A method for detecting cell surface n-glycan based on enzymatic digestion and fluorescent labeling
By optimizing the cell surface N-glycan detection method using enzymatic digestion and fluorescent labeling, the problems of sample loss and impurity interference in trace cell samples were solved, achieving efficient and low-cost N-glycan chain detection and improving the sensitivity and accuracy of the detection.
Patent Information
- Application Number
- CN202610248307.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-03-02
- Publication Date
- 2026-06-23
AI Technical Summary
Existing technologies for detecting N-glycan chains in trace cell samples suffer from problems such as large sample loss, low recovery rate, cumbersome operation, and severe interference from impurities, which limit the application of N-glycanomics in clinical diagnosis and basic research.
A cell surface N-glycan detection method based on enzymatic digestion and fluorescent labeling was adopted. The sample processing flow was optimized by tryingpase digestion, PNGase F digestion, magnetic bead purification and fluorescent labeling, which reduced multiple centrifugation and cumbersome operations. A magnetic bead-acetonitrile purification system was used to remove impurities.
It improves the extraction efficiency and sensitivity of the detection, reduces costs, simplifies the operation steps, and ensures the accuracy and signal-to-noise ratio of the detection results.
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Figure CN122256475A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioanalytical technology, and in particular to a method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling. Background Technology
[0002] N-glycomics is an important discipline that studies the structure, function, and dynamic changes of protein N-glycans, and it has significant implications for biomarker discovery, drug development, and basic biological research. The current standard analytical procedure for N-glycans includes steps such as enzymatic release of N-glycans from glycoproteins, purification, fluorescent labeling, separation, and detection. The most commonly used method is to use peptidyl-N-glycosidase F under denaturing conditions to cleave N-glycans from glycoproteins, followed by purification using methods such as graphitized carbon black columns.
[0003] Existing technologies face numerous challenges and limitations when processing trace amounts of cell samples. Large sample volumes are required; many traditional methods necessitate milligram-level protein levels to obtain reliable signals. Operations are cumbersome; classic glycan purification methods involve numerous steps, including multiple transfers and centrifugations, which can easily lead to sample loss. Significant interference from impurities is also present; salts, detergents, nucleic acids, and other impurities in cell lysates can interfere with enzyme digestion efficiency and fluorescent labeling reactions. Low labeling efficiency or poor stability of labeled products can also affect detection sensitivity and result reliability.
[0004] The high sample loss and low recovery rate in the detection of N-glycans in trace cell samples are key bottlenecks restricting the development of this technology. When processing precious samples such as rare cell populations, clinical puncture samples, and stem cell cultures, the multi-step operation of traditional methods leads to significant sample loss during transfer and purification, resulting in low recovery rates and poor reproducibility, which severely limits the application of N-glycanomics in clinical diagnosis and basic research. Summary of the Invention
[0005] Based on this, one or more embodiments of the present invention provide a method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling. This method greatly improves extraction efficiency and detection sensitivity, and is low in cost and simple in procedure.
[0006] The technical solution of the present invention includes the following contents: A method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling includes the following steps: Cells were enzymatically hydrolyzed using trypsin to obtain glycoproteins; The glycoprotein was denatured and then digested with PNGase F to obtain N-glycans; The N-glycan chain was purified to obtain purified N-glycan chain; The purified N-glycan was fluorescently labeled with 8-aminopyrene-1,3,6-trisulfonic acid to obtain fluorescently labeled N-glycans. The fluorescently labeled N-glycan chain was purified to obtain the purified fluorescently labeled N-glycan chain; The purified fluorescently labeled N-glycans were then detected.
[0007] In some embodiments, the cells are washed with PBS and collected by centrifugation before being digested with trypsin.
[0008] Furthermore, the steps of enzymatically digesting cells with trypsin to obtain glycoproteins include: Cells, trypsin, and buffer solution are mixed and processed at a first temperature and a first rotation speed for a first time to obtain an enzymatic hydrolysate. The enzymatic hydrolysate was centrifuged, and the supernatant was collected to obtain the glycoprotein. Optionally, the first temperature is 36.8℃-37.2℃, the first rotation speed is 145rpm-155rpm, and the first time is 14min-16min.
[0009] Further, the step of centrifuging the enzymatic hydrolysate includes: The enzymatic hydrolysate is centrifuged at a second temperature and a second rotation speed for a second time. The second temperature is 3.8℃-4.2℃, the second rotation speed is 14000rpm-16000rpm, and the second time is 14min-16min.
[0010] Further, the steps of denaturing the glycoprotein and then digesting it with PNGase F to obtain N-glycans include: The glycoprotein is heated at a third temperature for a third time to obtain a denatured glycoprotein; Optionally, the third temperature is 98.8℃-99.2℃, and the third time is 4.8min-5.2min; The denatured glycoprotein was digested with PNGase F to obtain the N-glycan.
[0011] Further, the step of digesting the denatured glycoprotein with PNGase F includes: The denatured glycoprotein, PNGase F enzyme, and buffer solution were mixed and treated at a fourth temperature for a fourth time. The fourth temperature is 36.8℃-37.2℃, and the fourth time is 12h-24h.
[0012] Further, the step of purifying the N-glycan chain to obtain purified N-glycan chains includes: The N-glycans, magnetic beads, and acetonitrile were mixed, stirred, and placed on a magnetic rack. The precipitate was collected to obtain the purified N-glycans.
[0013] Further, the purified N-glycan chain is fluorescently labeled with 8-aminopyrene-1,3,6-trisulfonic acid to obtain fluorescently labeled N-glycan chains, comprising the following steps: The purified N-glycan, 8-aminopyrene-1,3,6-trisulfonic acid, and buffer solution were mixed and treated at a fifth temperature for a fifth time to obtain the fluorescently labeled N-glycan. The fifth temperature is 54.8℃-55.2℃, and the fifth time is 50min-70min.
[0014] In some embodiments, the step of purifying the fluorescently labeled N-glycan chain includes: The fluorescently labeled N-glycans, magnetic beads, and acetonitrile were mixed, stirred, and placed on a magnetic rack. The precipitate was collected to obtain the purified fluorescently labeled N-glycans.
[0015] Further, the step of detecting the purified fluorescently labeled N-glycans includes: The purified fluorescently labeled N-glycans, magnetic beads, and water were mixed, stirred, and placed on a magnetic rack. The supernatant was then collected. The supernatant was subjected to capillary electrophoresis analysis and laser-induced fluorescence detection. Optionally, the conditions for the capillary electrophoresis analysis include: oven temperature 29.8℃-30.2℃; pre-electrophoresis voltage 14.58kV-15.2kV; pre-electrophoresis time 178s-182s; sample loading voltage 14.58kV-15.2kV; and electrophoresis time 2250s-2350s.
[0016] The cell surface N-glycan detection method based on enzymatic digestion and fluorescent labeling of the present invention greatly reduces sample loss during transfer and purification by combining optimized trypsin pretreatment with an efficient magnetic bead purification strategy. It adopts a unique "magnetic bead-acetonitrile" purification system, which performs two efficient purification processes after glycan release and after fluorescent labeling, effectively removing various impurities such as protein residues, salts, enzymes, detergents, and unbound fluorescent dyes.
[0017] The cell surface N-glycan detection method based on enzymatic digestion and fluorescent labeling of this invention is based on the core design of "enzyme digestion-magnetic bead purification-labeling", avoiding cumbersome and variable steps such as multiple centrifugation, column chromatography, and vacuum drying. Most operations are completed in a single tube through mixing and magnetic separation. By simplifying the steps, reducing reagent consumption, and saving manual time, the overall cost per sample is effectively controlled.
[0018] This invention employs a two-step purification process—purification after enzymatic digestion and purification after labeling. Through these two purification processes with different functions, the former provides a pure environment for the labeling reaction, while the latter ensures the signal-to-noise ratio of the detection signal, effectively improving the sensitivity and accuracy of the detection results. Attached Figure Description
[0019] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0020] Figure 1 This is a comparison chart of the detection results of capillary electrophoresis analysis of Examples 1-3 and Comparative Examples 1-2 of this application.
[0021] Figure 2 This is a comparison chart of the detection results of capillary electrophoresis analysis of Example 1 and Comparative Examples 3-5 of this application. Detailed Implementation
[0022] The present invention will be further described below with reference to embodiments and examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the protection scope of the appended claims.
[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
[0024] N-glycans are oligosaccharide chains linked to asparagine residues in proteins via nitrogen atoms. In this field, N-glycans share a common core structure (GlcNAc2-Man3), but exhibit high diversity in branching structures and terminal monosaccharide composition. The structural complexity of N-glycans determines their crucial functions in vital processes such as protein folding, stability, and cell recognition. Analysis of N-glycans is of great significance for understanding protein function, disease diagnosis, and drug development.
[0025] This invention provides a method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling, comprising the following steps: S1. Use trypsin to enzymatically digest cells to obtain glycoproteins; S2. The glycoprotein is denatured and then digested with PNGase F to obtain N-glycans; S3. The N-glycan chain is purified to obtain the purified N-glycan chain; S4. The purified N-glycan chain was fluorescently labeled with 8-aminopyrene-1,3,6-trisulfonic acid to obtain fluorescently labeled N-glycan chains; S5. The fluorescently labeled N-glycan chain is purified to obtain the purified fluorescently labeled N-glycan chain; S6. Detect the purified fluorescently labeled N-glycans.
[0026] Step S1: This step involves pretreating cells with proteases, a technique that uses proteases to treat cell samples under mild conditions to release glycoproteins. Protease pretreatment effectively disrupts cell membrane structures, releasing intracellular glycoproteins while maintaining the integrity of the glycan structure. Commonly used proteases in protease pretreatment include trypsin, proteinase K, and pepsin. This invention uses trypsin for treatment, which has the advantages of high specificity and efficiency.
[0027] In some embodiments, the cells are washed with PBS and collected by centrifugation before being digested with trypsin.
[0028] Furthermore, the steps of enzymatically digesting cells with trypsin to obtain glycoproteins include: Cells, trypsin, and buffer solution are mixed and processed at a first temperature and a first rotation speed for a first time to obtain an enzymatic hydrolysate. The enzymatic hydrolysate was centrifuged, and the supernatant was collected to obtain the glycoprotein. Optionally, the first temperature is 36.8℃-37.2℃, the first rotation speed is 145rpm-155rpm, and the first time is 14min-16min.
[0029] In this embodiment, by processing at appropriate temperature and time, excessive enzymatic hydrolysis leading to glycan degradation can be avoided.
[0030] Further, the step of centrifuging the enzymatic hydrolysate includes: The enzymatic hydrolysate is centrifuged at a second temperature and a second rotation speed for a second time. The second temperature is 3.8℃-4.2℃, the second rotation speed is 14000rpm-16000rpm, and the second time is 14min-16min.
[0031] Step S2: This step involves denaturing the glycoprotein and then using the PNGase F enzyme to specifically cleave the N-glycans to obtain the N-glycans. PNGase F cleavage refers to the technique of using peptide-N-glycosidase F (PNGase F) to specifically cleave the N-glycans linked to the protein. PNGase F is an enzyme that can recognize and hydrolyze the GlcNAc-Asn bonds in glycoproteins, releasing the N-glycans intact without damaging their structure.
[0032] In some embodiments, the step of denaturing the glycoprotein and then digesting it with PNGase F to obtain N-glycans includes: The glycoprotein is heated at a third temperature for a third time to obtain a denatured glycoprotein; Optionally, the third temperature is 98.8℃-99.2℃, and the third time is 4.8min-5.2min; The denatured glycoprotein was digested with PNGase F to obtain the N-glycan.
[0033] Further, the step of digesting the denatured glycoprotein with PNGase F includes: The denatured glycoprotein, PNGase F enzyme, and buffer solution were mixed and treated at a fourth temperature for a fourth time. The fourth temperature is 36.8℃-37.2℃, and the fourth time is 12h-24h.
[0034] Step S3: This step is to purify the N-glycan chain to remove residual proteins, salts, enzymes, and other impurities from the product after enzymatic digestion in step S2.
[0035] In some embodiments, the step of purifying the N-glycan chain to obtain the purified N-glycan chain includes: The N-glycans, magnetic beads, and acetonitrile were mixed, stirred, and placed on a magnetic rack. The precipitate was collected to obtain the purified N-glycans.
[0036] In this embodiment, the purification step is based on a magnetic bead-organic solvent purification system. In this art, magnetic beads, with their hydrophilic or hydrophobic surfaces, effectively adsorb sugar chains while repelling impurities, and organic solvents (such as acetonitrile) precipitate proteins, remove salts, and act as detergents. In this embodiment, the specific binding of the special functional groups on the surface of the magnetic beads to the target molecules (such as sugar chains) is utilized, and rapid separation is achieved through an external magnetic field.
[0037] Step S4: This step involves labeling N-glycan chains with fluorescent dyes to facilitate subsequent detection.
[0038] This step involved fluorescently labeling the purified N-glycans with 8-aminopyrene-1,3,6-trisulfonic acid (APTS). APTS is a highly sensitive fluorescent dye with three sulfonic acid groups, enabling it to specifically react with the reducing ends of the glycans.
[0039] In some embodiments, the purified N-glycan chains are fluorescently labeled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) to obtain fluorescently labeled N-glycan chains, comprising: The purified N-glycan, 8-aminopyrene-1,3,6-trisulfonic acid, and buffer solution were mixed and treated at a fifth temperature for a fifth time to obtain the fluorescently labeled N-glycan. The fifth temperature is 54.8℃-55.2℃, and the fifth time is 50min-70min.
[0040] Step S5: This step involves purifying the fluorescently labeled N-glycans. The main purpose is to remove excess fluorescent dye, salt, protein, and other reaction impurities to obtain a high-purity labeled glycan sample for subsequent detection and analysis, ensuring the signal-to-noise ratio of the detection signal.
[0041] In some embodiments, the step of purifying the fluorescently labeled N-glycan chain includes: The fluorescently labeled N-glycans, magnetic beads, and acetonitrile were mixed, stirred, and placed on a magnetic rack. The precipitate was collected to obtain the purified fluorescently labeled N-glycans.
[0042] Step S6: This step involves detecting fluorescently labeled N-glycans using methods including capillary electrophoresis and laser-induced fluorescence detection. Capillary electrophoresis is a highly efficient separation technique based on the capillary separation principle, utilizing the difference in migration of charged particles within a capillary driven by an electric field. For glycan analysis, coated capillaries are typically used to reduce adsorption of the glycans onto the capillary wall, and the separation medium is often a borate or glycine buffer. Laser-induced fluorescence detection is a highly sensitive optical detection technique that uses a laser to excite fluorescently labeled glycan molecules, detecting the emitted fluorescence signal using a photomultiplier tube.
[0043] In some embodiments, the fluorescently labeled N-glycan is purified before fluorescence detection of the fluorescently labeled N-glycan; Further, the purification step of the fluorescently labeled N-glycan chain includes: The fluorescently labeled N-glycans, magnetic beads, and acetonitrile were mixed, stirred, and placed on a magnetic rack. The precipitate was collected to obtain the purified fluorescently labeled N-glycans.
[0044] Further, the step of fluorescence detection of the fluorescently labeled N-glycan chain includes: Mix the fluorescently labeled N-glycan chain, magnetic beads, and water, stir, place on a magnetic rack, and collect the supernatant; The supernatant was subjected to capillary electrophoresis analysis and laser-induced fluorescence detection. Optionally, the conditions for the capillary electrophoresis analysis include: oven temperature 29.8℃-30.2℃; pre-electrophoresis voltage 14.58kV-15.2kV; pre-electrophoresis time 178s-182s; sample loading voltage 14.58kV-15.2kV; and electrophoresis time 2250s-2350s.
[0045] The buffer solutions of this invention include PBS buffer, 2-PB buffer system, etc., where the 2-PB buffer system is a buffer system with 2-pyridineboronic acid (2-PB) as the main component. The 2-PB buffer system plays a catalytic and stabilizing role in glycan fluorescent labeling reactions. 2-PB can form a stable borate ester complex with the cis-diol structure of the glycan, promoting the reaction between the fluorescent dye and the reducing end of the glycan. This buffer system is typically used under conditions of pH 4-6.
[0046] The following are some specific examples.
[0047] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this invention document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer; raw materials and reagents not specified can be obtained commercially, or can be prepared by those skilled in the art according to known means.
[0048] Example 1 This embodiment provides a method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling, including the following steps: (1) Cell treatment: Collect CAR-T cells and centrifuge at 15,000 rpm for 15 min at 4℃, discard the supernatant; resuspend in 500 μL of cold PBS containing 2.5 mg / mL trypsin, shake at 37℃ and 150 rpm for 15 min, then centrifuge at 1000 rpm for 5 min to collect the supernatant; finally wash three times with pre-cooled PBS (1000 rpm, 5 min).
[0049] (2) Enzyme digestion: Incubate the cell supernatant in a 99°C water bath for 5 min; add 0.5 U of PNGase F and digest overnight at 37°C for 16 h.
[0050] (3) Glycan purification: Take out the sample digested overnight, add 20 μL of magnetic beads and 400 μL of anhydrous acetonitrile, mix well and let stand for 1 min, place on a magnetic rack, and discard the supernatant after standing.
[0051] (4) Fluorescent labeling: Add 2 μL of APTS and 2 μL of 2-PB (2 mol / L), and heat at 55 °C for 1 h for labeling.
[0052] (5) Final purification: Add 160 μL of anhydrous acetonitrile, mix well, and then magnetically separate and discard the supernatant; add 180 μL of 96% acetonitrile, wash twice, and air dry for 10 min; add 50 μL of water, magnetically separate, and then take the supernatant.
[0053] (6) Capillary electrophoresis detection: 2 μL of labeled sample was mixed with 8 μL of HD formamide and analyzed by capillary electrophoresis. The electrophoresis conditions were: constant temperature chamber temperature 30℃; pre-electrophoresis voltage 15kV; pre-electrophoresis time 180s; sample loading voltage 15kV; electrophoresis time 2300s.
[0054] Example 2 This comparative example provides another method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling, which is basically the same as the method in Example 1, except that the concentration of trypsin is 0.5 mg / mL.
[0055] Example 3 This comparative example provides another method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling, which is basically the same as the method in Example 1, except that the concentration of trypsin is 5.0 mg / mL.
[0056] Comparative Example 1 This comparative example provides another method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling, which is basically the same as the method in Example 1, except that trypsin is replaced with proteinase K at an equal concentration (2.5 mg / mL).
[0057] Comparative Example 2 This comparative example provides another method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling, which is basically the same as the method in Example 1. The difference is that no enzymes are added during the cell treatment process, and 500 μL of cold PBS is used directly, and the cells are shaken at 37°C and 150 rpm for 15 min.
[0058] Comparative Example 3 This comparative example provides another method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling, which is basically the same as the method in Example 1. The difference is that after completing the fluorescent labeling, no final purification is performed, and capillary electrophoresis detection is performed directly.
[0059] Comparative Example 4 This comparative example provides another method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling, which is basically the same as the method in Example 1. The difference is that after enzymatic digestion, glycan purification is not performed, and fluorescent labeling is performed directly.
[0060] Comparative Example 5 This comparative example provides another method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling, which is basically the same as the method in Example 1. The difference is that after enzyme digestion, glycan purification is not performed, and fluorescent labeling is performed directly; and after fluorescent labeling, final purification is not performed, and capillary electrophoresis detection is performed directly.
[0061] The detection results of Examples 1-3 and Comparative Examples 1-2 are as follows: Figure 1 , Figure 1 The results show that the use of trypsin in Examples 1-3 effectively released glycoproteins from the cell surface. Specifically, Example 1 used trypsin at a concentration of 2.5 mg / mL, achieving the maximum recovery rate of N-glycans. Example 2 used a relatively low concentration of trypsin, resulting in less release than Example 1. Example 3 used a relatively high concentration of trypsin, failing to significantly improve the recovery rate and posing a risk of excessive hydrolysis. In contrast, Comparative Example 1, using proteinase K, disrupted the glycoprotein structure, leading to a significant decrease in recovery rate. Comparative Example 2, using no enzyme, showed no detectable signal.
[0062] The detection results of Examples 1 and Comparative Examples 3-5 are as follows: Figure 2 , Figure 2This indicates that the two purification steps, "purification after enzymatic digestion" and "purification after labeling," are both indispensable key steps for achieving high-sensitivity and high-accuracy detection results. The use of two-step purification can ensure the signal-to-noise ratio of the detection signal.
[0063] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0064] The embodiments described above merely illustrate several implementations of the present invention and should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification can be used to interpret the content of the claims.
Claims
1. A method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling, characterized in that, Includes the following steps: Cells were enzymatically hydrolyzed using trypsin to obtain glycoproteins; The glycoprotein was denatured and then digested with PNGase F to obtain N-glycans; The N-glycan chain was purified to obtain purified N-glycan chain; The purified N-glycan was fluorescently labeled with 8-aminopyrene-1,3,6-trisulfonic acid to obtain fluorescently labeled N-glycans. The fluorescently labeled N-glycan is purified to obtain purified fluorescently labeled N-glycan; the purified fluorescently labeled N-glycan is then detected.
2. The method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling according to claim 1, characterized in that, Before enzymatically digesting the cells with trypsin, the cells were washed with PBS and collected by centrifugation.
3. The method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling according to claim 1, characterized in that, The steps for obtaining glycoproteins by enzymatically digesting cells with trypsin include: Cells, trypsin, and buffer solution are mixed and processed at a first temperature and a first rotation speed for a first time to obtain an enzymatic hydrolysate. The enzymatic hydrolysate was centrifuged, and the supernatant was collected to obtain the glycoprotein. Optionally, the first temperature is 36.8℃-37.2℃, the first rotation speed is 145rpm-155rpm, and the first time is 14min-16min.
4. The method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling according to claim 3, characterized in that, The step of centrifuging the enzyme hydrolysate includes: The enzymatic hydrolysate is centrifuged at a second temperature and a second rotation speed for a second time. The second temperature is 3.8℃-4.2℃, the second rotation speed is 14000rpm-16000rpm, and the second time is 14min-16min.
5. The method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling according to claim 1, characterized in that, The steps of denaturing the glycoprotein and then digesting it with PNGase F to obtain N-glycans include: The glycoprotein is heated at a third temperature for a third time to obtain a denatured glycoprotein; Optionally, the third temperature is 98.8℃-99.2℃, and the third time is 4.8min-5.2min; The denatured glycoprotein was digested with PNGase F to obtain the N-glycan.
6. The method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling according to claim 5, characterized in that, The steps of digesting the denatured glycoprotein with PNGase F include: The denatured glycoprotein, PNGase F enzyme, and buffer solution were mixed and treated at a fourth temperature for a fourth time. The fourth temperature is 36.8℃-37.2℃, and the fourth time is 12h-24h.
7. The method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling according to claim 1, characterized in that, The steps for purifying the N-glycan to obtain purified N-glycans include: The N-glycans, magnetic beads, and acetonitrile were mixed, stirred, and placed on a magnetic rack. The precipitate was collected to obtain the purified N-glycans.
8. A method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling according to any one of claims 1-7, characterized in that, The steps of fluorescently labeling the purified N-glycans with 8-aminopyrene-1,3,6-trisulfonic acid to obtain fluorescently labeled N-glycans include: The purified N-glycan, 8-aminopyrene-1,3,6-trisulfonic acid, and buffer solution were mixed and treated at a fifth temperature for a fifth time to obtain the fluorescently labeled N-glycan. The fifth temperature is 54.8℃-55.2℃, and the fifth time is 50min-70min.
9. A method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling according to any one of claims 1-7, characterized in that, The purification steps for the fluorescently labeled N-glycans include: The fluorescently labeled N-glycans, magnetic beads, and acetonitrile were mixed, stirred, and placed on a magnetic rack. The precipitate was collected to obtain the purified fluorescently labeled N-glycans.
10. A method for detecting cell surface N-glycans based on enzymatic digestion and fluorescent labeling according to any one of claims 1-7, characterized in that, The steps for detecting the purified fluorescently labeled N-glycans include: The purified fluorescently labeled N-glycans, magnetic beads, and water were mixed, stirred, and placed on a magnetic rack. The supernatant was then collected. The supernatant was subjected to capillary electrophoresis analysis and laser-induced fluorescence detection. Optionally, the conditions for the capillary electrophoresis analysis include: oven temperature 29.8℃-30.2℃; pre-electrophoresis voltage 14.58kV-15.2kV; pre-electrophoresis time 178s-182s; sample loading voltage 14.58kV-15.2kV; and electrophoresis time 2250s-2350s.