Method for identifying glycosyltransferase or glycosidase-specific enzymes involved in DNA / RNA glycosylation

Abstract

The invention discloses a method for identifying glycosyltransferase or glycosidase specific enzyme participating in DNA / RNA glycosylation, which is characterized in that oxidation labeling is carried out on glycosylation modification of DNA / RNA molecules, and solid phase coupling and enzyme digestion strategies are combined, so that enzyme or sugar binding protein specifically bound with glycosylation DNA / RNA can be efficiently and accurately captured, and the specific enzyme of glycosyltransferase or glycosidase can be identified. The interference caused by non-specific binding is obviously reduced. The method is not only suitable for various biological samples including tissue slices, blood and cell samples, but also compatible with subsequent mass spectrometric analysis, and high-throughput identification and function annotation of targeted proteins are realized. The technology provides a brand new tool for analyzing the action mechanism of DNA / RNA sugar modification in epigenetic regulation and control, and has important value for promoting the research of related disease mechanisms and the development of targeted therapy strategies.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a method for identifying a glycosyltransferase or glycosidase specific enzyme involved in DNA / RNA glycosylation. BACKGROUND

[0002] Glycosylation is a key modification method for regulating the function of biomolecules, and its abnormal expression is closely related to the occurrence and development of various diseases. It is not only an important way for the function regulation of biological macromolecules, but also provides potential targets for disease diagnosis and treatment. At present, the research in this field mainly focuses on the field of protein glycosylation. The mechanism and regulation network of functional enzymes such as glycosyltransferases have been systematically analyzed, and the molecular correlation between them and major diseases such as tumors and neurodegenerative diseases has been established. With the advancement of technology, the scope of glycosylation research has gradually expanded to the level of RNA and DNA. In 2021, international research first confirmed the existence of RNA glycosylation modification, and revealed its function in important biological processes such as gene expression regulation. However, there are still significant technical bottlenecks in the research of DNA / RNA glycosylation functional enzymes. First, known glycosyltransferases or glycosidases all have protein as the action substrate, and there is a lack of specific catalytic enzyme system for DNA / RNA and other nucleic acid molecules. Second, the substrate recognition mechanism, catalytic activity and biological function of key glycosylases (such as glycosyltransferases and glycoside hydrolases) in the process of nucleic acid glycosylation have not been elucidated, making it difficult to establish the molecular correlation between them and the disease process. In addition, there is still a lack of identification technology for DNA / RNA specific glycosylation enzymes. These serious lags in enzymatic research have restricted the application of nucleic acid glycosylation related disease markers and targeted intervention strategies in the development, and have become a key obstacle to breakthroughs in this field.

[0003] Therefore, it is of great scientific value and application prospect to develop a method for accurately identifying a glycosyltransferase or glycosidase specific enzyme involved in DNA / RNA glycosylation. SUMMARY

[0004] The purpose of the present application is to provide a method for identifying a glycosyltransferase or glycosidase specific enzyme involved in DNA / RNA glycosylation, which solves the above problems.

[0005] The technical solution of the present application is as follows:

[0006] A method for identifying a glycosyltransferase or glycosidase specific enzyme involved in DNA / RNA glycosylation, comprising the following steps:

[0007] (1) selectively oxidizing the C6 hydroxyl group or cis-diol in the sugar residue of the glycosylated DNA / RNA fragment by using an oxidase or an oxidizing agent to generate an active aldehyde group, and covalently condensing the active aldehyde group with a hyperlinked hydrazide magnetic bead to achieve efficient capture of the oxidized glycosylated DNA / RNA;

[0008] (2) releasing the glycosylated DNA / RNA by glycosidase enzyme, identifying the sequence characteristics by NGS sequencing, and obtaining the glycosylated DNA / RNA fragments;

[0009] (3) synthesizing the glycosylated DNA / RNA fragments with biotin label by using biotin-DNA / RNA;

[0010] (4) incubating the glycosylated DNA / RNA fragments with biotin label with cell lysate, and enriching the glycosyl recognition enzyme or related protein specifically combined by using streptavidin magnetic beads, to obtain the magnetic bead-bound protein;

[0011] (5) heating to elute the magnetic bead-bound protein, and adding trypsin to incubate overnight at 37℃, so as to fully enzymatically digest the protein into peptide segments;

[0012] (6) purifying the enzymatically digested peptide segments by using C18 solid phase extraction, removing impurities and concentrating the sample, and completing the identification of the protein components by LC-MS / MS mass spectrometry.

[0013] Further, in step (1), when the DNA fragments, the length of the DNA fragments is 50-500 base pairs; when the RNA fragments, the length of the RNA fragments is 10-300 nt.

[0014] Further, in step (1), the oxidase is galactose oxidase, and the galactose oxidase specifically oxidizes galactose and N-acetylgalactosamine.

[0015] Further, in step (1), the oxidizing agent is sodium periodate, and the sodium periodate specifically oxidizes cis-structure monosaccharides.

[0016] Further, in step (2), the glycosidase is peptide-N-glycosidase F or O-glycosidase.

[0017] Further, in step (3), the synthesis of the glycosylated DNA / RNA fragments with biotin label by using biotin-DNA / RNA is specifically: preparing a DNA / RNA single-stranded solution with a concentration of 100 μmol / L by using an annealing buffer containing 10 mmol / L Tris-HCl, 50 mmol / L NaCl and 1 mmol / L EDTA, mixing equal volumes of complementary DNA / RNA single-stranded solutions with equal molar concentrations in a centrifuge tube, placing the centrifuge tube in 95℃ for 5 minutes, and then slowly cooling to room temperature to complete the annealing reaction, finally obtaining the glycosylated DNA / RNA fragments with biotin label.

[0018] Further, in step (4), the final concentration of the biotin-labeled glycosylated DNA / RNA fragments is 45-55 nM, and the incubation time is 20-40 minutes.

[0019] Further, in step (4), the cell lysis solution contains 1% NP-40, 150 millimoles of NaCl, 50 millimoles of Tris-HCl, and protease inhibitors.

[0020] Further, in step (5), the elution buffer contains 2% SDS and 100 millimoles of DTT.

[0021] Further, in step (5), the amount of trypsin added is 1.5-2.5 micrograms.

[0022] The present application provides a method for identifying glycosyltransferase or glycosidase specific enzymes involved in DNA / RNA glycosylation, which mainly includes two core stages: the first stage is based on the oxidation of glycan of glycosylated DNA / RNA to generate aldehyde group, and the specific capture and identification of glycosylated nucleic acid fragments are completed by hydrazine-aldehyde condensation reaction and enzymatic hydrolysis; the second stage is to synthesize glycosylated DNA / RNA probes and combine them to the solid phase medium, and then incubate with the lysed proteins of the biological sample, and then use the specific non-covalent binding property of glycosylated nucleic acid and target enzyme to accurately identify the DNA / RNA specific glycosyltransferase or glycosidase through purification, elution and mass spectrometry analysis. This technology realizes the systematic analysis of nucleic acid glycosylation modification and its acting enzyme for the first time through the multi-dimensional strategy of chemical modification, biological enrichment and mass spectrometry analysis, provides a high-throughput research tool for revealing the molecular mechanisms of glycosylated DNA / RNA in gene expression regulation, cell signal transduction and disease occurrence, and fills the technical gap in the field of nucleic acid epigenetic modification enzyme research. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The present application provides a method for identifying glycosyltransferase or glycosidase specific enzymes involved in DNA glycosylation. The flowchart is shown in the figure. In the figure, the colored double helix represents different sequence DNA fragments, the yellow dot represents galactose, the yellow square represents N-acetylgalactosamine, the green dot represents mannose, the black spherical ball represents hydrazine magnetic beads, the blue spherical ball represents biotin labeling, the multicolor irregular ellipse represents DNA binding protein, and the arrow direction indicates the sequence of the experimental process. DETAILED DESCRIPTION

[0024] Please refer to Figure 1 , Figure 1 The present application provides a method for identifying glycosyltransferase or glycosidase specific enzymes involved in DNA glycosylation. The flowchart is shown in the figure. In the figure, the colored double helix represents different sequence DNA fragments, the yellow dot represents galactose, the yellow square represents N-acetylgalactosamine, the green dot represents mannose, the black spherical ball represents hydrazine magnetic beads, the blue spherical ball represents biotin labeling, the multicolor irregular ellipse represents DNA binding protein, and the arrow direction indicates the sequence of the experimental process. Figure 1As shown, the purpose of the present application is to provide a method for identifying glycosyltransferase or glycosidase specific enzymes involved in DNA glycosylation, comprising the following steps:

[0025] Step one: using oxidase or oxidant to selectively oxidize the C6 hydroxyl group or cis-diol of the sugar residue in the glycosylated DNA fragment to generate an active aldehyde group, and covalently condensing the active aldehyde group with ultralink hydrazide magnetic beads to achieve efficient capture of oxidized glycosylated DNA.

[0026] 1. Sample preparation: Use ultrasonic disrupter or DNA degradation enzyme to fragment the long-chain DNA sample, control the length in the range of 50-500 base pairs, and optimize the subsequent reaction efficiency. Obtain DNA fragments for subsequent reactions;

[0027] 2. Coupling, experiment on DNA fragment sample: using galactose oxidase (GAO) or sodium periodate (NaIO4) and other oxidants to selectively oxidize the C6 hydroxyl group of galactose (Gal), N-acetylgalactosamine (GalNAc) or mannose (Mannose) and other sugar residues in the glycosylated DNA fragment (Note: sodium periodate specifically oxidizes cis-structure monosaccharides, and GAO specifically acts on Gal and GalNAc), to generate an active aldehyde group;

[0028] 3. Covalent condensation of aldehyde group with ultralink hydrazide magnetic beads to achieve efficient capture of glycosylated DNA fragments;

[0029] Step two: release N-glycosylated DNA by glycosidase enzyme digestion, and identify its sequence characteristics by NGS sequencing to obtain DNA fragments with glycosylation.

[0030] After releasing the solid-phase bound glycosylated DNA fragments by glycosidase (such as PNGase F or O-glycosidase) enzyme digestion, identify its sequence characteristics by NGS sequencing.

[0031] Step three: use biotin-DNA to synthesize glycosylated DNA fragments with biotin labeling.

[0032] Step four: After incubation of biotinylated fragments with cell lysate proteins by affinity purification method to simulate natural interaction, use streptavidin magnetic beads to enrich specific binding glycosyl recognition enzymes or related proteins.

[0033] Step five: elute the magnetic bead-bound proteins by heating, and add trypsin to incubate overnight at 37°C to fully enzymatically digest the proteins into peptide segments.

[0034] Step six: Purification of the enzymatic peptides by C18 solid phase extraction to remove impurities and concentrate the sample, and finally identify the protein components by LC-MS / MS mass spectrometry.

[0035] Similarly, the method for identifying the glycosyltransferase or glycosidase specific enzyme involved in RNA glycosylation is also similar, the only difference is that the length of the RNA fragment is in the range of 10-300 nt. In this paper, DNA / RNA represents DNA or RNA.

[0036] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the technical solutions of the present application will be further described below combined with specific examples. However, the present application is not limited to the listed examples, and any known changes within the scope of the claimed rights of the present application should also be included.

[0037] The "one embodiment" or "embodiment" referred to herein means that the specific features, structures or characteristics can be included in at least one implementation of the present application. The "in one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. It should be noted that in the present application, the large end is the relatively large end of the tapered artificial blood vessel, and the small end is the relatively small end of the tapered artificial blood vessel.

[0038] Example 1

[0039] The following example demonstrates a method for identifying a glycosyltransferase or glycosidase specific enzyme involved in DNA / RNA glycosylation, the specific steps are as follows:

[0040] Step one: using oxidase or oxidant to selectively oxidize the C6 hydroxyl group or cis diol of the sugar residue in the glycosylated DNA / RNA fragment to generate an active aldehyde group, and covalently condensing the active aldehyde group with super-hyperlinked hydrazine magnetic beads to achieve efficient capture of the oxidized glycosylated DNA / RNA.

[0041] 1. Sample preparation:

[0042] (1) For extracting DNA / RNA from biological samples, when the sample source is paraffin-embedded tissue sections, select three tumor sections with a thickness of about 4 microns and an area of about 2 square centimeters, use a sterile blade to scrape the tumor area tissue, and transfer it to a 1.5 milliliter centrifuge tube. Ensure that the total section thickness is controlled below 100 microns to improve the DNA / RNA extraction effect. According to the thickness of the tissue, add an appropriate amount of mineral oil (add 300 microliters when less than or equal to 50 microns, and add 500 microliters when greater than 50 microns), incubate at 80°C for 1 minute, and mix well. Then add 200 microliters of lysis buffer, centrifuge at 10,000 x g for 15 seconds to separate, add 20 microliters of proteinase K from the lower aqueous phase, incubate at 56°C for 1 hour, then warm up to 80°C for 2 hours for complete lysis. After the sample cools to room temperature, add 10 microliters of RNase A and incubate for 5 minutes, then add 220 microliters of BL buffer and 240 microliters of ethanol in sequence to allow the nucleic acid to bind.

[0043] (2) For nucleic acid extraction from blood and cell samples, the following operation scheme is recommended: accurately measure 200 microliters of the sample to be tested (if the sample volume is insufficient, supplement with ddH2O) and place it in a 1.5 milliliter centrifuge tube. Add 20 microliters of proteinase K, 4 microliters of RNase A, and 200 microliters of Buffer AL solution in sequence. Mix the system by vortexing for 15 seconds, then transfer it to a 56°C constant temperature environment for incubation for 10 minutes. After the reaction system cools naturally, add 200 microliters of absolute ethanol and mix thoroughly. All types of samples after lysis treatment need to be purified by adsorption column, using specific formula washing buffer for multiple washing, and finally using 50 microliters of DEPC water for efficient DNA / RNA elution.

[0044] The purified DNA / RNA above needs to be treated with non-contact ultrasonic treatment: accurately take 2000 ng of DNA / RNA sample, adjust the total volume to 40 microliters with 1 x TE buffer, and transfer to a low-adsorption 0.2 milliliter special breaking tube. The ultrasonic parameters are set to 30 seconds of work / intermittent time alternation cycle. After completing 5-10 cycles, the sample is taken out for vortex mixing and transient centrifugation. A total of 13 cycles are performed to achieve a genomic fragmentation target of 100-300 bp. Subsequently, magnetic bead purification method is used: add 1.8 times the volume of DNA / RNA Clean Beads, stand at room temperature for 5 minutes, then place on a magnetic stand to separate. After removing the supernatant, wash twice with 80% (v / v) ethanol, dry, and resuspend in 45 microliters of DEPC water to obtain 40 microliters of high-purity DNA / RNA fragments.

[0045] 2. Coupling: 2 micrograms of lyophilized DNA / RNA fragment sample was dissolved in 200 microliters of 100 millimolar sodium acetate buffer, and 20 millimolar sodium periodate was added at a final concentration (1 / 10 volume of 200 millimolar stock solution was added), and the reaction was shaken at room temperature for 2 hours in the dark. After the reaction was terminated, the system was diluted to 600 microliters using 1x PBS buffer. Then solid-phase coupling was performed: 100 microliters of 50% (w / v) hydrazide magnetic bead suspension was injected into a 1.5 milliliter spin column, which was washed with DEPC water and centrifuged at 6000 rpm for 5 minutes. The oxidized DNA / RNA sample and 60 microliters of 100 millimolar aniline solution were sequentially added to the pretreated magnetic beads, and the coupling was shaken at room temperature for 3 hours.

[0046] Step two: N-glycosylated DNA / RNA was released by glycosidase enzyme digestion, and its sequence characteristics were identified by NGS sequencing to obtain DNA / RNA fragments with glycosylation.

[0047] After centrifugation to remove unbound components, 1.5 millimolar NaCl solution (shaken vigorously for three times), DEPC water (three times), and freshly prepared 25 millimolar NH4HCO3 buffer (three times) were sequentially used for deep purification. Finally, the magnetic beads were resuspended in 200 microliters of ammonium bicarbonate buffer, and 1-2 microliters of PNGase F enzyme solution was added (vortexing was prohibited), and the glycosylated DNA / RNA was released by gentle shaking at 37°C overnight.

[0048] After collecting the N-glycosylated glycoDNA / RNA in the centrifugal supernatant, it was eluted three times with DEPC water containing 10-30% (v / v) acetonitrile (100 microliters each time), and about 400 microliters of sample solution was obtained by combining. After vacuum centrifugal concentration and drying, the DNA / RNA sequencing was performed by resuspending in an appropriate amount of DEPC water, and the DNA / RNA sequencing results were obtained.

[0049] Step three: biotin-DNA / RNA was used to synthesize glycosylated DNA / RNA fragments with biotin labeling.

[0050] After DNA / RNA sequencing was completed, specific DNA / RNA fragments with biotin were synthesized according to the sequencing results.

[0051] DNA / RNA fragments to subsequently capture the glycosyl recognition enzymes or other sugar binding proteins that bind to them via streptavidin magnetic beads. The synthetic DNA / RNA single-stranded complementary fragments were annealed to form double-stranded with biotin labeling by mixing equal volume and molarity of the DNA / RNA single-stranded solutions at a concentration of 100 μmol / L in an Eppendorf tube, incubating the Eppendorf tube at 95 °C for 5 min, and then slowly cooling to room temperature to complete the annealing reaction. The final DNA / RNA double-stranded with biotin labeling was obtained. Next, the biotinylated DNA / RNA double-stranded was combined with streptavidin magnetic beads (Dynabeads M-280 Streptavidin). Fifty microliters of magnetic beads were taken and washed three times with 1 x PBS (containing 0.1% (v / v) Tween-20) using a magnetic stand to separate and discard the supernatant each time. The washed magnetic beads were resuspended in 100 microliters of 1 x PBS, and biotinylated DNA / RNA (final concentration of about 50 nM) was added. The mixture was incubated at room temperature for 30 min with gentle rotation to ensure sufficient binding. After the binding was completed, the magnetic beads were separated using a magnetic stand, and the supernatant was discarded. The magnetic beads were washed three times with 1 x PBS (containing 0.1% (v / v) Tween-20) to remove non-specifically bound molecules.

[0052] Step four: Simulate natural interaction by affinity purification method - after incubation of biotinylated fragments with cell lysate proteins, streptavidin magnetic beads were used to enrich the specifically bound glycosyl recognition enzymes or related proteins.

[0053] The treated magnetic beads were added to the cell lysate containing the target proteins, usually 500 microliters of lysate containing 1% (v / v) NP-40, 150 millimolar NaCl, 50 millimolar Tris-HCl (pH 7.5), and a protease inhibitor cocktail. The mixture was incubated at 4 °C for 2 hours with gentle rotation to promote the binding of target proteins to DNA / RNA.

[0054] Step five: The proteins bound to the magnetic beads were eluted by heating, and trypsin was added to incubate overnight at 37 °C to fully enzymatically digest the proteins into peptide segments.

[0055] After the incubation, the magnetic beads were separated using a magnetic stand and washed three times with lysis buffer to remove non-specifically bound proteins. Finally, the magnetic beads were resuspended in 50 microliters of elution buffer (containing 2% (w / v) SDS, 100 millimolar DTT) and heated at 95 °C for 5 minutes to release the bound proteins, and the supernatant was collected. Subsequently, the protein-containing supernatant was collected and 2 micrograms of trypsin was added thereto, and incubated at 37 °C overnight to ensure that the proteins were completely digested into peptide fragments.

[0056] Step six: Purification of the digested peptides using C18 solid phase extraction to remove impurities and concentrate the sample, and finally identifying the protein components through LC-MS / MS mass spectrometry.

[0057] After the enzyme digestion, the resulting peptide solution needs to be purified by C18 solid phase extraction to remove salt ions, detergents and other interfering substances, to ensure the sensitivity and accuracy of downstream mass spectrometry analysis.

[0058] Before C18 purification, first use 1 milliliter of 80% (v / v) acetonitrile (ACN) / 0.1% (v / v) trifluoroacetic acid (TFA) to condition the C18 column, activate it to enhance the subsequent binding capacity. Then, adjust the pH of the enzyme digestion product to less than 3 (usually by adding TFA to a final concentration of 1% (v / v)) to ensure that the peptides can be efficiently adsorbed on the C18 filler. Then wash the column twice with 1 milliliter of 0.1% (v / v) TFA to balance the filler, and then slowly load the sample into the C18 column. After loading the sample, use a syringe or rubber dropper to collect the first effluent and re-load it into the column to improve recovery. Then, use 1 milliliter of 0.1% (v / v) TFA to wash the column 5 times in succession to remove impurities. After washing, elute the peptides with 800 μL of 80% (v / v) ACN solution, repeat this step twice, a total of 1600 microliters of eluent is used to collect the target peptides. Finally, transfer the eluent to a new centrifuge tube, use a vacuum centrifuge (SpeedVac) to dry the sample, obtain a concentrated and pure peptide sample, ready for subsequent liquid chromatography-mass spectrometry (LC-MS / MS) analysis.

[0059] Note: All solutions used in the entire analysis process need to be prepared with DEPC water. This method effectively realizes the recognition and enrichment of proteins binding to DNA / RNA glycosylation modification sites through a series of highly specific and systematic steps, including oxidative modification of DNA / RNA fragments, covalent coupling with hydrazine-based magnetic beads, release and recovery of bound proteins, and subsequent biotin labeling and streptavidin magnetic bead enrichment strategy. By combining specific enzyme digestion, C18 solid phase extraction purification and high sensitivity mass spectrometry analysis, this method can accurately capture and identify functional proteins binding to glycosylated DNA / RNA, greatly improving the resolution and depth of DNA / RNA glycosylation regulation research, and providing a powerful technical platform and theoretical support for exploring the function of DNA / RNA glycosylation in development, disease occurrence and cell fate regulation.

[0060] Example 2

[0061] The following examples demonstrate the experimental part of a method for identifying glycosyltransferases or glycosidases specific enzymes involved in DNA / RNA glycosylation.

[0062] The DNA / RNA fragments verified in the previous sequencing that can be glycosylated in MCF-7 cells were selected as the experimental group, and the DNA / RNA fragments that cannot be glycosylated were selected as the control group, and the MCF-7 cell lysate was used for co-incubation.

[0063]

[0064] Table 1

[0065] Please refer to Table 1, which is the synthesis information of biotin-labeled glycosylated DNA / RNA fragments synthesized in step three of Example 1. As shown in Table 1, these DNA / RNA fragments with glycosylation sites were synthesized to provide substrates for identifying glycosyltransferases or glycosidases involved in DNA / RNA glycosylation.

[0066]

[0067] Table 2

[0068] After steps four and five, the obtained peptide samples were subjected to step seven sequence analysis. Please refer to Table 2, which is the cell lysate proteins identified by GREnzyme, and the following are the top thirty proteins with fold difference in the analysis results. As shown in Table 2, glycosyltransferases or glycosidases involved in DNA / RNA glycosylation were identified through glycosylated DNA / RNA fragment substrates.

[0069] In summary, the method for identifying the specific enzyme of glycosyltransferase or glycosidase involved in DNA / RNA glycosylation has the technical advantages of high specificity and high sensitivity of the constructed DNA / RNA glycosylation enrichment and binding enzyme analysis platform. The platform not only provides innovative methodological support for analyzing the molecular mechanism of DNA / RNA sugar modification, but also significantly improves the identification efficiency of interacting proteins through the two-stage enrichment strategy (magnetic bead capture and biotin affinity purification), which has important application value in the development of disease biomarkers, epigenetic regulation research and glyco biology. The method is not only suitable for various biological sample types, including tissue sections, blood and cell samples, but also compatible with subsequent mass spectrometry analysis, realizing high-throughput identification and functional annotation of target proteins. The technology provides a new tool for studying the mechanism of DNA / RNA sugar modification in epigenetic regulation, and helps to promote the research and development of targeted intervention strategies for related disease mechanisms.

[0070] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. A method for identifying a glycosyltransferase or glycosidase specific enzyme involved in DNA / RNA glycosylation, characterized in that, The method comprises the following steps: (1) selectively oxidizing the C6 hydroxyl group or cis-diol of the sugar residue in the oxidized DNA / RNA fragments by using an oxidase or an oxidizing agent to generate an active aldehyde group, and covalently condensing the active aldehyde group with super-linking hydrazine magnetic beads to realize efficient capture of the oxidized glycosylated DNA / RNA; (2) releasing the glycosylated DNA / RNA by enzymatic hydrolysis of glycosidase, identifying the sequence characteristics of the glycosylated DNA / RNA by NGS sequencing, and obtaining the glycosylated DNA / RNA fragments; (3) synthesizing the glycosylated DNA / RNA fragments with biotin labeling by using biotin-DNA / RNA; (4) after incubating the glycosylated DNA / RNA fragments with biotin labeling with a cell lysate, the specific binding glycosyl recognition enzyme or related protein is enriched by using streptavidin magnetic beads to obtain the magnetic bead-bound protein; (5) heating and eluting the magnetic bead-bound protein, and adding trypsin to incubate overnight at 37°C to fully enzymatically hydrolyze the protein into peptide segments; (6) purifying the enzymatically hydrolyzed peptide segments by using C18 solid-phase extraction, removing impurities and concentrating the sample, and identifying the protein components by LC-MS / MS mass spectrometry.

2. The method of identifying a specific enzyme of a glycosyltransferase or glycosidase involved in DNA / RNA glycosylation according to claim 1, characterized in that: In step (1), when the DNA fragments, the length of the DNA fragments is 50-500 base pairs; when the RNA fragments, the length of the RNA fragments is 10-300 nt.

3. The method of identifying a specific enzyme of a glycosyltransferase or glycosidase involved in DNA / RNA glycosylation according to claim 1, wherein: In step (1), the oxidase is galactose oxidase, and the galactose oxidase specifically oxidizes galactose and N-acetylgalactosamine.

4. The method of identifying a specific enzyme of a glycosyltransferase or glycosidase involved in DNA / RNA glycosylation according to claim 1, characterized in that: In step (1), the oxidizing agent is sodium periodate, and the sodium periodate specifically oxidizes cis-structure monosaccharides.

5. The method of identifying a specific enzyme of a glycosyltransferase or glycosidase involved in DNA / RNA glycosylation according to claim 1, wherein: In step (2), the glycosidase is peptide-N-glycosidase F or O-glycosidase.

6. The method of identifying a specific enzyme of a glycosyltransferase or glycosidase involved in DNA / RNA glycosylation according to claim 1, wherein, In step (3), the synthesis of the glycosylated DNA / RNA fragments with biotin labeling by using biotin-DNA / RNA is specifically: preparing a DNA / RNA single-stranded solution with a concentration of 100 μmol / L by using an annealing buffer containing 10 mmol / L Tris-HCl, 50 mmol / L NaCl and 1 mmol / L EDTA, mixing equal volumes of complementary DNA / RNA single-stranded solutions with equal molar concentrations in a centrifuge tube, placing the centrifuge tube in 95°C for 5 minutes, and then slowly cooling to room temperature to complete the annealing reaction, thereby obtaining the glycosylated DNA / RNA fragments with biotin labeling.

7. The method of identifying a specific enzyme of a glycosyltransferase or glycosidase involved in DNA / RNA glycosylation according to claim 1, wherein: In step (4), the final concentration of the glycosylated DNA / RNA fragments with biotin labeling is 45-55 nM, and the incubation time is 20-40 minutes.

8. The method of identifying a specific enzyme of a glycosyltransferase or glycosidase involved in DNA / RNA glycosylation according to claim 1, wherein: In step (4), the cell lysate is a mixed solution containing 1% NP-40, 150 mmol NaCl, 50 mmol Tris-HCl and protease inhibitors.

9. The method of identifying a specific enzyme of a glycosyltransferase or glycosidase involved in DNA / RNA glycosylation according to claim 1, wherein: In step (5), the elution buffer used in the elution contains 2% SDS and 100 mmol DTT.

10. The method of identifying a specific enzyme of a glycosyltransferase or glycosidase involved in DNA / RNA glycosylation according to claim 1, wherein: In step (5), the amount of trypsin added is 1.5-2.5 μg.

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