Magnetic bead for enriching tyrosine N-acetylgalactosamine modification sites as well as preparation method and application of magnetic bead

Abstract

The invention belongs to the technical field of biochemical analysis, discloses a magnetic bead for enriching tyrosine N-acetylgalactosamine modification sites as well as a preparation method and application of the magnetic bead, and particularly discloses a recombinant antibody which comprises a Fab fragment of a G10C antibody, an immunoglobulin Fc region and an AviTag tag. The invention provides a recombinant antibody capable of enriching a tyrosine-O-GalNAc glycosylated peptide fragment in a specific targeting manner. By coupling the recombinant antibody to a magnetic bead, enrichment of tyrosine-O-GalNAc glycosylation peptide fragments can be effectively improved, interference and signal shielding of glycosylation modification on serine and threonine on site identification can be avoided, and more tyrosine glycosylation sites can be identified in a single sample.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical analysis, and specifically relates to a magnetic bead for enriching tyrosine N-acetylgalactosamine modification sites, and a preparation method and application thereof. Background Art

[0002] Protein O-GalNAc glycosylation is a complex post-translational modification of proteins. It is connected to proteins through the oxygen atoms of serine, threonine or tyrosine side chains, and plays a regulatory role in many important physiological and pathological processes, including congenital glycosylation diseases, infectious diseases, immune-related diseases, cancer, etc. Therefore, identifying and monitoring O-GalNAc glycosylation is of great value for disease diagnosis and treatment. For example, in the occurrence and development of cancer, truncated O-GalNAc glycosylation (such as Tn antigen and T antigen) is widely expressed in tumor tissues and is considered to be a potential biomarker for cancer detection and diagnosis.

[0003] Traditional O-GalNAc glycosylation-related research focuses on glycosylation sites modified on serine and threonine in proteins, and relatively few studies have been conducted on tyrosine O-GalNAc glycosylation sites. Studies have shown that tyrosine O-GalNAc glycosylation modified on amyloid precursor protein (APP) is associated with Alzheimer's disease (AD), suggesting that the role of tyrosine O-GalNAc glycosylation in physiological and pathological processes needs to be further discovered. Therefore, the discovery and identification of protein tyrosine O-GalNAc glycosylation sites has potential value for the diagnosis and treatment of related diseases.

[0004] Currently, the identification of O-glycosylation sites relies on the weak affinity of lectins. The lectins commonly used to enrich O-sugars include: VVA (Vicia Villosa Lectin) recognizes N-acetylgalactosamine residues at the end of sugar chains, especially Tn antigen; PNA (Peanut Agglutinin) preferentially binds to T antigen (DOI: 10.1038 / nmeth.1731); Jacalin (Jacaranda lectin) recognizes O-linked oligosaccharides, especially T antigen structures (flow chart as shown in the figure). Figure 1, DOI: 10.1038 / s41467-022-33806-8). However, since O-GalNAc glycosylation is modified on serine and threonine in proteins in large quantities in cells and tissues, this method of using lectins to enrich and identify tyrosine O-GalNAc glycosylation sites is greatly interfered with and has low identification efficiency. That is, the affinity of lectins is weak, the enrichment efficiency is not high enough, and there is a large amount of interference from non-O-glycopeptides. Only 3.7% of the identified glycosylation sites are on tyrosine (DOI: 10.1038 / s41467-022-33806-8), and tyrosine glycosylated peptides cannot be specifically enriched.

[0005] Therefore, there is an urgent need to develop a biological material or method that can specifically enrich tyrosine glycosylated peptides, so as to avoid the interference of glycosylation modifications on serine and threonine on site identification and signal masking. Summary of the invention

[0006] The first aspect of the present invention aims to provide a recombinant antibody.

[0007] The purpose of the second aspect of the present invention is to provide the use of the recombinant antibody of the first aspect of the present invention in enriching O-GalNAc glycosylated peptides or preparing a product for enriching O-GalNAc glycosylated peptides.

[0008] The third aspect of the present invention is to provide an immunomagnetic bead.

[0009] The purpose of the fourth aspect of the present invention is to provide the use of the immunomagnetic beads of the third aspect of the present invention in enriching O-GalNAc glycosylated peptides or detecting O-GalNAc glycosylation sites.

[0010] The fifth aspect of the present invention aims to provide a method for enriching and analyzing tyrosine O-GalNAc glycosylation sites.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is:

[0012] The first aspect of the present invention provides a recombinant antibody, comprising a Fab fragment of a G10C antibody, an immunoglobulin Fc region and an AviTag tag.

[0013] In some embodiments of the present invention, the Fab fragment of the G10C antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:1, and the amino acid sequence of the light chain is shown in SEQ ID NO:2.

[0014] In some embodiments of the present invention, the Fab fragment of the G10C antibody is hinged to the immunoglobulin Fc region.

[0015] In some embodiments of the present invention, the immunoglobulin is a combination of one or more selected from IgG, IgA1, IgA2, IgD, IgE, and IgM.

[0016] In some embodiments of the present invention, the IgG is selected from one or more combinations of IgG1, IgG2, IgG3 or IgG4 subtypes.

[0017] In some embodiments of the present invention, the amino acid sequence of the immunoglobulin Fc region is as shown in SEQ ID NO: 3; or an amino acid sequence that is modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 3 and has the same or similar function.

[0018] In some embodiments of the present invention, the AviTag tag is connected to the C-terminus of the Fc region of the immunoglobulin via a connecting peptide.

[0019] In some embodiments of the present invention, the connecting peptide is a flexible linker.

[0020] In some embodiments of the present invention, the flexible linker amino acid sequence includes but is not limited to GSAS, (GGCAGCGCCAGC) n 、(GGCGGCGGCAGC) n 、(GGCGGCGGCGGCAGC) n 、YAPVDV、(GGGS) n 、(GGSG) n 、(GGGGS) n 、(G) n , where 1≤n≤5, and n is an integer.

[0021] In some embodiments of the present invention, the amino acid sequence of the AviTag tag is shown in SEQ ID NO:4.

[0022] In some embodiments of the present invention, the recombinant antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:5, and the amino acid sequence of the light chain is shown in SEQ ID NO:2.

[0023] The second aspect of the present invention provides the use of the recombinant antibody of the first aspect of the present invention in enriching O-GalNAc glycosylated peptides or preparing a product for enriching O-GalNAc glycosylated peptides.

[0024] In some embodiments of the present invention, the O-GalNAc glycosylated peptide segment includes a tyrosine-O-GalNAc glycosylated peptide segment.

[0025] The third aspect of the present invention provides an immunomagnetic bead, comprising the recombinant antibody of the first aspect of the present invention and streptavidin magnetic beads, wherein the recombinant antibody is coupled to the surface of the streptavidin magnetic beads via streptavidin-biotin.

[0026] In some embodiments of the present invention, the method for preparing the immunomagnetic beads comprises the following steps:

[0027] The recombinant antibody is mixed with ATP, biotin and biotin ligase, and incubated at 3-5°C for 8-14 hours to obtain a biotin-modified recombinant antibody;

[0028] The biotin-modified recombinant antibody is mixed with streptavidin magnetic beads and incubated at 3-5° C. for 3-5 hours to obtain immunomagnetic beads.

[0029] In the presence of ATP and biotin, biotin ligase (BirA) connects a biotin to the lysine residue of the AviTag tag, thereby achieving biotin labeling of the target antibody.

[0030] The fourth aspect of the present invention provides the use of the immunomagnetic beads according to the third aspect of the present invention in enriching O-GalNAc glycosylated peptides or detecting O-GalNAc glycosylation sites.

[0031] In some embodiments of the present invention, the O-GalNAc glycosylation includes tyrosine-O-GalNAc glycosylation.

[0032] A fifth aspect of the present invention provides a method for enriching and analyzing tyrosine-O-GalNAc glycosylation sites, comprising the following steps:

[0033] Using the immunomagnetic beads of the third aspect of the present invention to mix with the peptide sample, incubate, and elute to obtain tyrosine-O-GalNAc glycosylated peptides;

[0034] Mass spectrometry analysis of tyrosine-O-GalNAc glycosylated peptides.

[0035] In some embodiments of the present invention, the incubation condition is 3-5° C. for 3-5 hours.

[0036] In some embodiments of the present invention, the peptide samples are derived from cells and / or tissues.

[0037] In some embodiments of the present invention, when the peptide sample is derived from cells, the peptide sample is obtained by the following method:

[0038] Lyse cells: Mix cells with RapiGest and NH4HCO3 solution, sonicate, and cook the sample. Repeat the sonication and cooking several times to obtain a lysate.

[0039] Reduction / alkylation treatment: add dithiothreitol to the lysate, treat at 55-65°C for 40-50 min, add iodoacetamide, incubate in dark for 25-35 min, and obtain solution A;

[0040] Enzyme digestion: Add trypsin to solution A, shake at 37°C for 8-13 hours, add TFA to terminate the enzyme digestion reaction, and collect the supernatant;

[0041] Desialylation: adjust the pH of the supernatant to 5-6, add ceramidase, and shake at 37°C for 5-7h;

[0042] Sep-Pak desalting treatment: Use C18 column for desalting to obtain peptide samples.

[0043] In some embodiments of the present invention, the mass spectrometry analysis comprises the following steps: dissolving the tyrosine-O-GalNAc glycosylated peptide fragments with 0.1% FA, loading onto a mass spectrometer, and analyzing the data using software Proteome Discoverer 2.2.

[0044] During the mass spectrometry analysis, mobile phase A: 0.1%-0.3% FA, mobile phase B: 70%-80% acetonitrile and 0.1%-0.3% FA, flow rate: 2-4 μL / min, scanning range: 355-1700 m / z, resolution: 70000, automatic gain control (AGC) target value: 5e5.

[0045] The beneficial effects of the present invention are:

[0046] The present invention provides a recombinant antibody capable of specifically targeting and enriching tyrosine-O-GalNAc glycosylated peptides. By coupling the recombinant antibody to magnetic beads, the enrichment of tyrosine-O-GalNAc glycosylated peptides can be effectively improved, and the interference and signal masking of glycosylation modifications on serine and threonine on site identification can be avoided, so that more tyrosine glycosylation sites can be identified in a single sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is a schematic diagram of the lectin enrichment process in the prior art (DOI: 10.1038 / s41467-022-33806-8).

[0048] Figure 2 This is the recombinant plasmid map of the recombinant G10C antibody.

[0049] Figure 3 This is the electrophoresis diagram of the recombinant G10C antibody.

[0050] Figure 4Magnetic beads enriched in tyrosine-O-GalNAc prepared by the present invention and a schematic diagram of the enrichment of the magnetic beads.

[0051] Figure 5 Schematic diagram of biotin labeling reaction. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below through specific examples.

[0053] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0054] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0055] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0056] Terminology explanation:

[0057] O-GalNAc glycosylation: A post-translational modification of proteins that attaches N-acetylgalactosamine (GalNAc) to the side chain oxygen atom of serine, threonine or tyrosine.

[0058] Example 1 Expression and purification of recombinant G10C antibody

[0059] The heavy chain / light chain amino acid sequence of the Fab in the antigen binding region of the G10C antibody (PDB: 7UT3) is shown in SEQ ID NO: 1-2, which is connected to the homologous mouse antibody IgG2a subtype Fc (shown in SEQ ID NO: 3), and the company is responsible for optimizing the sequence and synthesizing the gene, which is then recombined into a dual-promoter expression plasmid. The antibody is further modified by adding an AviTag tag (GLNDIFEAQKIEWHE (SEQ ID NO: 4)) to the C-terminus of the heavy chain for fixing the antibody. Subsequently, the plasmid was electroporated into Chinese hamster ovary cells (CHO cells), and the enzyme-linked immunosorbent assay (ELISA) was used to screen cell lines that stably and highly expressed G10C. After 4 days of cell culture, the culture supernatant was collected and purified using Protein A+G agarose (Biyuntian, P2019) to obtain the recombinant G10C antibody, which was stored at 4°C. The recombinant plasmid map of the recombinant G10C antibody is shown below. Figure 2 As shown, the electrophoresis diagram of the purified recombinant G10C antibody is as follows Figure 3 shown.

[0060] EVKLVESGGVLVKPGGSLKLSCAASGFTFRNYDMSWVRQTPEKRLEWVASISGGYTTYYPDIMRGRFTISRDNVRNILYLEMRSLRSEDTAMYYCARNYGHDAMDYWGQGT SVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPR(SEQID NO:1).

[0061] The amino acid sequence of the heavy chain of the recombinant G10C antibody is shown in SEQ ID NO:5, and the amino acid sequence of the light chain is shown in SEQ ID NO:2.

[0062] Amino acid sequence of the heavy chain of the recombinant G10C antibody:

[0063] EVKLVESGGVLVKPGGSLKLSCAASGFTFRNYDMSWVRQTPEKRLEWVASISGGYTTYYPDIMRGRFTISRDNVRNILYLEMRSLRSEDTAMYYCARNYGHDAMDYWGQGTSVTVSSA KTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLG GPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPP EEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGSGGGSGLNDIFEAQKIEWHE(SEQ ID NO:5).

[0064] Amino acid sequence of the light chain of the recombinant G10C antibody:

[0065] QIVLTQSPAIMSASPGEKVTLTCSASSGIGFIHWYQQKPGTSPKRWIYDTSILASGVPARFSGSGSETSYSLTITIMEAEDAATYYCHQRSSYPTFGGGTKLEIKRAD AAAPVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ IDNO:2).

[0066] Example 2 Preparation of antibody-coupled streptavidin magnetic beads

[0067] This example is used to prepare magnetic beads for enriching tyrosine-O-GalNAc. The recombinant G10C antibody purified in Example 1 is labeled with biotin using biotin ligase BirA through an in vitro reaction, and further connected to streptavidin magnetic beads to obtain magnetic beads enriched with tyrosine-O-GalNAc (the preparation flow chart is shown in FIG. Figure 4 ). The specific preparation method is as follows:

[0068] (1) Biotin labeling of recombinant G10C antibody

[0069] The AviTag tag is a short peptide tag consisting of 15 amino acids (GLNDIFEAQKIEWHE). In the presence of ATP and biotin, biotin ligase (BirA) connects a biotin to the lysine residue of the AviTag tag, thereby achieving biotin labeling of the target protein (biotin labeling reaction diagram as shown in Figure 5 ). The purified recombinant G10C antibody with AviTag tag was labeled with biotin (P0630S) using the BioTec Biotin Labeling Kit and reacted at 4°C overnight. The system is shown in Table 1.

[0070] Table 1 Biotin labeling reaction system

[0071]

[0072]

[0073] (2) Biotin-labeled recombinant G10C antibody linked to streptavidin magnetic beads

[0074] The non-covalent interaction between biotin and streptavidin is the strongest known protein-ligand interaction and is a good way to immobilize antibodies. The biotin-labeled recombinant G10C antibody was incubated with streptavidin magnetic beads (MCE, HY-K0208) at an equivalent ratio of 50 μg to 100 μL. The specific operation is as follows:

[0075] 1) Suspend the magnetic beads thoroughly and take 200 μL of streptavidin magnetic bead suspension;

[0076] 2) Wash twice with 1 mL of phosphate buffered saline (PBS), mixing by inversion at 4°C for 5 min each time;

[0077] 3) Add recombinant G10C antibody labeled with phycocyanin (100 μg, final concentration 0.1 μg / μL), adjust the volume to 1 mL with PBS so that the final concentration of streptavidin magnetic beads is 2 mg / mL, and incubate at 4°C with rotation for 4 hours;

[0078] 4) Wash three times with 1 mL of PBS, rotating at 4°C for 5 min each time to remove non-specifically bound proteins;

[0079] 5) Further add 100 μL of 50 mM glycine (pH 2.4) to wash away the weakly binding antibodies, and obtain magnetic beads for enriching tyrosine-O-GalNAc. Recorded as antibody-coupled streptavidin magnetic beads;

[0080] 6) Add PBS to the magnetic beads and store at 4°C for later use.

[0081] Example 3

[0082] A method for detecting and analyzing tyrosine glycosylation sites comprises the following steps:

[0083] (1) Enrichment of tyrosine glycosylation sites

[0084] 1) Cell sample pretreatment

[0085] Collect cell pellets: The volume is about 100 μL, and the cell pellets can be stored in a -80°C refrigerator (use low protein adsorption centrifuge tubes throughout the process).

[0086] Lyse cells: Take a 1mg tube of RapiGest (a surfactant), add 500μL 50mM NH4HCO3 to dissolve to 0.2% (W / V) concentration, add to the cell pellet to resuspend. Ultrasonic power AMP 25%, on 5s, off 5s, ultrasonic for 3min for each sample; then boil at 98℃ for 5min. Then, add 50mM NH4HCO3 to make the lysate 1mL, the final concentration of RapiGest is 0.1%, repeat the ultrasonic and boiling operations. Use BCA quantification to calculate the protein amount.

[0087] Reduction / alkylation treatment: Add dithiothreitol (DTT) at a final concentration of 10 mM to the above lysate and treat at 60°C for 45 min. After standing at room temperature, add iodoacetamide (IAM) at a final concentration of 20 mM and incubate at room temperature in the dark for 30 min.

[0088] Enzyme digestion: Add trypsin (enzyme source) according to the amount of protein, the ratio of enzyme: protein = 1:100, shake at 37°C overnight. Add 5 μL TFA to make the pH less than 2 to terminate the enzyme digestion reaction, centrifuge at 18000g at 4°C for 30 minutes, and transfer the supernatant to another centrifuge tube.

[0089] Desialylation: Adjust the pH to 5-6 with 1 M sodium hydroxide, add ceramidase (Sigma, N3001) at a final concentration of 0.1 U / mL, and shake at 37° C. for 6 h.

[0090] Sep-Pak desalting treatment: Take a 3CC C18 column (Waters, WAT054945), add 8mL methanol to activate the column; add 8mL 0.1% TFA to balance the column; add the peptide fragment after enzyme digestion, repeat the loading twice; add 4mL 0.1% TFA and 4mL 0.1% FA to wash the salt; finally, use 1.5mL 50% methanol + 0.1% FA to elute the peptide fragment. First use a rotary evaporator to remove the methanol for about 2 to 3 hours, and then freeze-dry overnight.

[0091] 2) Enrichment of tyrosine glycosylated peptides

[0092] The dried peptide was resuspended with 500 μL PBS, the pH was adjusted to about neutral, and the peptide was quantified (13 μg / μL, 6.5 mg in total). The peptide was mixed with the antibody-coupled streptavidin magnetic beads of Example 2, incubated at 4°C for 4 h, then washed 3 times with 1 mL PBS, once with 1 mL of grade 3 water, and finally eluted with 100 μL 0.1% TFA 2 times, the 2 eluates were mixed, and dried with a rotary evaporator.

[0093] (2) Mass spectrometry

[0094] The mass spectrometer model was Nano LC-Q Exactive Plus (Thermo Fisher). The sample dissolved in 0.1% FA was separated on a C18 column, with mobile phase A: 0.1% FA, mobile phase B: 80% acetonitrile and 0.1% FA, flow rate 3 μL / min, time 120 min. The scan range was 355-1700 m / z, the resolution was 70000, and the automatic gain control (AGC) target value was 5e5. MS2 analysis used data-dependent acquisition, and dynamic exclusion was set to 60s. Within each cycle time, the most abundant multiply charged precursors were selected from the full scan and fragmented by high-energy collision dissociation (HCD) in the orbital trap, with a resolution of 35000 and an AGC target set to 5e4.

[0095] The results were analyzed using the software Proteome Discoverer 2.2 and compared with human sequences in the UniProt database. For the trypsin digestion mode, the cleavage sites were set to lysine and arginine, and the maximum number of missed cleavage sites allowed was 2. The variable modifications set included methionine oxidation (+15.995Da), N-terminal acetylation (+42.011Da), HexNAc (serine, threonine or tyrosine, +203.079Da); the fixed modification was carbamidomethylation of cysteine ​​(+57.021Da).

[0096] Example 4

[0097] The detection and analysis method of Example 3 was used to detect and analyze the tyrosine glycosylation sites in HEK293 wild-type cells and cells in which N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GalT1) was knocked out by CRISPR / Cas9 technology. Among them, the cells in which N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GalT1) was knocked out by CRISPR / Cas9 technology made the O-sugar chain truncated, both of which were Tn antigen and STn antigen structures, which is called SimpleCell cell line. The cells were lysed, sialic acid was removed using ceramidase, and mass spectrometry was performed after desalting (the specific process is the same as in Example 3).

[0098] The results showed that 5 tyrosine-O-GalNAc peptides were enriched and identified in HEK 293WT cells, and the sites identified on tyrosine accounted for 28% of the total glycosylation sites; 15 tyrosine-O-GalNAc peptides were enriched and identified in HEK 293SimpleCell cells, and the sites identified on tyrosine accounted for 65% of the total glycosylation sites. The site information is shown in Table 2.

[0099] Table 2 Tyrosine glycosylation site information in each cell

[0100]

[0101] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A recombinant antibody comprising a Fab fragment of a G10C antibody, an immunoglobulin Fc region and an AviTag tag.

2. The recombinant antibody according to claim 1, characterized in that The Fab fragment of the G10C antibody includes a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:1, and the amino acid sequence of the light chain is shown in SEQ ID NO:

2.

3. The recombinant antibody according to claim 1, characterized in that The Fab fragment of the G10C antibody is hingedly connected to the immunoglobulin Fc region; and / or, the AviTag tag is connected to the C-terminus of the immunoglobulin Fc region via a connecting peptide; Preferably, the amino acid sequence of the AviTag tag is shown in SEQ ID NO:

4.

4. The recombinant antibody according to any one of claims 1 to 3, characterized in that The amino acid sequence of the heavy chain of the recombinant antibody is shown in SEQ ID NO:

5.

5. Use of the recombinant antibody according to any one of claims 1 to 4 in enriching O-GalNAc glycosylated peptides or preparing a product for enriching O-GalNAc glycosylated peptides.

6. The use according to claim 5, characterized in that: The O-GalNAc glycosylated peptide segment includes a tyrosine-O-GalNAc glycosylated peptide segment.

7. An immunomagnetic bead comprising the recombinant antibody according to any one of claims 1 to 4 and streptavidin magnetic beads, wherein the recombinant antibody is coupled to the surface of the streptavidin magnetic beads via streptavidin-biotin.

8. Use of the immunomagnetic beads according to claim 7 in enriching O-GalNAc glycosylated peptides or detecting O-GalNAc glycosylation sites.

9. A method for enriching and analyzing tyrosine-O-GalNAc glycosylation sites, comprising the following steps: Mixing the immunomagnetic beads according to claim 7 with the peptide sample, incubating, and eluting to obtain tyrosine-O-GalNAc glycosylated peptides; Mass spectrometry analysis of tyrosine-O-GalNAc glycosylated peptides.

10. The enrichment and analysis method according to claim 9, characterized in that: The incubation condition is 3-5° C. for 3-5 hours.

Images