Method for preparing, separating and purifying fluorescently-labeled oligosaccharide

Abstract

The invention relates to the field of fluorescence-labeled oligosaccharides, in particular to a method for preparing, separating and purifying fluorescence-labeled oligosaccharides. The invention provides a method for preparing, separating and purifying fluorescently-labeled oligosaccharides, and a relatively pure single oligosaccharide can be obtained by the method and can be used as a built-in standard substance for detecting protein N oligosaccharides such as plasma, serum, IgG and the like. According to the method, the separation operation of the oligosaccharide can be completed without expensive high performance liquid chromatography, the operation method is simple and convenient, and the cost is relatively low.

Description

Technical Field

[0001] The present invention relates to the field of fluorescently labeled oligosaccharides, and particularly to a method for preparing, separating and purifying fluorescently labeled oligosaccharides. Background Art

[0002] Oligosaccharides, also known as oligosaccharides, are linear or branched polymers formed by the glycosidic bond binding of 2 to 10 monosaccharide molecules. However, the composition of the oligosaccharide mixture is relatively complex, which is not conducive to subsequent research. Therefore, it is necessary to separate and purify it to obtain a pure oligosaccharide product with a single component for structure identification, and finally obtain an oligosaccharide sample with a clear structure and a single component for convenient in-depth research and development and utilization.

[0003] Commonly used methods for oligosaccharide separation and purification include capillary electrophoresis, liquid chromatography, membrane separation technology, etc. Due to reasons such as operation complexity and sensitivity, liquid chromatography combined with mass spectrometry is currently used more for sugar structure separation and identification. However, due to the large sample volume required for liquid chromatography and the relatively complex analysis when combined with mass spectrometry, and the high cost, many people have also turned to using laser-induced fluorescence excitation high-performance capillary gel electrophoresis to separate and detect oligosaccharides. With the wide use of fluorescent labeling reagents (such as APTS), combined with capillary electrophoresis technology, picomole-level APTS-derivatized oligosaccharide samples can be detected, and oligosaccharides linked by α-2,6 glycosidic bonds and α-2,3 glycosidic bonds at the terminal sialic acid (Neu5Ac) can also be separated. Due to the high sensitivity of the CGE-LIF (laser-induced fluorescence excitation high-performance capillary gel electrophoresis technology), only a very small amount of APTS-labeled oligosaccharides (50 pmol) is required for separation and fluorescence detection, but only the corresponding peak elution order and the fluorescence signal intensity can be detected. Since the content of the separated oligosaccharides is low, it is difficult to collect a single component for structure identification. Summary of the Invention

[0004] In view of the above existing technical problems, it is necessary for the present invention to invent a method for preparing, separating and purifying fluorescently labeled oligosaccharides to make up for the disadvantages of the low content of oligosaccharides with a single structure separated by CGE-LIF electrophoresis and the inconvenience of purification.

[0005] The present invention discloses a method for preparing fluorescently labeled oligosaccharides, comprising the following steps:

[0006] SS1 Preparation of fluorescently labeled sample: Dissolve the sample with ultrapure water, shake, mix well and centrifuge, take the supernatant into a PCR reaction tube, place it on a PCR instrument and evaporate to dryness with the lid open, then add an APTS solution and a DMSO solution of NaCNBH3 to the PCR reaction tube, mix well and centrifuge, and then place the above-mentioned PCR reaction tube after mixing and centrifuging into a PCR instrument for heating reaction. After the reaction is completed, add ultrapure water to the PCR reaction tube, mix well and centrifuge to obtain a fluorescently labeled sample;

[0007] Preparation of S2 polyacrylamide gel double-layer glass plate: prepare polyacrylamide solution by taking acrylamide, N,N-methylenebisacrylamide and double distilled water; prepare ammonium persulfate solution by taking ammonium persulfate and ultrapure water; take tris(hydroxymethyl)aminomethane, Na2EDTA·2H2O and sterilized double distilled water, stir and dissolve them thoroughly, then add glacial acetic acid to dissolve them thoroughly, and then add sterilized double distilled water to make up to volume to prepare TAE buffer solution; take TAE solution and add agar powder, mix well and heat to dissolve to prepare agarose gel, and pour it into the sealing groove of the glass plate to seal the bottom of the double-layer glass plate before solidification. After the agarose gel solidifies, mix the polyacrylamide solution, TAE buffer solution, sterilized double distilled water, ammonium persulfate and tetramethylethylenediamine to obtain a mixed solution, and introduce the mixed solution into the assembled double-layer glass plate. After the double-layer glass plate is filled with polyacrylamide gel, insert a 1.5mm 26-foot comb into the gel above the glass plate, and pull out the comb after the polyacrylamide is fully solidified to obtain a polyacrylamide gel double-layer glass plate;

[0008] S3 sample loading, photo taking and gel cutting: fix the double-layer glass plate of polyacrylamide gel prepared in step S2 in a vertical electrophoresis tank, fill the upper and lower tanks of the vertical electrophoresis tank with TAE buffer, set the voltage for pre-electrophoresis, take the fluorescent labeled sample prepared in step S1, add glycerol and ethylenediaminetetraacetic acid, mix well, add to the gel wells on the double-layer glass plate of polyacrylamide gel, set the electrophoresis voltage, turn off the electrophoresis instrument when the front fluorescent dye reaches the bottom edge of the polyacrylamide gel, remove the double-layer glass plate of polyacrylamide gel, and then place it on a blue light gel cutting instrument for detection, mark the position of each sugar band and save it; cut each sugar band and place it in a 1.5 ml centrifuge tube and number it to obtain fluorescent labeled oligosaccharide gel strips.

[0009] Preferably, in the S1 fluorescent labeling sample preparation step, the APTS concentration in the APTS solution is 19-21 mM; the APTS solution also contains a citric acid buffer with a concentration of 1.1-1.3 M.

[0010] Preferably, in the step of preparing the S2 polyacrylamide gel double-layer glass plate, the mass fraction of the prepared polyacrylamide solution is 29-31%; the mass fraction of the prepared ammonium persulfate solution is 9-11%.

[0011] Preferably, in the step of preparing the S2 polyacrylamide gel double-layer glass plate, the prepared TAE buffer has two concentrations, one is 5xTAE buffer and the other is 1xTAE buffer.

[0012] Preferably, in the step of preparing the S2 polyacrylamide gel double-layer glass plate, the mass fraction of the agarose gel is 1.5-2.5%.

[0013] Preferably, in the step of preparing the double-layer glass plate of S2 polyacrylamide gel, the volume of the polyacrylamide solution in the mixed solution is 33-34 ml; the TAE buffer solution is 5x TAE buffer solution, with a volume of 9.5-10.5 ml; the volume of sterilized double-distilled water is 6.5-7 ml; the volume of ammonium persulfate is 250 μl, and the volume of tetramethylethylenediamine is 25 μl.

[0014] Preferably, in the step of S3 loading, photographing and cutting the gel, the pre-electrophoresis time is 15-30 min; the volume ratio of glycerol to ethylenediaminetetraacetic acid is 1:(0.95-1.05); the concentration of ethylenediaminetetraacetic acid is 0.09-0.11 mol / L; after the gel holes on the double-layer glass plate of polyacrylamide gel, set the electrophoresis voltage to 195-205 V. After the sample electrophoreses out of the holes, adjust the electrophoresis voltage to 115-125 V.

[0015] A method for separating and purifying fluorescently labeled oligosaccharides, comprising the following steps:

[0016] (1) The fluorescently labeled oligosaccharide gel strip prepared by the above method for preparing fluorescently labeled oligosaccharides is ground with a grinding rod and added to a No. 1 centrifuge tube. Then, 1 mmol / L EDTA (pH = 8.0) buffer solution is added to the No. 1 centrifuge tube, and then the centrifuge tube lid is covered and incubated on a constant temperature oscillator. The incubated sample is centrifuged in a high-speed centrifuge. After the centrifugation is completed, the supernatant in the No. 1 centrifuge tube is transferred to a No. 2 centrifuge tube, and the above centrifugation operation is repeated twice, and the supernatants are combined;

[0017] (2) Equilibrate the silica hydroxyl magnetic beads at room temperature, add the equilibrated silica hydroxyl magnetic beads to the No. 2 centrifuge tube described in (1), then add 95% acetonitrile and shake well, and then place it on a magnetic rack and let it stand for 1 min. After the solution becomes clear, discard the supernatant. Then add 95% aqueous acetonitrile solution, shake well for 10 s, then place it on a magnetic rack and let it stand for 30 s until the solution becomes clear, discard the supernatant, and then repeat the above steps 2 times, discard the supernatant, and place the No. 2 centrifuge tube with the supernatant removed on the magnetic rack to dry;

[0018] (3) Add sterilized double-distilled water to the dried No. 2 centrifuge tube in (2), shake well and let it stand, transfer the supernatant to a No. 3 centrifuge tube, and then perform vacuum centrifugal concentration and drying to obtain highly purified fluorescently labeled oligosaccharides.

[0019] Preferably, in step (1), the rotation speed of the constant temperature oscillator is 500-1000 rpm; the volume ratio of the fluorescently labeled oligosaccharide gel strip to 1 mmol / L EDTA (pH = 8.0) buffer solution is 1:(1-2).

[0020] Preferably, in step (2), the equilibration time of the silica hydroxyl magnetic beads is 25 - 35 min; the volume ratio of the silica hydroxyl magnetic beads to 95% acetonitrile is 1:(15 - 20); the storage temperature of the high-purity fluorescently labeled oligosaccharide is -15 - -25 °C.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The present invention provides a method for the preparation, separation and purification of fluorescently labeled oligosaccharides. This method can complete the separation operation of oligosaccharides without the need for expensive high-performance liquid chromatography. The operation method is simple and the cost is low.

[0023] Through this method, relatively pure single oligosaccharides can be obtained, which can be used as internal standards for the detection of protein N-glycans in plasma, serum, IgG, etc.

[0024] In addition, since this electrophoresis method has a separation principle similar to that of high-performance capillary electrophoresis, for the highly abundant unknown N-glycans on unknown proteins, the elution order obtained is basically the same. It can make up for the deficiency that high-performance capillary electrophoresis cannot collect and separate single oligosaccharides. Thus, the purified sample can be further used for mass spectrometry analysis to identify the oligosaccharide structures in the same order as high-performance capillary electrophoresis. This is conducive to the high-throughput detection of N-glycan abundances in various types of samples by the high-performance capillary electrophoresis system and expands its application and promotion. Description of the Drawings

[0025] Figure 1 : Electrophoresis separation result diagram of fluorescently labeled water-soluble starch polyacrylamide

[0026] Figure 2 : Electrophoresis separation result diagram of fluorescently labeled IgG FA2 N-glycan polyacrylamide

[0027] Figure 3 : Electrophoresis separation result diagram of fluorescently labeled plasma protein A2G2 N-glycan polyacrylamide

[0028] Figure 4 : Capillary electrophoresis detection and separation diagram of plasma samples before and after separation by polyacrylamide gel. P4 is the A2G2 N-glycan peak; the upper part is the N-glycan sample after IgG separation; the lower part is the N-glycan sample before IgG separation

[0029] Figure 5 : Capillary electrophoresis detection and separation diagram of the sample after IgG immunoglobulin separation by polyacrylamide gel. P1 is the FA2 N-glycan peak

[0030] Figure 6 : Capillary electrophoresis detection and separation diagram of the sample after amylomaltose separation by polyacrylamide gel. P3 is the amylomaltose 5 (DP5) sugar peak Detailed Embodiments

[0031] The following embodiments are provided to better further understand the present invention, which are not limited to the described optimal embodiments, and do not constitute a limitation to the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other prior arts falls within the protection scope of the present invention.

[0032] For those embodiments where specific experimental steps or conditions are not indicated, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments whose manufacturers are not indicated, they are all conventional reagent products that can be obtained through commercial purchase.

[0033] Example 1: A method for preparing a fluorescently labeled oligosaccharide, comprising the following steps:

[0034] S1 Preparation of fluorescently labeled sample: Weigh 20 mg of soluble starch, dissolve it in 3.2 ml of ultrapure water, shake well, mix and centrifuge; Take 2 μl of the starch supernatant solution into a 200 μl PCR reaction tube, place it on a PCR instrument, and evaporate it to dryness at 65 °C with the lid open for 20 min. Add 2 μl of an APTS solution (19 mM APTS, 1.1 M citrate buffer) and 2 μl of a DMSO solution containing 1 M NaCNBH3 to the sample, mix well for 30 s and centrifuge for 10 s; Place the PCR tube in a PCR instrument and heat it at 90 °C for 2 h. After the reaction is completed, add 50 μl of ultrapure water to the PCR tube, mix well for 30 s, and centrifuge for 10 s to terminate the reaction.

[0035] Preparation of double-layer glass plate of S2 polyacrylamide gel: Weigh 28 g of acrylamide, 1 g of N,N'-methylenebisacrylamide, and add 100 ml of double-distilled water to prepare a 29% polyacrylamide solution; weigh 0.9 g of ammonium persulfate and 10 ml of water to prepare a 9% ammonium persulfate solution; weigh 24.2 g of tris(hydroxymethyl)aminomethane and 3.72 g of Na2EDTA·2H2O, add 800 ml of sterilized double-distilled water and stir well to dissolve, then add 2.71 ml of glacial acetic acid and dissolve completely, and finally add sterilized double-distilled water to make up to 1 L to prepare 5x TAE buffer. Take 200 ml of 5x TAE buffer, add 800 ml of sterilized double-distilled water to prepare 1000 ml of 1x TAE buffer and mix well; take 100 ml of 1x TAE buffer and add 1.5 g of agarose powder, mix well and heat in a microwave oven at the highest power for 3 min to completely dissolve the agarose powder in TAE to prepare a 1.5% agarose gel by mass fraction. Pour it into the sealing groove of the glass plate before solidification to seal the bottom of the double-layer glass plate. Wait for 15 min until the 1.5% agarose gel solidifies. Take 33 ml of 29% polyacrylamide solution, 9.5 ml of 5x TAE buffer, 6.5 ml of sterilized double-distilled water and mix well, then add 250 μl of 9% ammonium persulfate and 25 μl of tetramethylethylenediamine and mix well to obtain a mixed solution. Pour the mixed solution into the assembled double-layer glass plate (note that no bubbles should be generated). After filling the double-layer glass plate with polyacrylamide gel, insert a 1.5-mm 26-tooth comb into the gel above the glass plate and wait for 30 min until the polyacrylamide solidifies completely; gently remove the comb after the gel solidifies to obtain a double-layer glass plate of polyacrylamide gel.

[0036] Loading, photographing and gel cutting: Fix the double-layer glass plate of polyacrylamide gel prepared in step S2 in a vertical electrophoresis tank, and fill both the upper and lower tanks of the vertical electrophoresis tank with 1x TAE buffer; connect the electrophoresis tank to the electrophoresis instrument, set the voltage to 120 v and pre-electrophorese for 15 min; add 1 μl of glycerol and 0.95 μl of 0.95 mol / L EDTA (pH = 8.0) to the APTS-labeled starch product and mix well. Turn off the power of the electrophoresis instrument, and add the mixed APTS-labeled sample to the gel wells; set the electrophoresis voltage to 195 V. After the sample electrophoreses out of the wells, adjust the electrophoresis voltage to 115 V for electrophoresis; electrophorese for 4 hours until the frontmost fluorescent dye reaches the lower edge of the polyacrylamide gel, then turn off the electrophoresis instrument. After electrophoresis, remove the gel plate of the electrophoresis instrument, and then place it on a blue light gel cutting instrument for detection. Mark the position of each sugar band and save it; Cut each sugar band and place it in a 1.5-ml centrifuge tube and number it to obtain a fluorescently labeled oligosaccharide gel strip.

[0037] A method for separating and purifying fluorescently labeled oligosaccharides, comprising the following steps:

[0038] (1) The fluorescently labeled oligosaccharide gel strip prepared by the above method for preparing fluorescently labeled oligosaccharides is ground with a grinding rod and added to a centrifuge tube. Then, 1 mmol / L EDTA (pH 8.0) buffer solution is added to centrifuge tube No. 1. The volume ratio of the fluorescently labeled oligosaccharide gel strip to 1 mmol / L EDTA (pH = 8.0) buffer solution is 1:1. Then, cover the centrifuge tube lid and incubate at 37 °C for 30 minutes on a thermostatic shaker. The rotation speed of the thermostatic shaker is 500 rpm; place the incubated sample in a high-speed centrifuge with a 1.5 ml rotor and centrifuge at a maximum speed of 12,000 rpm for 1 min; transfer the supernatant in centrifuge tube No. 1 to centrifuge tube No. 2; add 0.5 times the volume of 1 mmol / L EDTA (pH = 8.0) buffer solution to the precipitate, shake and mix evenly for 10 s, and centrifuge again at 12,000 rpm for 1 min. Combine the supernatants of the two times.

[0039] (2) Take out the silica hydroxyl magnetic beads in advance and equilibrate at room temperature for 25 min; add the equilibrated silica hydroxyl magnetic beads to centrifuge tube No. 2 described in (1), then add 95% acetonitrile and shake evenly, and then place it on a magnetic rack and let it stand for 1 min. The volume ratio of the silica hydroxyl magnetic beads to 95% acetonitrile is 1:15; after the solution is clarified, discard the supernatant, then add 15 times the volume of 95% aqueous acetonitrile solution of the magnetic beads, take it off the magnetic rack, mix evenly for 10 s, centrifuge briefly, and then place it on the magnetic rack again and let it stand for 30 s until the solution is clarified; discard the supernatant, and then repeat the above steps 2 times; discard the supernatant, and place centrifuge tube No. 2 with the supernatant removed on the magnetic rack to dry.

[0040] (3) Add 50 μl of sterile double-distilled water to centrifuge tube No. 2 dried in (2), take it off the magnetic rack, shake and mix evenly for 10 s, and let it stand for 5 min; centrifuge the sample briefly and place it on the magnetic rack for 2 min. After the solution is clarified, transfer all the supernatant to centrifuge tube No. 3; then perform vacuum centrifugal concentration and drying to obtain high-purity fluorescently labeled oligosaccharides, and store the obtained fluorescently labeled oligosaccharides at -15 °C.

[0041] Example 2: A method for preparing fluorescently labeled oligosaccharides, comprising the following steps:

[0042] S1 Preparation of fluorescently labeled sample: Weigh 20 mg of soluble starch, dissolve it with 3.2 ml of ultrapure water, shake and mix evenly and centrifuge; take 2 μl of the starch supernatant solution into a 200 μl PCR reaction tube, place it on a PCR instrument and evaporate to dryness at 65 °C with the lid open for 20 min. Add 2 μl of APTS solution (20 mM APTS, 1.2 M citrate buffer) and 2 μl of DMSO solution containing 1 M NaCNBH3 to the sample, mix evenly for 30 s and centrifuge for 10 s; place the PCR tube in the PCR instrument and heat at 90 °C for 2 h. After the reaction is completed, add 50 μl of ultrapure water to the PCR tube, mix evenly for 30 s, and centrifuge for 10 s to terminate the reaction.

[0043] Preparation of double-layer glass plate of S2 polyacrylamide gel: Take 29 g of acrylamide, 1 g of N,N-methylenebisacrylamide, and 100 ml of double-distilled water to prepare a 30% polyacrylamide solution; take 1 g of ammonium persulfate and 10 ml of water to prepare a 10% ammonium persulfate solution; take 24.2 g of tris(hydroxymethyl)aminomethane and 3.72 g of Na2EDTA·2H2O, add 800 ml of sterilized double-distilled water and stir well to dissolve, then add 2.71 ml of glacial acetic acid and dissolve fully, and then add sterilized double-distilled water to make up to 1 L to prepare 5x TAE buffer. Take 200 ml of 5x TAE buffer, add 800 ml of sterilized double-distilled water to prepare 1000 ml of 1x TAE buffer and mix well; take 100 ml of 1x TAE buffer and add 2 g of agarose powder, mix well and heat in the microwave oven at the highest power for 3 min to make the agarose powder fully dissolve in TAE to prepare a 2% agarose gel by mass fraction, and pour it into the sealing groove of the glass plate before solidification to seal the bottom of the double-layer glass plate. Wait for 15 min until the 2% agarose gel solidifies. Take 33.33 ml of 30% polyacrylamide solution, 10 ml of 5x TAE buffer, and 6.77 ml of sterilized double-distilled water and mix well, then add 250 μl of 10% ammonium persulfate by mass fraction and 25 μl of tetramethylethylenediamine and mix well to obtain a mixed solution, and pour the mixed solution into the assembled double-layer glass plate (pay attention not to generate bubbles). After the double-layer glass plate is filled with polyacrylamide gel, insert a 1.5 mm 26-tooth comb into the gel above the glass plate and wait for 30 min until the polyacrylamide solidifies fully; gently pull out the comb after the gel solidifies, and pull out the comb after the polyacrylamide solidifies fully to obtain a double-layer glass plate of polyacrylamide gel.

[0044] S3 Loading, photographing and gel cutting: Fix the double-layer glass plate of polyacrylamide gel prepared in step S2 in a vertical electrophoresis tank, and fill both the upper and lower tanks of the vertical electrophoresis tank with 1x TAE buffer; connect the electrophoresis tank to the electrophoresis instrument, set the voltage to 120 v, and pre-electrophorese for 15 - 30 min; add 1 μl of glycerol and 1.0 μl of 0.95 mol / L EDTA (PH = 8.0) to the APTS-labeled starch product and mix well, turn off the power of the electrophoresis instrument, and add the mixed APTS-labeled sample to the gel wells; set the electrophoresis voltage to 200 V, and after the sample electrophoreses out of the wells, adjust the electrophoresis voltage to 120 V for electrophoresis; Electrophorese for 4 hours until the frontmost fluorescent dye reaches the lower edge of the polyacrylamide gel, then turn off the electrophoresis instrument. After electrophoresis, remove the gel plate of the electrophoresis instrument, and then place it on a blue light gel cutting instrument for detection, mark the position of each sugar band and save it; Cut each sugar band and place it in a 1.5 ml centrifuge tube and number it to obtain a fluorescently labeled oligosaccharide gel strip.

[0045] A method for separating and purifying fluorescently labeled oligosaccharides, comprising the following steps:

[0046] (1) The fluorescently labeled oligosaccharide gel strip prepared by the above method for preparing a fluorescently labeled oligosaccharide is ground with a grinding rod and added to a centrifuge tube. Then, 1 mmol / L EDTA (pH 8.0) buffer is added to centrifuge tube No. 1. The volume ratio of the fluorescently labeled oligosaccharide gel strip to 1 mmol / L EDTA (pH = 8.0) buffer is 1:1.5. Then, the centrifuge tube lid is covered, and it is incubated at 37 °C for 30 minutes on a constant temperature shaker. The rotation speed of the constant temperature shaker is 750 rpm; the incubated sample is centrifuged at a maximum speed of 12,000 rpm for 1 min in a high-speed centrifuge with a 1.5 ml rotor; the supernatant in centrifuge tube No. 1 is transferred to centrifuge tube No. 2; 0.5 times the volume of 1 mmol / L EDTA (pH = 8.0) buffer is added to the precipitate, shaken and mixed evenly for 10 s, and then centrifuged at 12,000 rpm for 1 min again. The supernatants of the two times are combined.

[0047] (2) The silica hydroxyl magnetic beads are taken out in advance and equilibrated at room temperature for 30 min; the equilibrated silica hydroxyl magnetic beads are added to centrifuge tube No. 2 described in (1), and 95% acetonitrile is added and shaken evenly, then placed on a magnetic stand and left to stand for 1 min. The volume ratio of the silica hydroxyl magnetic beads to 95% acetonitrile is 1:17; after the solution is clarified, the supernatant is discarded, and then 17 times the volume of the magnetic beads of 95% aqueous acetonitrile solution is added. It is taken off the magnetic stand, mixed evenly for 10 s, centrifuged briefly, and then placed on the magnetic stand and left to stand for 30 s until the solution is clarified; the supernatant is discarded, and then the above steps are repeated 2 times; the supernatant is discarded, and centrifuge tube No. 2 with the supernatant removed is placed on the magnetic stand to dry.

[0048] (3) 50 μl of sterile double-distilled water is added to centrifuge tube No. 2 dried in (2), taken off the magnetic stand, shaken and mixed evenly for 10 s, and left to stand for 5 min; the sample is centrifuged briefly and placed on the magnetic stand for 2 min. After the solution is clarified, all the supernatant is transferred to centrifuge tube No. 3; then vacuum centrifugation concentration and drying are carried out to obtain highly pure fluorescently labeled oligosaccharide, and the obtained fluorescently labeled oligosaccharide is stored at -20 °C.

[0049] Example 3: A method for preparing a fluorescently labeled oligosaccharide, comprising the following steps:

[0050] S1 Fluorescently labeled sample preparation: Weigh 20 mg of soluble starch, dissolve it in 3.2 ml of ultrapure water, shake and mix evenly and centrifuge; take 2 μl of the starch supernatant solution and place it in a 200 μl PCR reaction tube, place it on a PCR instrument and evaporate to dryness at 65 °C with the lid open for 20 min. Add 2 μl of APTS solution (21 mM APTS, 1.3 M citrate buffer) and 2 μl of DMSO solution containing 1 M NaCNBH3 to the sample, mix evenly for 30 s and centrifuge for 10 s; place the PCR tube in the PCR instrument and heat at 90 °C for 2 h. After the reaction is completed, 50 μl of ultrapure water is added to the PCR tube, mixed evenly for 30 s, and centrifuged for 10 s to terminate the reaction.

[0051] Preparation of double-layer glass plate of S2 polyacrylamide gel: Take 30 g of acrylamide, 1 g of N,N'-methylenebisacrylamide, and 100 ml of double-distilled water to prepare a 31% polyacrylamide solution; take 1.1 g of ammonium persulfate and 10 ml of water to prepare an 11% ammonium persulfate solution; take 24.2 g of tris(hydroxymethyl)aminomethane and 3.72 g of Na2EDTA·2H2O, add 800 ml of sterilized double-distilled water and stir well to dissolve, then add 2.71 ml of glacial acetic acid and dissolve fully, and then add sterilized double-distilled water to make up to 1 L to prepare 5x TAE buffer. Take 200 ml of 5x TAE buffer, add 800 ml of sterilized double-distilled water to prepare 1000 ml of 1x TAE buffer and mix well; take 100 ml of 1x TAE buffer and add 2.5 g of agar powder, mix well and heat in a microwave oven at the highest power for 3 min to make the agar powder fully dissolve in TAE to prepare a 2.5% agarose gel by mass fraction, and pour it into the sealing groove of the glass plate before solidification to seal the bottom of the double-layer glass plate. Wait for 15 min until the 2.5% agarose gel solidifies. Take 34 ml of 31% polyacrylamide solution, 10.5 ml of 5x TAE buffer, and 7 ml of sterilized double-distilled water and mix well, then add 250 μl of 11% ammonium persulfate by mass fraction and 25 μl of tetramethylethylenediamine and mix well to obtain a mixed solution. Pour the mixed solution into the assembled double-layer glass plate (note that no bubbles should be generated). After the double-layer glass plate is filled with polyacrylamide gel, insert a 1.5-mm 26-tooth comb into the gel above the glass plate and wait for 30 min until the polyacrylamide solidifies fully; after the gel solidifies, gently pull out the comb. After the polyacrylamide solidifies fully, pull out the comb to obtain a double-layer glass plate of polyacrylamide gel.

[0052] S3 Loading, photographing and gel cutting: Fix the double-layer glass plate of polyacrylamide gel prepared in step S2 in a vertical electrophoresis tank, and fill both the upper and lower tanks of the vertical electrophoresis tank with 1x TAE buffer; connect the electrophoresis tank to the electrophoresis instrument, set the voltage to 120 v, and pre-electrophorese for 15 - 30 min; add 1 μl of glycerol and 1.05 μl of 0.95 mol / L EDTA (pH = 8.0) to the APTS-labeled starch product and mix well. Turn off the power of the electrophoresis instrument, and add the mixed APTS-labeled sample to the gel wells; set the electrophoresis voltage to 205 V. After the sample electrophoreses out of the wells, adjust the electrophoresis voltage to 125 V for electrophoresis; electrophorese for 4 hours until the fluorescent dye at the front end reaches the lower edge of the polyacrylamide gel, then turn off the electrophoresis instrument. After electrophoresis, remove the gel plate of the electrophoresis instrument, and then place it on a blue light gel cutting instrument for detection, mark the position of each sugar band and save it; cut each sugar band and place it in a 1.5-ml centrifuge tube and number it to obtain a fluorescently labeled oligosaccharide gel strip.

[0053] A method for separating and purifying fluorescently labeled oligosaccharides, comprising the following steps:

[0054] (1) The fluorescently labeled oligosaccharide gel strip prepared by the above method for preparing a fluorescently labeled oligosaccharide is ground with a grinding rod and added to a centrifuge tube. Then, 1 mmol / L EDTA (pH 8.0) buffer is added to centrifuge tube No. 1. The volume ratio of the fluorescently labeled oligosaccharide gel strip to 1 mmol / L EDTA (pH = 8.0) buffer is 1:2. Then, the centrifuge tube lid is covered, and it is incubated at 37 °C for 30 minutes on a thermostatic shaker with a rotation speed of 1000 rpm; the incubated sample is centrifuged at a maximum speed of 12000 rpm for 1 minute in a high-speed centrifuge with a 1.5 ml rotor; the supernatant in centrifuge tube No. 1 is transferred to centrifuge tube No. 2; 0.5 times the volume of 1 mmol / L EDTA (pH = 8.0) buffer is added to the precipitate, shaken and mixed evenly for 10 s, and then centrifuged at 12000 rpm for 1 minute again. The supernatants of the two times are combined.

[0055] (2) The silica hydroxyl magnetic beads are taken out in advance and equilibrated at room temperature for 35 minutes; the equilibrated silica hydroxyl magnetic beads are added to centrifuge tube No. 2 described in (1), and then 95% acetonitrile is added and shaken evenly, and then placed on a magnetic rack and left standing for 1 minute. The volume ratio of the silica hydroxyl magnetic beads to 95% acetonitrile is 1:20; after the solution becomes clear, the supernatant is discarded, and then 95% acetonitrile aqueous solution with 20 times the volume of the magnetic beads is added, taken off the magnetic rack, mixed evenly for 10 s, centrifuged instantaneously, and then placed on the magnetic rack and left standing for 30 s until the solution becomes clear; the supernatant is discarded, and then the above steps are repeated 2 times; the supernatant is discarded, and centrifuge tube No. 2 with the supernatant removed is placed on the magnetic rack to dry.

[0056] (3) 50 μl of sterilized double-distilled water is added to centrifuge tube No. 2 dried in (2), taken off the magnetic rack, shaken and mixed evenly for 10 s, and left standing for 5 minutes; the sample is centrifuged instantaneously and placed on the magnetic rack for 2 minutes. After the solution becomes clear, all the supernatant is transferred to centrifuge tube No. 3; then vacuum centrifugation concentration and drying are carried out to obtain highly pure fluorescently labeled oligosaccharide, and the obtained fluorescently labeled oligosaccharide is stored at -17 °C.

[0057] Example 4: A method for preparing a fluorescently labeled oligosaccharide, comprising the following steps:

[0058] S1 Fluorescent-labeled sample preparation: Take 0.5 mg of IgG N-glycoprotein, dissolve it in 20 μl of ultrapure water, mix well by shaking and centrifuge. Take 2 μl of the IgG glycoprotein supernatant solution into a 200 μl PCR reaction tube, place it on a PCR instrument, denature at 95 °C for 5 min, and then digest with enzymes at 37 °C for 3 h. Add 2 μl of APTS solution (20 mM APTS, 1.2 M citrate buffer) and 2 μl of DMSO solution containing 1 M NaCNBH3 to the sample, mix well for 30 s and centrifuge for 10 s. Place the PCR tube in the PCR instrument and heat at 90 °C for 2 h. After the reaction, add 50 μl of ultrapure water to the PCR tube, mix well for 30 s, and centrifuge for 10 s to terminate the reaction.

[0059] The preparation steps of the double-layer glass plate of polyacrylamide gel in S2, the steps of loading samples, photographing and cutting the gel in S3, and the subsequent separation and purification methods are the same as those in Example 2.

[0060] Example 5: A method for preparing fluorescent-labeled oligosaccharides, comprising the following steps:

[0061] S1 Fluorescent-labeled sample preparation: Take 2 μl of serum solution into a 200 μl PCR reaction tube, place it on a PCR instrument, denature at 95 °C for 5 min, and then digest with enzymes at 37 °C for 3 h. Add 2 μl of APTS solution (20 mM APTS, 1.2 M citrate buffer) and 2 μl of DMSO solution containing 1 M NaCNBH3 to the sample, mix well for 30 s and centrifuge for 10 s. Place the PCR tube in the PCR instrument and heat at 90 °C for 2 h. After the reaction, add 50 μl of ultrapure water to the PCR tube, mix well for 30 s, and centrifuge for 10 s to terminate the reaction.

[0062] The preparation steps of the double-layer glass plate of polyacrylamide gel in S2, the steps of loading samples, photographing and cutting the gel in S3, and the subsequent separation and purification methods are the same as those in Example 2.

[0063] The purity of the fluorescent-labeled oligosaccharides prepared in Examples 1 to 5 was detected after 1, 5, 10, 20, and 30 days of storage, and the detection results are shown in the following table:

[0064]

[0065] It can be seen from the data in the above table that the fluorescent-labeled oligosaccharides prepared by the preparation and purification method provided by the present invention have high purity, can be stored for a long time, and during long-term storage, the purity will not change greatly and the properties are stable.

[0066] From Figures 1 to 6 it can be seen that the oligosaccharide gel strips obtained by the preparation, separation and purification method of a fluorescent-labeled oligosaccharide provided by the present invention are clear, the oligosaccharide content is high, and it is easy to collect single components for structure identification.

[0067] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A method for preparing fluorescently labeled oligosaccharides, characterized in that: The following steps are involved: S1 Preparation of fluorescent labeled samples: Dissolve the sample with ultrapure water, shake and mix, centrifuge, take the supernatant into a PCR reaction tube, place it on a PCR instrument, open the cover and evaporate to dryness, then add APTS solution and NaCNBH3 DMSO solution into the PCR reaction tube, mix and centrifuge, then place the above mixed and centrifuged PCR reaction tube in a PCR instrument for heating reaction, after the reaction is completed, add ultrapure water into the PCR reaction tube, mix and centrifuge to obtain the fluorescent labeled sample; Preparation of S2 polyacrylamide gel double-layer glass plate: prepare polyacrylamide solution by taking acrylamide, N,N-methylenebisacrylamide and double distilled water; prepare ammonium persulfate solution by taking ammonium persulfate and ultrapure water; take tris(hydroxymethyl)aminomethane, Na2EDTA·2H2O and sterilized double distilled water, stir and dissolve them thoroughly, then add glacial acetic acid to dissolve them thoroughly, and then add sterilized double distilled water to make up to volume to prepare TAE buffer solution; take TAE solution and add agar powder, mix well and heat to dissolve to prepare agarose gel, and pour it into the sealing groove of the glass plate to seal the bottom of the double-layer glass plate before solidification. After the agarose gel solidifies, mix the polyacrylamide solution, TAE buffer solution, sterilized double distilled water, ammonium persulfate and tetramethylethylenediamine to obtain a mixed solution, and introduce the mixed solution into the assembled double-layer glass plate. After the double-layer glass plate is filled with polyacrylamide gel, insert a 1.5mm 26-foot comb into the gel above the glass plate, and pull out the comb after the polyacrylamide is fully solidified to obtain a polyacrylamide gel double-layer glass plate; S3 sample loading, photo taking and gel cutting: fix the double-layer glass plate of polyacrylamide gel prepared in step S2 in a vertical electrophoresis tank, fill the upper and lower tanks of the vertical electrophoresis tank with TAE buffer, set the voltage for pre-electrophoresis, take the fluorescent labeled sample prepared in step S1, add glycerol and ethylenediaminetetraacetic acid, mix well, add to the gel wells on the double-layer glass plate of polyacrylamide gel, set the electrophoresis voltage, turn off the electrophoresis instrument when the front fluorescent dye reaches the bottom edge of the polyacrylamide gel, remove the double-layer glass plate of polyacrylamide gel, and then place it on a blue light gel cutting instrument for detection, mark the position of each sugar band and save it; cut each sugar band and place it in a 1.5 ml centrifuge tube and number it to obtain fluorescent labeled oligosaccharide gel strips.

2. The method for preparing a fluorescently labeled oligosaccharide according to claim 1, characterized in that: In the S1 fluorescent labeling sample preparation step, the APTS concentration in the APTS solution is 19-21 mM; the APTS solution also contains a citric acid buffer solution with a concentration of 1.1-1.3 M.

3. The method for preparing a fluorescently labeled oligosaccharide according to claim 1, characterized in that: In the step of preparing the S2 polyacrylamide gel double-layer glass plate, the mass fraction of the prepared polyacrylamide solution is 29-31%; the mass fraction of the prepared ammonium persulfate solution is 9-11%.

4. The method for preparing a fluorescently labeled oligosaccharide according to claim 1, characterized in that: In the step of preparing the S2 polyacrylamide gel double-layer glass plate, the prepared TAE buffer has two concentrations, one is 5xTAE buffer and the other is 1xTAE buffer.

5. The method for preparing a fluorescently labeled oligosaccharide according to claim 1, characterized in that: In the step of preparing the S2 polyacrylamide gel double-layer glass plate, the mass fraction of the agarose gel is 1.5-2.5%.

6. The method for preparing a fluorescently labeled oligosaccharide according to claim 1, characterized in that: In the S2 polyacrylamide gel double-layer glass plate preparation step, the volume of the polyacrylamide solution in the mixed liquid is 33-34 ml; the TAE buffer is 5xTAE buffer, the volume is 9.5-10.5 ml; the volume of sterile double distilled water is 6.5-7 ml; the volume of ammonium persulfate is 250 μl, and the volume of tetramethylethylenediamine is 25 μl.

7. The method for preparing a fluorescently labeled oligosaccharide according to claim 1, characterized in that: In the S3 sample loading, photo-taking and gel cutting steps, the pre-electrophoresis time is 15 to 30 minutes; the volume ratio of glycerol to ethylenediaminetetraacetic acid is 1:(0.95 to 1.05); the concentration of ethylenediaminetetraacetic acid is 0.09 to 0.11 mol / L; when the polyacrylamide gel is in the gel well on the double-layer glass plate, the electrophoresis voltage is set to 195 to 205 V, and after the sample is electrophoresed out of the well, the electrophoresis voltage is adjusted to 115 to 125 V.

8. A method for separating and purifying fluorescently labeled oligosaccharides, characterized in that: The following steps are involved: (1) The fluorescently labeled oligosaccharide gel strip prepared by the above-mentioned method for preparing fluorescently labeled oligosaccharides is ground with a grinding rod and added to centrifuge tube No. 1, and then 1mmol / LEDTA (PH=8.0) buffer is added to centrifuge tube No. 1, and then the centrifuge tube cover is closed and incubated on a constant temperature oscillator. The incubated sample is placed in a high-speed centrifuge for centrifugation. After the centrifugation is completed, the supernatant in centrifuge tube No. 1 is transferred to centrifuge tube No. 2, and the above centrifugation operation is repeated twice to combine the supernatants; (2) The silicon hydroxy magnetic beads were balanced at room temperature, and the balanced silicon hydroxy magnetic beads were added to the No. 2 centrifuge tube described in (1), and then 95% acetonitrile was added and shaken evenly, and then placed on a magnetic rack for 1 min. After the solution was clarified, the supernatant was discarded, and then 95% acetonitrile aqueous solution was added and shaken for 10 seconds, and then placed on a magnetic rack for 30 seconds until the solution was clarified, and the supernatant was discarded. Then, the above steps were repeated twice, and the supernatant was discarded. The No. 2 centrifuge tube with the supernatant removed was placed on a magnetic rack to dry; (3) Add sterile double distilled water to the No. 2 centrifuge tube dried in (2), shake and mix well, let stand, transfer the supernatant to the No. 3 centrifuge tube, and then perform vacuum centrifugation concentration and drying to obtain high-purity fluorescent labeled oligosaccharides.

9. The method for separating and purifying fluorescently labeled oligosaccharides according to claim 8, characterized in that: In step (1), the rotation speed of the constant temperature oscillator is 500-1000 rpm; the volume ratio of the fluorescent labeled oligosaccharide gel strip to 1 mmol / L EDTA (PH=8.0) buffer is 1:(1-2).

10. The method for separating and purifying fluorescently labeled oligosaccharides according to claim 8, characterized in that: In step (2), the equilibration time of the silicon hydroxy magnetic beads is 25 to 35 minutes; the volume ratio of the silicon hydroxy magnetic beads to 95% acetonitrile is 1:(15 to 20); and the storage temperature of the high-purity fluorescently labeled oligosaccharide is -15 to -25°C.

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