N-carbohydrate chain detection method based on in-gel digestion and electrophoresis technology and application of N-carbohydrate chain detection method

Abstract

The invention discloses an N-carbohydrate chain detection method based on an in-gel enzyme digestion and electrophoresis technology and application. The method comprises the following steps: separating a glycoprotein sample through gel electrophoresis and cutting a target strip; carrying out decoloration and dehydration treatment on the target glycoprotein colloidal particles; adding an enzyme solution, and directly performing enzymolysis in the gel to release an N-carbohydrate chain; after ultrasonic treatment, carrying out transferase liquid cutting, drying and marking an N-carbohydrate chain sample; and finally, carrying out separation, detection and analysis by utilizing capillary electrophoresis. According to the method, the in-gel enzyme digestion and the capillary electrophoresis technology are creatively combined, the complex sample transfer and pretreatment steps in a traditional method are avoided, the operation process is remarkably simplified, the sample loss is reduced, and the method is particularly suitable for efficient detection of the low-abundance N-carbohydrate chain. The method has the characteristics of high sensitivity, high resolution and high throughput, can accurately analyze the N-carbohydrate chain in a complex biological sample, and has wide application in disease marker screening, clinical diagnosis and glycomics research.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an N-sugar chain detection method and application based on in-gel enzyme cutting and electrophoresis technology. Background Art

[0002] Glycosylation is one of the important post-translational modifications of proteins, among which N-glycosylation plays a key role in a variety of biological processes and disease development. The sugar chains modified by N-glycosylation have diverse structures and can affect the stability, function, folding and intercellular signaling of proteins. Abnormal N-glycosylation patterns are often closely related to diseases, especially in the research of cancer, neurodegenerative diseases, immune diseases, etc., N-glycans are considered to be potential biomarkers. Therefore, the efficient detection and analysis of N-glycans is of great significance for revealing disease mechanisms, discovering diagnostic markers and developing therapeutic methods.

[0003] At present, the commonly used methods for N-glycan detection mainly include mass spectrometry (MS), liquid chromatography (LC) and capillary electrophoresis (CE). MS has become the main tool for N-glycan analysis due to its high sensitivity and high resolution, and can accurately identify the composition and structure of glycans. However, MS usually requires more complicated sample pretreatment steps, such as the release, purification and derivatization of glycans. Especially for glycoproteins in complex biological samples (such as serum and cell lysates), specific glycoproteins or glycopeptides need to be enriched first. In this process, the sample loss is large, the operation steps are cumbersome, and the analysis efficiency is reduced, especially for the detection of N-glycans of low-abundance glycosylated proteins.

[0004] In order to simplify the process of N-glycan detection, gel electrophoresis separation technology (such as SDS-PAGE) has become one of the preliminary separation steps for N-glycan detection. Through SDS-PAGE, proteins can be separated according to molecular weight and visualized in the gel. This process can not only effectively reduce the complexity of the sample, but also separate and enrich specific target proteins, providing a basis for subsequent N-glycan analysis. However, despite the wide application of SDS-PAGE in protein-related analysis, the technology for directly detecting N-glycans from gels is not yet mature. How to efficiently release and detect N-glycans directly from the gel after protein separation has become a key problem in glycomics research.

[0005] In-gel digestion is a technique widely used in proteomics research. It digests proteins in gel to generate peptide fragments for subsequent mass spectrometry analysis. This technique can avoid the difficulty of protein re-dissolution in solution digestion and is particularly suitable for the analysis of hydrophobic proteins. However, although in-gel digestion has been widely used in proteomics, its application in N-glycan detection has not been fully developed. The main technical difficulties are as follows: (1) Complex sample pretreatment: Although in-gel digestion has less difficulty in protein re-dissolution, the separation and detection of N-glycans still face great challenges due to the different distribution of N-glycans in glycoproteins. Existing in-gel digestion techniques require more complex sample pretreatment steps (such as removing protein contaminants, purifying glycans, etc.) when processing glycoproteins, which increases the complexity and time cost of the detection process. (2) Limited detection of low-abundance glycans: Although in-gel digestion has advantages in peptide generation, the detection of low-abundance glycans is still a problem in glycan analysis. Existing methods are not sensitive enough to low-abundance N-glycans, especially in complex biological samples. The extraction and enrichment process of sugar chains is often limited by the sample type and the type of sugar chains, which affects the efficiency of detection. (3) Limitations of mass spectrometry: In-gel enzyme cleavage technology usually needs to be combined with mass spectrometry for analysis. However, mass spectrometry has high requirements for sample pretreatment, especially in sugar chain analysis. The sensitivity and resolution of the mass spectrometer are affected by sample purity, impurities and detection conditions. Therefore, when processing complex glycoprotein samples, existing technologies may cause signal overlap or background noise, affecting the accuracy and efficiency of the analysis.

[0006] Although gel electrophoresis and in-gel digestion techniques have significant advantages in protein analysis, their combination for N-glycan detection still faces some challenges: (1) Complex operation procedures and high risk of sample loss: Traditional in-gel digestion techniques require the extraction of peptide fragments from the gel, transfer to a solution for enzymatic hydrolysis, and then perform glycan analysis. However, such steps increase the complexity of sample processing, and each sample transfer and extraction process will result in a certain amount of sample loss. For complex samples (such as serum, cell lysates, etc.), these steps will significantly affect the recovery rate of glycans, thereby affecting the accuracy and reliability of glycan analysis. (2) Poor technical compatibility, affecting high-throughput analysis: After using gel electrophoresis to separate proteins, additional protein transfer and purification steps are usually required for subsequent glycan analysis. These steps not only increase experimental time and operational difficulty, but also affect the throughput of the technology. In practical applications, especially when a large number of samples need to be processed, the poor compatibility of existing methods makes rapid and high-throughput glycan detection difficult. (3) It is difficult to combine separation and sugar chain analysis: Gel electrophoresis separation and in-gel enzyme digestion are mainly used for protein analysis, while the separation and analysis of sugar chains usually require different technical support. When these two technologies are combined, how to effectively release sugar chains from proteins and ensure that sugar chains can be successfully separated, identified and quantitatively analyzed is still a technical difficulty. Existing technologies often face problems with separation efficiency and accuracy of sugar chain quantitative analysis, especially when there are many types of sugar chains and large differences in molecular weight.

[0007] In summary, existing N-glycan detection technologies have problems such as complex sample processing, low efficiency, and insufficient sensitivity, especially in the detection of low-abundance glycosylated proteins and the effective release, separation, and identification of sugar chains on in-gel proteins. Therefore, it is of great significance to develop an N-glycan detection method that can simplify the sample pretreatment process and improve detection efficiency and sensitivity. Although gel electrophoresis and in-gel enzyme digestion techniques have been widely used in protein analysis, their application in N-glycan detection still needs further exploration and optimization. Summary of the invention

[0008] In view of the shortcomings of the prior art, the present invention provides an N-sugar chain detection method and application based on in-gel enzyme cleavage and electrophoresis technology, and provides an efficient, sensitive and easy-to-operate N-sugar chain detection method by innovatively combining in-gel enzyme cleavage and electrophoresis technology. This method significantly simplifies the operation process, improves the detection efficiency and accuracy, is particularly suitable for the analysis of complex biological samples, and has a wide range of application potential in disease research and clinical diagnosis.

[0009] The present invention is achieved through the following technical solutions:

[0010] A method for detecting N-glycans based on in-gel digestion and electrophoresis technology comprises the following steps:

[0011] Step 1) separating the glycoprotein sample by gel electrophoresis technology to prepare a gel;

[0012] Step 2) cutting the target glycoprotein band on the gel prepared in step 1) to obtain the target glycoprotein micelles;

[0013] Step 3) placing the target glycoprotein micelles obtained in step 2) in an EP tube, adding a decolorizing solution to decolorize the target glycoprotein micelles, and repeating the decolorization 1 to 3 times until the blue color fades away;

[0014] Step 4) After completely discarding the decolorizing solution in the EP tube, add 100% acetonitrile solution into the EP tube to dehydrate the target glycoprotein micelles;

[0015] Step 5) After completely discarding the 100% acetonitrile solution in the EP tube, add enzyme solution to the EP tube and incubate at 37°C overnight to release the N-sugar chain;

[0016] Step 6) placing the EP tube treated in step 5 in an ultrasonic cleaner, and after ultrasonic treatment, transferring the enzyme digestion solution to a new EP tube, drying and labeling, and preparing an N-sugar chain sample;

[0017] Step 7) separating, detecting and analyzing the N-sugar chain sample prepared in step 6) on a capillary electrophoresis instrument.

[0018] Preferably, the specific steps of preparing the gel in step 1) are as follows: after separating the glycoprotein sample according to molecular weight, placing the gel on a shaker, staining with 2 to 5 mL of Coomassie Brilliant Blue staining solution for at least 2 hours, then decolorizing the stained gel with 2 to 5 mL of decolorizing solution for 30 to 60 minutes, and repeating the decolorization 3 to 4 times until the protein bands are clearly displayed.

[0019] Preferably, in step 2), after the target glycoprotein band is cut, it needs to be cut again into 1-2 mm 2 The pellets were transferred into EP tubes and stored at 4°C.

[0020] Preferably, the specific steps of the decolorization in step 3) are as follows: add 100-300 μL of decolorization solution to the EP tube containing the target glycoprotein micelles, place it in a constant temperature mixer at 800 rpm and 37°C for decolorization for 30-60 minutes, discard the liquid, and repeat 1-3 times until the blue color fades away.

[0021] Preferably, the specific steps of the dehydration treatment in step 4) are as follows: add 100-300 μL of 100% acetonitrile solution into the EP tube containing the target glycoprotein micelles, shake at 37° C. for about 10-30 minutes, wait until the micelles are dehydrated and turn white, discard the liquid, and evaporate the remaining acetonitrile solution.

[0022] Preferably, the specific steps of incubation in step 5) are as follows: add 100-300 μL of enzyme solution to the EP tube containing the target glycoprotein micelles, incubate at 37° C. for 30-60 min, and if the micelles cannot be completely immersed at this time, add 25 mM NH 4 HCO 3 The solution was made to completely immerse the particles and incubated at 37°C overnight.

[0023] Preferably, the enzyme solution is formulated as follows: PNGase F enzyme and sialidase are dissolved in 25 mM NH 4 HCO 3 The solution has a final concentration of PNGase F of 1000-1500 U / mL and a final concentration of sialidase of 100-150 U / mL.

[0024] Preferably, the specific steps of preparing the N-sugar chain sample in step 6) are as follows: placing the EP tube after overnight enzyme digestion in an ultrasonic cleaner, ultrasonically treating for 30 to 60 minutes, vortexing for 10 to 30 seconds, centrifuging for 10 to 30 seconds, and repeating the operation twice; transferring all the liquid in the ultrasonically treated EP tube to a new EP tube, centrifuging for 10 to 30 seconds, drying at 80°C with the lid open for 1 to 2 hours until completely dry, and then cooling to 4°C; then adding 10 to 20 μL of the dried EP tube Add purified water, repeatedly blow around the inner wall, vortex for 1-2 minutes, centrifuge for 10-30 seconds, dry at 80℃ with the cover open for 30-60 minutes until completely dry, and then cool to 4℃; continue to add 2-5μL fluorescent marker solution to the dried EP tube, cover the cap, number it, centrifuge for 10-30 seconds, place it at 90℃ for 1-2 hours, cool to 4℃, add 10-30μL stop solution, vortex for 10-30 seconds to mix thoroughly, and centrifuge for 10-30 seconds to obtain the N-sugar chain sample.

[0025] Preferably, in step 7), the amount of the N-sugar chain sample is 5-10 μL, and the model of the capillary electrophoresis instrument is ABI 3500dx gene sequencer.

[0026] Application of the above-mentioned N-sugar chain detection method based on in-gel enzyme cleavage and electrophoresis technology in the preparation of an N-sugar chain detection kit.

[0027] The beneficial effects of the present invention are as follows:

[0028] (1) The present invention innovatively performs N-glycan cleavage reaction directly in the gel and ensures that the effectively released N-glycans can be smoothly separated, identified and quantitatively analyzed, while avoiding the frequent sample transfer and complicated pretreatment steps in traditional methods. This integrated technology significantly simplifies the operation process and reduces sample loss, especially for the detection of low-abundance N-glycans. By combining the enzymatic cleavage reaction with gel electrophoresis separation, the present invention realizes an integrated operation from sample separation to sugar chain release, greatly improving the detection efficiency.

[0029] (2) The present invention combines in-gel digestion and capillary electrophoresis (CE) technology to achieve efficient separation and sensitive detection of N-glycans. CE technology, with its advantages of high resolution and low sample consumption, can accurately distinguish N-glycans of different structures. In addition, by introducing fluorescent labeling, the sensitivity of detection is further improved, making the analysis of low-abundance glycans more accurate. This combination of high sensitivity and high resolution significantly improves the accuracy of N-glycan detection.

[0030] (3) The detection method of the present invention is particularly suitable for the detection of N-glycans in complex biological samples (such as serum, tissue samples, etc.). Through gel electrophoresis technology, the target glycoprotein can be effectively enriched and the N-glycans can be released, thereby achieving highly selective and highly sensitive detection in complex samples. This technology provides a powerful tool for the screening of disease markers, clinical diagnosis, and glycomics research, and has a wide range of application potential.

[0031] (4) Traditional N-glycan detection methods are prone to sample loss during sample pretreatment and transfer, especially for the detection of low-abundance sugar chains. The present invention avoids the sample transfer step by directly performing the enzyme cleavage reaction in the gel, significantly reduces sample loss, and improves the recovery rate of sugar chains. This improvement is particularly suitable for the detection of low-abundance sugar chains and provides more reliable technical support for related research.

[0032] (5) The detection method of the present invention has the characteristics of high throughput and high compatibility, can process multiple samples at the same time, and is suitable for large-scale glycomics research. In addition, the method is compatible with existing mass spectrometry analysis technology and can be further combined with mass spectrometry for accurate identification of sugar chain structure, providing a more comprehensive solution for glycomics research.

[0033] (6) Since the present invention can efficiently and sensitively detect N-glycans in complex biological samples, it has important application value in the screening of disease markers and clinical diagnosis. Abnormal N-glycosylation patterns are closely related to a variety of diseases (such as cancer, neurodegenerative diseases, immune diseases, etc.), and the technology of the present invention provides a new research tool for the early diagnosis and treatment of these diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 4 bands with different molecular weights in SDS-PAGE gel electrophoresis in Example 1;

[0035] Figure 2 The N-glycan maps corresponding to the serum in the range of 35-100 KDa (A) and four different molecular weight bands (B, C, D, E) in Example 1. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] Unless otherwise specified, the technical means used in the following examples are all conventional means well known to those skilled in the art, and the experimental methods without specifying the specific conditions are all conventional methods in the art.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] Example 1

[0040] A method for detecting N-glycans based on in-gel digestion and electrophoresis technology, by which the N-glycan profile in human serum is detected, with four different molecular weight bands in the range of 35 to 100 KDa being detected, and the specific steps are as follows:

[0041] (1) After separating the human serum samples according to molecular weight by gel electrophoresis (SDS-PAGE) technology, place the gel on a shaker and stain it with 2-5 mL of Coomassie Brilliant Blue staining solution for at least 2 hours. Then use 2-5 mL of decolorizing solution A (water: anhydrous ethanol: glacial acetic acid = (25-50): (20-40): (5-10), volume ratio) to decolorize the stained gel for 30-60 minutes. Repeat the decolorization 3-4 times until the protein bands are clearly displayed. Figure 1 Shown is a destained human serum protein gel showing four bands of different molecular weights.

[0042] (2) After the target glycoprotein band is cut from the gel, it needs to be cut again into 1-2 mm 2 Transfer the pellets to EP tubes and store at 4°C.

[0043] (3) Add 100-300 μL of decolorizing solution B (100% ACN: 50-100 mM NH 4 HCO 3=1:1, volume ratio) was added to the EP tube containing the target glycoprotein micelles, placed in a constant temperature mixer, decolorized at 800 rpm and 37°C for 30-60 min, the liquid was discarded, and the process was repeated 1-3 times until the blue color faded away.

[0044] (4) Add 100-300 μL of 100% acetonitrile (ACN) solution to the EP tube containing the target glycoprotein micelles, shake at 37°C for about 10-30 min, wait until the micelles are dehydrated and turn white, discard the liquid, and evaporate the remaining ACN (it can be placed in a constant temperature incubator for appropriate heating).

[0045] (5) Add 100-300 μL of enzyme solution to the EP tube containing the target glycoprotein particles and incubate at 37°C for 30-60 min. If the particles cannot be completely immersed at this time, add 25 mM NH 4 HCO 3 The solution was made to completely immerse the particles and incubated at 37°C overnight.

[0046] The enzyme solution was formulated as follows: PNGase F (final concentration 1000-1500 U / mL) and sialidase (final concentration 100-150 U / mL) were dissolved in 25 mM NH 4 HCO 3 Solution.

[0047] (6) Place the EP tube after overnight enzyme digestion in an ultrasonic cleaner, ultrasonically treat for 30 to 60 minutes, vortex and oscillate for 10 to 30 seconds, centrifuge for 10 to 30 seconds, and repeat this operation twice; transfer all the liquid in the ultrasonically treated EP tube to a new EP tube, centrifuge for 10 to 30 seconds, dry at 80°C with the lid open for 1 to 2 hours until completely dry, and then cool to 4°C; then add 10 to 20 μL of purified water to the dried EP tube, repeatedly blow around the inner wall, and vortex for 1 to 2 minutes after blowing is complete. , centrifuge for 10-30s, open the cover and dry at 80℃ for 30-60min until completely dry, then cool to 4℃; continue to add 2-5μL fluorescent labeling solution (5-10mM8-aminopyrene-1,3,6-trisulfonic acid) to the dried EP tube, cover the cap, number it, centrifuge for 10-30s, place it at 90℃ for 1-2h, cool to 4℃, add 10-30μL stop solution (ultrapure water), vortex oscillation for 10-30s to mix thoroughly, centrifuge for 10-30s to prepare N-sugar chain samples.

[0048] (7) 5 to 10 μL of the obtained sugar chain sample was placed in an ABI dedicated 96-well plate and then detected using an ABI 3500dx sequencer.

[0049] like Figure 2As shown, the sugar chains include P1 (NGA2F), P2 (NGA2FB), P3 (NG1A2F-1), P4 (NG1A2F-2), P5 (NA2), P6 (NA2F), P7 (NA2FB), P8 (NA3), P9 (NA3Fb), P10 (NA4) and P11 (NA4Fb), among which P3 (NG1A2F-1) and P4 (NG1A2F-2) are isomers, and the peak height represents the relative content of the sugar chains.

[0050] Through Figure 2 After analysis, the following conclusions can be drawn: (1) Clarifying the distribution characteristics of sugar chains in human serum: The overall sugar chains were classified by bands, and the proportion of each sugar chain in different molecular weight bands was further clarified. Taking P9 as an example, its peak proportions in bands 1, 2, 3, and 4 were 5.4%, 7.4%, 23.3%, and 52.4%, respectively. This result shows that P9 in human serum mainly comes from the proteins in bands 3 and 4, which can provide a basis for subsequent sugar chain tracing. (2) Detection and enrichment of low-abundance sugar chains: Taking P11 (NA4Fb) as an example, its abundance in total serum is low, with a peak value of only 159. However, after further purification and enrichment of the sugar chains in band 4 by the detection method of the present invention, the peak value of P11 in the band 4 spectrum was significantly increased to 1217, and the peak signal was enhanced by about 7 times.

[0051] The above results fully demonstrate the high sensitivity and high resolution advantages of the present invention in the detection of low-abundance sugar chains. The present invention successfully achieved high-sensitivity and high-resolution analysis of multiple sugar chains in human serum samples, especially the significant enrichment and detection of low-abundance sugar chains (such as P11), which fully demonstrated the technical advantages of the present invention. These results provide important experimental basis for glycosylation research and lay a solid foundation for subsequent sugar chain function research and clinical application.

[0052] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope required by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

Claims

1. A method for detecting N-glycans based on in-gel digestion and electrophoresis, characterized in that: The following steps are involved: Step 1) separating the glycoprotein sample by gel electrophoresis technology to prepare a gel; Step 2) cutting the target glycoprotein band on the gel prepared in step 1) to obtain the target glycoprotein micelles; Step 3) placing the target glycoprotein micelles obtained in step 2) in an EP tube, adding a decolorizing solution to decolorize the target glycoprotein micelles, and repeating the decolorization 1 to 3 times until the blue color fades away; Step 4) After completely discarding the decolorizing solution in the EP tube, add 100% acetonitrile solution into the EP tube to dehydrate the target glycoprotein micelles; Step 5) After completely discarding the 100% acetonitrile solution in the EP tube, add enzyme solution to the EP tube and incubate at 37°C overnight to release the N-sugar chain; Step 6) placing the EP tube treated in step 5 in an ultrasonic cleaner, and after ultrasonic treatment, transferring the enzyme digestion solution to a new EP tube, drying and labeling, to prepare an N-sugar chain sample; Step 7) separating, detecting and analyzing the N-sugar chain sample prepared in step 6) on a capillary electrophoresis instrument.

2. The method for detecting N-sugar chains based on in-gel digestion and electrophoresis according to claim 1, characterized in that: The specific steps of preparing the gel in step 1) are as follows: after separating the glycoprotein sample according to molecular weight, the gel is placed on a shaker and stained with 2 to 5 mL of Coomassie Brilliant Blue staining solution for at least 2 hours, and then the stained gel is destained with 2 to 5 mL of decolorizing solution for 30 to 60 minutes, and the decolorization is repeated 3 to 4 times until the protein bands are clearly displayed.

3. The method for detecting N-sugar chains based on in-gel digestion and electrophoresis according to claim 1, characterized in that: In step 2), after the target glycoprotein band is cut, it needs to be cut again into 1-2 mm 2 The pellets were transferred into EP tubes and stored at 4°C.

4. The method for detecting N-sugar chains based on in-gel digestion and electrophoresis according to claim 1, characterized in that: Step 3) The specific steps of the decolorization are as follows: add 100-300 μL of decolorization solution to the EP tube containing the target glycoprotein micelles, place it in a constant temperature mixer at 800 rpm and 37°C for decolorization for 30-60 minutes, discard the liquid, and repeat 1-3 times until the blue color fades away.

5. The method for detecting N-sugar chains based on in-gel digestion and electrophoresis according to claim 1, characterized in that: Step 4) The specific steps of the dehydration treatment are as follows: add 100-300 μL of 100% acetonitrile solution into the EP tube containing the target glycoprotein micelles, shake at 37° C. for about 10-30 minutes, wait until the micelles are dehydrated and turn white, discard the liquid, and evaporate the remaining acetonitrile solution.

6. The method for detecting N-sugar chains based on in-gel digestion and electrophoresis according to claim 1, characterized in that: Step 5) The specific steps of the incubation are as follows: add 100-300 μL of enzyme solution to the EP tube containing the target glycoprotein micelles, incubate at 37°C for 30-60 min. If the micelles cannot be completely immersed at this time, add 25 mM NH4HCO3 solution to completely immerse the micelles, and incubate at 37°C overnight.

7. The method for detecting N-sugar chains based on in-gel digestion and electrophoresis according to claim 6, characterized in that: The formula of the enzyme solution is as follows: PNGase F enzyme and sialidase are dissolved in 25mM NH4HCO3 solution, the final concentration of the PNGase F enzyme is 1000-1500U / mL, and the final concentration of the sialidase is 100-150U / mL.

8. The method for detecting N-sugar chains based on in-gel digestion and electrophoresis technology according to claim 1, characterized in that: Step 6) The specific steps of preparing the N-sugar chain sample are as follows: place the EP tube after overnight enzyme digestion in an ultrasonic cleaner, ultrasonically treat for 30 to 60 minutes, vortex oscillate for 10 to 30 seconds, centrifuge for 10 to 30 seconds, and repeat the operation twice; transfer all the liquid in the ultrasonically treated EP tube to a new EP tube, centrifuge for 10 to 30 seconds, dry at 80°C with the lid open for 1 to 2 hours until completely dry, and then cool to 4°C; then add 10 to 20 μL of purified Add water, repeatedly blow around the inner wall, vortex for 1-2 minutes, centrifuge for 10-30 seconds, dry at 80℃ with the cover open for 30-60 minutes until completely dry, and then cool to 4℃; continue to add 2-5μL fluorescent marker solution to the dried EP tube, cover the cap, number it, centrifuge for 10-30 seconds, place it at 90℃ for 1-2 hours, cool to 4℃, add 10-30μL stop solution, vortex for 10-30 seconds to mix thoroughly, and centrifuge for 10-30 seconds to obtain the N-sugar chain sample.

9. The method for detecting N-sugar chains based on in-gel digestion and electrophoresis according to claim 1, characterized in that: Step 7) The amount of the N-sugar chain sample used is 5-10 μL, and the model of the capillary electrophoresis instrument is ABI 3500dx gene sequencer.

10. Use of the N-sugar chain detection method based on in-gel digestion and electrophoresis technology as claimed in any one of claims 1 to 9 in the preparation of an N-sugar chain detection kit.

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