealing epoxy groups which are not reacted with serotonin on the surfaces of the magnetic particles, and after the reaction is finished, using a binding buffer solution (20 mMNa)3PO4pH6.0) for 3 times, adding the mixture into a serotonin-magnetic particle compound according to the volume ratio of milk to a combined buffer solution of 1:8, 1:4, 1:1, 1:0.25 and 1:0 to ensure that the final volume of the mixture (the mixed milk and the combined buffer solution) is 1mL, uniformly mixing, shaking in the dark (160-210 rpm) for 3 hours at room temperature, carrying out magnetic separation for 3 minutes, collecting supernatant (the non-combined component of the magnetic particle compound), and storing at 4 ℃. Washing the serotonin-magnetic particle complex with a buffer solution for 5-6 times until the absorbance value of the solution at 280nm is close to zero to determine that no protein exists in the solution, carrying out magnetic separation for 3min, collecting the supernatant (washing components, wherein the purpose of washing is to remove the non-specifically bound protein on the surface of the serotonin-magnetic particle complex), and storing at 4 ℃. With elution solution A (0.1M NaHCO)3And 0.5M NaCl, pH8.3) or elution solution B (20mM Na)3PO4And 0.5M NaCl, pH6.0), shaking and eluting at 160-210 rpm (preferably 180rpm) for 30min at room temperature, magnetically separating for 3min, collecting supernatant (sialoglycoprotein bound to the magnetic particle complex), and storing at 4 deg.C.
2.5 SDS-PAGE and silver staining (silver staining)
Taking milk solution diluted by 9 times, unbound components, washing components and each elution component, respectively adding 10 μ L of 5 Xloading buffer solution, boiling in boiling water for 5-8 min, and performing vertical SDS-PAGE electrophoresis on 5% concentrated gel and 10% separation gel until bromophenol blue comes out of the boundary. Developing by silver staining method.
2.6 verification of sialoprotein by Lectin-blot
After protein samples are subjected to SDS-PAGE electrophoretic separation, a PVDF membrane is taken and placed in methanol for soaking for 10-30 seconds, then the PVDF membrane is quickly transferred into a membrane transferring buffer solution for balancing for 1 minute, and a membrane transferring device is installed according to the following sequence: the negative electrode plate-spongy cushion-three layers of filter paper-separation glue-PVDF membrane-three layers of filter paper-spongy cushion-positive electrode plate, and no air bubbles can appear between each layer. The membrane is rotated for 80 minutes under the condition of 100V constant pressure, the protein on the glue can be transferred to the PVDF membrane, and the specific membrane rotating time is properly adjusted by the molecular weight of the protein. After the membrane transfer is finished, putting PVDF into 1 x carbon free aqueous solution, sealing for 1h at room temperature, respectively adding SNA and MAL-II marked by Cy-5 fluorescent dye after sealing is finished, incubating overnight at 4 ℃ in a dark place, taking out the PVDF membrane, putting the PVDF membrane into PBST buffer solution, washing for three times, and detecting a fluorescent signal by using a fluorescent scanner. Second, result and discussion
1. Coupling of serotonin with epoxidized magnetoparticles
Serotonin can be fixed on the surface of the magnetic particle by the reaction of the amino group of the serotonin and the epoxy group on the magnetic particle, and the result is 580cm by infrared spectrum detection-1The left and right peaks are Fe-O oscillations, which indicate that the core structure of the composite is Fe3O4(ii) a After epoxidation modification, at 1400cm-1The wider peaks shown at the left and right are related to the vibration of the epoxy group, which indicates that the epoxy modification of the magnetic particles by the epoxy reagent is successful; coupling of epoxidized magnetic particles with serotonin at 1350cm-1The left and right peaks are C-N vibration on secondary amino (-NH-) formed after the ring opening reaction of the epoxy group and the amino group, which shows that the serotonin is coupled to the surface of the magnetic particle through the reaction of the amino group and the epoxy group; at 3300cm-1And 1600cm-1The left and right peaks are the vibrations associated with hydroxyl groups on the surface of the magnetic particles (FIG. 2 shows the results for the 1# complex).
2. Relationship between coupling amount and coupling time of serotonin and epoxy magnetic particles
Because the extraction (enrichment) efficiency of the compounds with different carbon arm lengths is different, the extraction efficiency of the epoxidized magnetic particles (1 # epoxidized magnetic particles for short) corresponding to the 1# compound is the best, so the optimization condition is mainly performed on the 1# epoxidized magnetic particles.
When 1mg of the epoxy magnetic particles were reacted with 1mL of 0, 1, 2, 4, 10 and 50 μ M serotonin hydrochloride solution, respectively, infrared spectroscopy was used to detect the coupling effect of serotonin and 1mg of the epoxy magnetic particles at different concentrations, and the results showed that the coupling effect of the epoxy magnetic particles and serotonin was better at 10 μ M concentration, no characteristic infrared absorption peak was shown after the reaction at 0, 1, 2 and 4 μ M concentration, and the characteristic peaks formed after the reaction at 50 μ M and 10 μ M concentrations were not different, so that it was determined that the optimal coupling amount of serotonin for 1mg of the epoxy magnetic particles was 10 μ M × 1mL, i.e. 10nmol (about 2 μ g), as shown in fig. 3.
Taking 1mg of epoxy magnetic particles and 1mL of 10 mu M serotonin hydrochloride solution to carry out vibration reaction in a dark place at room temperature, and detecting the reaction effect by utilizing infrared spectroscopy after the reaction is carried out for 0h, 2h, 4h and 8h respectively, wherein the result shows that serotonin can be coupled to the surface of the epoxy magnetic particles after the reaction is carried out for 2h, and the reaction is carried out for 2h without difference from the reaction for 4h and 8h, so that the optimal coupling time is determined to be 2h, as shown in FIG. 4.
3. Combining the optimal ratio of buffer solution to milk volume
Taking 3mg of a blocked serotonin-magnetic particle compound, washing the compound for 3 times by using a binding buffer solution, respectively adding a mixed solution of milk and the binding buffer solution with the volume ratio of 1:8, 1:4, 1:1, 1:0.25 and 1:0, wherein the final volume is 1mL, uniformly mixing, carrying out light-shielding oscillation reaction for 3 hours at room temperature, washing the magnetic particle compound for 5-6 times by using the binding buffer solution after the reaction is finished until the light absorption value of the solution at 280nm is close to zero, eluting by using an elution solution A, carrying out oscillation elution at room temperature for 30 minutes, carrying out magnetic separation for 3 minutes, collecting supernatant, detecting the protein content in the final elution solution by using a BCA protein concentration determination kit, and the result shows that the highest final protein content is obtained by elution when the volume ratio of the milk to the binding buffer solution is 1:1 along with the increase of the milk content in the binding system, when the milk content was further increased, the amount of finally eluted protein did not increase but decreased, and therefore, the optimal ratio of milk to the binding buffer solution was determined to be 1:1 (shown in FIG. 5). The binding buffer solution is preferably 20mM sodium phosphate solution with pH6.0, and can protect the sialic acid structure on the protein in the sample from being complete under the condition of pH6.0, so as to facilitate the mutual binding of sialic acid and serotonin. Besides milk, sialic acid glycoprotein contained in soybean, peanut homogenate, pig serum, and the like can be detected (enriched).
4. Verification of enrichment efficiency of different serotonin-magnetic particle compounds and different elution solutions
After binding the different types of magnetic particle complexes to milk, after washing, the complexes were washed separately with elution solution A (0.1M NaHCO)3And 0.5M NaCl, pH8.3) and elution solution B (20mM Na)3PO4And 0.5M NaCl, pH6.0), and detecting the obtained protein by SDS-PAGE and silver staining, the results show that the amount of protein obtained after elution of the 1# serotonin-magnetic particle complex by the elution solution a is higher than the amount of protein obtained after elution of the elution solution B, according to previous research reports, the binding between serotonin and sialoglycoprotein is not related to the pH value in the system but is related to the ionic strength, and therefore it is presumed that the high concentration salt solution can more effectively dissociate the binding between serotonin and sialoglycoprotein, so that more effective elution can be achieved. Under the same elution conditions, the enrichment effect of the 1# serotonin-magnetic particle composite is found to be better than that of the 2# and 3# (shown in fig. 6) compared with the 1#, 2#, and 3# serotonin-magnetic particle composites.
5. Identification of sialoglycoproteins
Lectin MAL-II and SNA can respectively recognize sialic acid connected with alpha 2-3 and alpha 2-6 at the tail end of a sialoglycoprotein sugar chain, and a protein sample extracted by a serotonin-magnetic particle complex is incubated with fluorescently-labeled MAL-II and SNA on a PVDF membrane and then scanned by a fluorescence scanner, and the result shows (shown in figure 7) that in four bands (the molecular weights Mw are respectively near 80, 35, 30 and 20 KDa) of the protein silver staining, two protein bands (35, 30KDa) can be combined with MAL-II, which indicates that the protein at the two bands contains sialic acid connected with alpha 2-3, and the protein band at 80KDa has weaker combination with MAL-II, which indicates that the protein at the molecular weight possibly contains a small amount of sialic acid connected with alpha 2-3; the binding signal of the 80kDa protein band to SNA is strong, indicating that the protein in this band contains mainly sialic acid linked by alpha 2-6. However, the results of lectin-blot did not show whether the protein at 20kDa contained sialic acid, and could not show the detection results due to the low content of the protein with this molecular weight in the whole extracted protein sample and the sensitivity of the detection method.
Claims (8)
1. A serotonin-magnetic microparticle complex, comprising: the compound is magnetic particles with serotonin fixed on the surface;
the complex comprises binding to Fe3O4Silicon atoms on the surfaces of the magnetic particles and a functional group connected with the silicon atoms, wherein the structure of the functional group is shown as formula 1:
in the formula 1, n is 3-5, and R is the residual part of a hydrogen atom removed from the amino group of serotonin.
2. The serotonin-magnetic microparticle complex according to claim 1, wherein: the value of n is 3.
3. The serotonin-magnetic microparticle complex according to claim 1, wherein: said Fe3O4The magnetic particles are prepared by a chemical coprecipitation method.
4. A method for preparing the serotonin-magnetic microparticle complex according to claim 1, wherein: the preparation method comprises the following steps:
using epoxy modified magnetic particles as a carrier for fixing serotonin, and covalently coupling the serotonin on the surface of the carrier through the ring-opening reaction of amino and epoxy under an alkaline condition;
the preparation method specifically comprises the following steps:
1) 0.4-1.2 g Fe uniformly dispersed3O4Adding 3-7 mL of an epoxidation reagent and 0.2-0.6 mL of glacial acetic acid into the magnetic particles, then reacting in a water bath at 20-30 ℃ for 2-4 h, cleaning with absolute ethyl alcohol after the reaction is finished, and then drying at 60-80 ℃ to obtain epoxidation modified magnetic particles;
2) mixing 1mg of epoxy modified magnetic particles with 1mL of a serotonin hydrochloride solution with the concentration of 10-50 mu M, and reacting for 2-8 h in a dark place, wherein the serotonin hydrochloride solution adopts a coupling buffer solution as a solvent, and the coupling buffer solution is 0.1-0.3M NaHCO3And (3) reacting the magnetic particles with a mixed solution of 0.2-0.8M NaCl, washing the magnetic particles by using a coupling buffer solution, and drying at 50-80 ℃.
5. A method for enriching sialylated glycoproteins, characterized by: the method comprises the following steps:
mixing the collected sample with a binding buffer solution and a serotonin-magnetic particle compound, then oscillating the mixture for 2 to 4 hours at 20 to 25 ℃ in the dark, then sequentially washing and oscillating and eluting the mixture, and collecting eluent; the serotonin-magnetic particle compound is a magnetic particle with serotonin fixed on the surface; the complex comprises binding to Fe3O4Silicon atoms on the surfaces of the magnetic particles and a functional group connected with the silicon atoms, wherein the structure of the functional group is shown as formula 1:
in the formula 1, n is 3-5, and R is the residual part of a hydrogen atom removed from the amino group of serotonin.
6. The method of enriching sialylated glycoproteins according to claim 5, characterized in that: the conditions for shaking elution include: the elution temperature is 20-25 ℃, and the elution time is 20-60 min.
7. The method of enriching sialylated glycoproteins according to claim 5, characterized in that: the sample is selected from animal or human milk and a separation product thereof, blood and a separation product thereof or a plant homogenate, the dosage of the serotonin-magnetic particle compound is 1-5 mg, the total volume of the sample and the binding buffer solution is 0.5-1.5 mL, and the volume ratio of the sample to the binding buffer solution is 1: 0.25-8.
8. The method of enriching sialylated glycoproteins according to claim 5, characterized in that: the cleaning method comprises the steps of adopting a combined buffer solution which is a 10-30 mM sodium phosphate solution, and adopting 0.05-0.15M NaHCO as an eluant for shaking elution3Mixed with 0.2-0.8M NaCl, or 10-30 mM Na3PO4And 0.2-0.8M NaCl.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011091982A1 (en) * | 2010-01-28 | 2011-08-04 | Glycotope Gmbh | Process for the purification of glycoproteins |
| CN102336838A (en) * | 2011-06-27 | 2012-02-01 | 中国科学院长春应用化学研究所 | Paramagnetic metal complex and synthetic method and application thereof |
| CN103357359A (en) * | 2013-05-16 | 2013-10-23 | 英科新创(厦门)科技有限公司 | Complex immunity magnetic particle and preparation method thereof |
| CN103386282A (en) * | 2013-07-17 | 2013-11-13 | 江阴泽成生物技术有限公司 | Method for synthesizing superparamagnetic microspheres with epoxy-activated surfaces and connecting epoxy groups and proteins |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011091982A1 (en) * | 2010-01-28 | 2011-08-04 | Glycotope Gmbh | Process for the purification of glycoproteins |
| CN102336838A (en) * | 2011-06-27 | 2012-02-01 | 中国科学院长春应用化学研究所 | Paramagnetic metal complex and synthetic method and application thereof |
| CN103357359A (en) * | 2013-05-16 | 2013-10-23 | 英科新创(厦门)科技有限公司 | Complex immunity magnetic particle and preparation method thereof |
| CN103386282A (en) * | 2013-07-17 | 2013-11-13 | 江阴泽成生物技术有限公司 | Method for synthesizing superparamagnetic microspheres with epoxy-activated surfaces and connecting epoxy groups and proteins |
Non-Patent Citations (2)
| Title |
|---|
| 糖蛋白质组学中基于化学反应的富集方法研究进展;包慧敏 等;《色谱》;20161231;第1145-1153页 * |
| 靶向 MPO 的磁性纳米粒子的制备与表征;刘环 等;《高分子学报》;20150331;第330-336页（参见摘要） * |
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