N-glycan analysis kit based on capillary electrophoresis

Abstract

The invention provides a pre-treatment kit for quantitative analysis of glycoprotein N-glycan, which can be used for sample pre-treatment of glycosylation analysis based on capillary electrophoresis on a biological sample. According to the reagent combination provided by the kit, N-glycan can be separated from glycoprotein of a biological sample to be detected in a non-fixed mode, the separated N-glycan is marked and purified, and the treated sample can be directly used for glycosyl quantitative analysis based on capillary electrophoresis. Therefore, the invention is helpful for obtaining sensitive and reliable capillary electrophoresis detection signals of N-glycans, improving the analysis flux and reducing the test cost.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a pretreatment kit for protein N-glycan analysis in biological samples, which is used for N-glycan detection based on capillary electrophoresis. Background Art

[0002] Glycosylation is one of the ubiquitous post-translational modifications of proteins. It is the process in which polysaccharides (or oligosaccharides or sugar groups) are attached to specific sites of proteins under the action of enzymes. More than half of eukaryotic organisms undergo protein glycosylation, and almost all membrane proteins and secretory proteins are involved in glycosylation in their physiological functions. Protein glycosylation affects the structure of the protein, thereby determining the function of the protein, and abnormal glycosylation is closely related to the pathological process of the disease. Studies have shown that N-glycosylation of proteins plays an important regulatory role in the occurrence and development of various diseases such as inflammatory diseases, autoimmune diseases, tumors and metabolic diseases; and affects the susceptibility of viruses to human infection and clinical outcomes. By detecting the glycosylation characteristics of human-related proteins, it can be used as a biomarker for disease diagnosis and prognosis evaluation, and as an effective target for disease prevention and treatment, with broad and far-reaching application prospects.

[0003] Due to the great complexity of glycans in their structure (straight chain or branched chain), the charge (from neutral to negatively charged), polarity, and sensitivity to pH and temperature, the analysis of different glycans is very challenging. The current methods for glycan analysis mainly include mass spectrometry, liquid chromatography, and capillary electrophoresis. The sugar analysis test method based on capillary electrophoresis has the advantages of high precision, high throughput, low cost, and low sample loss, and will become one of the main methods for sugar sequencing in the foreseeable future. The development of sample pretreatment kits based on capillary electrophoresis is particularly important for improving sugar analysis technology. The currently published patents and publicly published sample processing steps can be summarized as follows: release oligosaccharide chains by chemical means or enzymatic cleavage; attach ultraviolet or fluorescent groups to sugar chains by derivatization; use sialidase to remove sialic acid from the oligosaccharides to be tested; and detect on the machine.

[0004] However, the above methods have the following defects: (1) Sialic acid in the sugar chain to be tested is removed during sample pretreatment, so the sugar chain structure in the test result cannot reflect the sugar modification structure in the actual sample to be tested. In addition, sialic acid in glycoproteins is related to the disease susceptibility of humans and other primates, and has attracted much attention in evolutionary medicine and cancer treatment. (2) The sensitivity of existing methods is low. In the published patents, the sensitivity of the test results is low, such as: ≤12 sugar chains detected. (3) The published detection methods do not have clear repeatability data. Summary of the invention

[0005] The present invention provides a pretreatment kit for quantitative analysis of N-glycans in biological samples, which is used for pretreatment of quantitative analysis of protein N-glycans, helps to obtain sensitive and reliable capillary electrophoresis detection signals of N-glycans, improves the analysis throughput and reduces the test cost. It includes the following components:

[0006] (1) Denaturation buffer: protein denaturing reagent;

[0007] (2) Enzymatic reaction solution: a reagent for hydrolyzing and separating N-glycans from glycoproteins;

[0008] (3) Fluorescent labeling solutions A and B: labeling solutions for labeling glycans;

[0009] (4) Purification reagents: reagents and materials for purifying polysaccharides.

[0010] It can be used to test samples containing glycoproteins, such as serum, plasma, aqueous humor, and other human tissue protein grinding fluids.

[0011] Specifically, the protein denaturing reagent is a denaturing solution containing sodium dodecyl sulfate SDS and NH4HCO3; the reagent for hydrolyzing and separating N-glycans from glycoproteins is a separation solution containing polypeptide N-glycosidase F; the fluorescent labeling solution A is a labeling solution containing 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt APTS, and the fluorescent labeling solution B is a labeling solution containing NaCNBH 3 The reagents for purifying polysaccharides are magnetic beads and acetonitrile washing solution.

[0012] Preferably, the denaturation buffer contains 5-15 mM NH 4 HCO 3 1-3% SDS solution, pH 8.0-8.6; preferably: the denaturing buffer contains 10mM NH 4 HCO 3 2% SDS solution, pH 8.3;

[0013] Specifically, the enzymatic hydrolysis reaction solution comprises 10-30 units of PNGase F and 5-15% NP-40 in a PBS solution; preferably, the enzymatic hydrolysis reaction solution comprises 20 units of PNGase F and 10% NP-40 in a PBS solution;

[0014] In a specific embodiment, the fluorescent labeling liquid A is a 1.0-1.4M citric acid solution containing 10-30mM APTS; the fluorescent labeling liquid B is a 0.5-1.5M NaCNBH 3Preferably, the fluorescent labeling solution A is a 1.2M citric acid solution containing 20mM APTS; the fluorescent labeling solution B is a 1M NaCNBH 3 of DMSO solution.

[0015] In a specific embodiment, the reagents for purifying polysaccharides are silanol-coated magnetic beads, 100% acetonitrile and 96% acetonitrile washing solution.

[0016] The present invention provides a method for quantitatively analyzing N-glycans derived from glycoproteins in a biological sample based on capillary electrophoresis, which comprises the following steps:

[0017] S1 Protein denaturation: Take the sample to be tested and add it to the PCR tube, add the denaturation buffer to mix, and centrifuge for 5-10 seconds; heat the PCR tube in the PCR instrument at 60℃ for 10 minutes, place it at room temperature, and then centrifuge it at low speed for 5-10 seconds to ensure that the small droplets condensed on the tube wall enter the solution at the bottom of the tube;

[0018] S2 enzymatic polysaccharide decomposition: add enzymatic reaction solution, mix well, incubate at 37℃ for 3h, and place at 4℃ for 15min to terminate the reaction;

[0019] S3 fluorescence labeling: centrifuge the incubated sample PCR tube, add fluorescent labeling solution A and fluorescent labeling solution B, and heat at 37°C for 16 hours to label; after labeling, add ultrapure water to each PCR tube to terminate the reaction;

[0020] S4 glycan purification: add magnetic bead suspension to a centrifuge tube and then add the labeled sample; add acetonitrile, mix well and place on a magnetic rack, let stand and discard the supernatant; add washing solution, mix well and place on a magnetic rack, let stand and discard the supernatant; optionally, repeat this step three times and air dry;

[0021] S5 performs quantitative analysis of N-glycans.

[0022] Specifically, the sample to be tested in S1 is not less than 40ug, and is selected from serum, plasma, aqueous humor of the eyeball, and other human tissue protein grinding fluid;

[0023] Add magnetic bead suspension to S4 and then add the labeled sample to the centrifuge tube, add 100% acetonitrile, mix well and place on the magnetic rack, let stand for 1 min, and discard the supernatant; add 96% acetonitrile washing solution, mix well and place on the magnetic rack, let stand for 1 min, and discard the supernatant; repeat this step three times and dry; add water to the purified sample, mix well and place on the magnetic rack, let stand for 1 min, and take the supernatant to a new centrifuge tube;

[0024] S5 uses a capillary electrophoresis instrument for detection: the labeled solution is added to the reaction plate and then the HD formamide mixture is added, capillary electrophoresis is performed on the glycomic analyzer, and the detected results are analyzed using analysis software to obtain a spectrum with the horizontal axis being the peak time and the vertical axis being the fluorescence intensity, and the N-glycan is quantitatively analyzed.

[0025] The reagent combination provided by the kit provided by the present invention can separate N-glycans from the glycoproteins of the biological sample to be tested in a non-fixed manner, and label and purify the separated N-glycans. The samples processed as above can be directly used for quantitative analysis of sugar groups based on capillary electrophoresis. Therefore, the present invention helps to obtain sensitive and reliable capillary electrophoresis detection signals of N-glycans, improve the throughput of analysis and reduce the cost of testing.

[0026] The experimental method of the present invention has the following advantages: (1) the sugar structure containing sialic acid is retained, and various types of sialic acid sugar chain structures can be detected, enriching the diversity of N-sugar structures; (2) the operation steps are simplified and the use of sialidase is reduced, thereby reducing the reagent cost; (3) magnetic bead purification is introduced to facilitate the realization of high-throughput automated solutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Shown is the capillary electrophoresis profile of human serum N-glycans.

[0028] Figure 2 Shows the results of the repeatability experiment.

[0029] Figure 3 Shows the results of the repeatability experiment for Days 1-3. DETAILED DESCRIPTION

[0030] The present invention is described below through specific implementation modes in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0031] Embodiment 1

[0032] (1) Protein denaturation:

[0033] The reagent is a denaturing buffer containing 10 mM NH 4 HCO 3 2% SDS solution, pH 8.3. The reagent formula is:

[0034] Reagent name Required amount 2% SDS 10ml <![CDATA[10mM NH 4 HCO 3 ]]> 0.79g NaOH Adjust to pH 8.3 with NaOH

[0035] Take 2 μL of the test serum (40ug) and add it to the PCR tube, add 4 μL of denaturing buffer and blow it 10 times with a gun to mix, and centrifuge for 5-10 seconds; place the PCR tube in the PCR instrument and heat it at 60℃ for 10 minutes, place it at room temperature and then centrifuge it at a low speed for 5-10 seconds to ensure that the small droplets condensed on the tube wall enter the solution at the bottom of the tube.

[0036] (2) Enzymatic decomposition of polysaccharides:

[0037] The reagent is an enzymatic reaction solution, containing PNGase F and 10% NP-40. The reagent formula is:

[0038]

[0039] Add 4 μl of enzymatic reaction solution to the PCR tube in step (1), pipette 10 times to mix, incubate at 37°C for 3 h, and place at 4°C for 15 min to terminate the reaction.

[0040] (3) Fluorescent labeling:

[0041] The reagents are: 1) Fluorescent labeling solution A, containing 20 mM APTS in 1.2 M citric acid solution; 2) Fluorescent labeling solution B, containing 1 M NaCNBH 3 of DMSO solution.

[0042] The formula of fluorescent labeling solution A reagent is:

[0043] Reagent name Required amount APTS 1mg 1.2M citric acid solution 95.6μl

[0044] The formula of fluorescent labeling solution B reagent is:

[0045] Reagent name Required amount <![CDATA[NaCNBH 3 ]]> 0.05g DMSO solution 796μl

[0046] Centrifuge the incubated sample PCR tube, take 2μl of the solution and put it into a new PCR tube, then add 2μl of fluorescent labeling solution A and 2μl of fluorescent labeling solution B, and heat at 37℃ for 16 hours to label. After labeling, add 50μL of ultrapure water to each PCR tube to terminate the reaction.

[0047] (4) Polysaccharide purification

[0048] a. Take out the silanol-coated magnetic beads purification reagent in advance and equilibrate it at room temperature for 10 minutes. Vortex mix before use.

[0049] b. Add 5 μl of magnetic bead suspension to the centrifuge tube and then add 10 μl of labeled sample.

[0050] c. Add 180 μl of 100% acetonitrile, mix well, place on a magnetic rack, let stand for 1 min, and discard the supernatant.

[0051] d. Add 100 μl of washing solution (96% acetonitrile), mix well, place on the magnetic rack, let stand for 1 min, discard the supernatant. Repeat this step three times and let dry (about 10 min).

[0052] e. Add 25 μl of water to the purified sample, mix well and place on a magnetic rack, let stand for 1 min, and take the supernatant to a new centrifuge tube.

[0053] (5) Capillary electrophoresis detection: Add 2 μl of labeled solution and then 8 μl of HD formamide mixture (OR600LIZ 1 μl + HD formamide 160 μl, the amount of OR600LIZ can be adjusted appropriately according to the sample situation) to a 96-well reaction plate, and perform capillary electrophoresis on a glycomic analyzer. The electrophoresis conditions are as follows: constant temperature 60°C; pre-electrophoresis voltage 15 kV; pre-electrophoresis time 180 sec; sample loading voltage 2-6 kV (depending on the sample concentration); sample loading time 18 sec; electrophoresis voltage 15 kV; electrophoresis time 2200 sec.

[0054] Serum N-glycans subjected to capillary electrophoresis can obtain an N-glycan electrophoresis spectrum. The coordinates of the spectrum can be adjusted and saved using software, and the height value corresponding to the peak can be obtained.

[0055] (6) Result analysis: The glycans were qualitatively and quantitatively analyzed using standard products, and the detected results were analyzed using analysis software to obtain a graph with the peak time as the abscissa and the fluorescence intensity as the ordinate ( Figure 1 ). In the comparative analysis of these glycan profiles, the detected sugar peaks can be numbered one by one according to the standard that the sugar peak elution time is consistent with that of the sugar standard, and the structure of the sugar to be tested can be determined. Among them, the following Table 1 represents the chemical structure corresponding to each sugar peak in the sugar test map.

[0056] Table 1N Sugar structure list

[0057]

[0058] Example 2: Repeatability Analysis

[0059] For the same sample in Example 1, the measurement was repeated 3 times, and the results did not change ( Figure 2 , Table 2) shows that the method of the present invention has strong repeatability.

[0060] Table 2 Repeatability experimental data

[0061]

[0062]

[0063] Embodiment 2: Reliability analysis.

[0064] The same sample in Example 1 was placed at 4°C and tested again after 3 days. There was no significant change in the results (see Figure 3 , Table 3), indicating that the method of the present invention has strong reliability.

[0065] Table 3 Results of repeatability experiment on days 1-3

[0066] Peak number Day 1 the next day Day 3 CV 1 1.11％ 1.20％ 1.16％ 3.77％ 3 3.40％ 3.33％ 3.34％ 1.11％ 4 26.87％ 27.77％ 27.51％ 1.69％ 6 2.26％ 2.20％ 2.21％ 1.41％ 7 4.90％ 5.01％ 4.97％ 1.18％ 9 2.01％ 2.07％ 2.09％ 2.21％ 15 0.79％ 0.78％ 0.78％ 0.62％ 16 0.34％ 0.35％ 0.34％ 1.77％ 17 1.26％ 1.32％ 1.30％ 2.04％ 19 9.92％ 9.94％ 9.89％ 0.25％ 20 8.26％ 8.16％ 8.12％ 0.88％ 21 2.09％ 2.08％ 2.11％ 0.77％ 22 0.51％ 0.49％ 0.50％ 2.15％ 24 9.21％ 9.23％ 9.24％ 0.18％ 25 1.47％ 1.46％ 1.46％ 0.08％ 26 9.03％ 8.77％ 8.76％ 1.72％ 27 3.77％ 3.59％ 3.66％ 2.35％ 28 2.35％ 2.20％ 2.30％ 3.23％ 29 0.59％ 0.56％ 0.61％ 3.98％ 30 9.07％ 8.68％ 8.76％ 2.35％ 31 0.80％ 0.80％ 0.84％ 3.14％ .

Claims

1. A pre-treatment kit for quantitative analysis of N-glycans derived from glycoproteins in biological samples based on capillary electrophoresis, comprising: (1) Denaturation buffer: contains protein denaturing reagent; (2) Enzymatic reaction solution: a reagent for hydrolyzing and separating N-glycans from glycoproteins; (3) Fluorescent labeling solutions A and B: Labeling solutions for labeling glycans; (4) Purification reagents: reagents for purifying polysaccharides.

2. The kit according to claim 1, characterized in that The protein denaturing reagent is a denaturing solution containing sodium dodecyl sulfate SDS and NH4HCO3; the reagent for hydrolyzing and separating N-glycans from glycoproteins is a separation solution containing polypeptide N-glycosidase F; the fluorescent labeling solution A is a labeling solution containing 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt APTS, and the fluorescent labeling solution B is a labeling solution containing NaCNBH3; the reagents for purifying polysaccharides are magnetic beads and acetonitrile washing solution.

3. The kit according to claim 1, characterized in that The denaturation buffer is a 1-3% SDS solution containing 5-15 mM NH4HCO3, with a pH of 8.0-8.6; preferably, the denaturation buffer is a 2% SDS solution containing 10 mM NH4HCO3, with a pH of 8.

3.

4. The kit according to claim 1, characterized in that The enzymatic hydrolysis reaction solution comprises a PBS solution containing 10-30 units of PNGase F and 5-15% NP-40; preferably, the enzymatic hydrolysis reaction solution comprises a PBS solution containing 20 units of PNGase F and 10% NP-40.

5. The kit according to claim 1, characterized in that The fluorescent labeling liquid A is a 1.0-1.4 M citric acid solution containing 10-30 mM APTS; the fluorescent labeling liquid B is a DMSO solution containing 0.5-1.5 M NaCNBH3; preferably, the fluorescent labeling liquid A is a 1.2 M citric acid solution containing 20 mM APTS; the fluorescent labeling liquid B is a DMSO solution containing 1 M NaCNBH3.

6. The kit according to claim 1, characterized in that The reagents for purifying polysaccharides are silanol-coated magnetic beads, 100% acetonitrile and 96% acetonitrile washing solution.

7. A method for quantitative analysis of N-glycans derived from glycoproteins in biological samples based on capillary electrophoresis, characterized in that: The sample processing is performed using the kit according to any one of claims 1 to 6, comprising the following steps: S1 Protein denaturation: Take the sample to be tested and add it to a PCR tube, add denaturation buffer to mix, and centrifuge for 5-10 seconds; heat the PCR tube in a PCR instrument at 60°C for 10 minutes, place it at room temperature, and then centrifuge it at a low speed for 5-10 seconds to ensure that the small droplets condensed on the tube wall enter the solution at the bottom of the tube; S2 enzymatic polysaccharide decomposition: add enzymatic reaction solution, mix well, incubate at 37℃ for 3h, and place at 4℃ for 15min to terminate the reaction; S3 fluorescence labeling: centrifuge the incubated sample PCR tube, add fluorescent labeling solution A and fluorescent labeling solution B, and heat at 37°C for 16 hours for labeling; after labeling, add ultrapure water to each PCR tube to terminate the reaction; S4 glycan purification: add magnetic bead suspension to a centrifuge tube and then add the labeled sample; Add acetonitrile, mix well, place on a magnetic rack, let stand, and discard the supernatant; add washing solution, mix well, place on a magnetic rack, let stand, and discard the supernatant; optionally, repeat this step three times and air dry; S5 performs quantitative analysis of N-glycans.

8. The method for quantitative analysis of N-glycans according to claim 7, wherein: The sample to be tested in S1 is no less than 40ug and is selected from serum, plasma, aqueous humor, and other human tissue protein grinding fluid; Add magnetic bead suspension to S4 and then add the labeled sample to the centrifuge tube, add 100% acetonitrile, mix well and place on the magnetic rack, let stand for 1 min, and discard the supernatant; add 96% acetonitrile washing solution, mix well and place on the magnetic rack, let stand for 1 min, and discard the supernatant; repeat this step three times and let it dry; add water to the purified sample, mix well and place on the magnetic rack, let stand for 1 min, and take the supernatant to a new centrifuge tube.

9. The method for quantitative analysis of N-glycans according to claim 7, characterized in that: S5 uses a capillary electrophoresis instrument for detection: the labeled solution is added to the reaction plate and then the HD formamide mixture is added, capillary electrophoresis is performed on the glycomic analyzer, and the detected results are analyzed using analysis software to obtain a spectrum with the horizontal axis being the peak time and the vertical axis being the fluorescence intensity, and the N-glycan is quantitatively analyzed.

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