A magnetic bead for enriching a tyrosine N-acetylgalactosamine modification site, a preparation method and application thereof

Abstract

This invention belongs to the field of biochemical analysis technology, and discloses a magnetic bead for enriching tyrosine N-acetylgalactosamine modification sites, its preparation method, and its application. Specifically, it discloses a recombinant antibody, including the Fab fragment of a G10C antibody, the Fc region of an immunoglobulin, and an AviTag tag. This invention provides a recombinant antibody capable of specifically targeting and enriching tyrosine-O-GalNAc glycosylated peptides. By coupling this recombinant antibody to magnetic beads, the enrichment of tyrosine-O-GalNAc glycosylated peptides can be effectively improved, and interference and signal masking by glycosylation modifications on serine and threonine can be avoided, enabling the identification of more tyrosine glycosylation sites in a single sample.

Description

Technical Field

[0001] This invention belongs to the field of biochemical analysis technology, specifically relating to a magnetic bead for enriching tyrosine N-acetylgalactosamine modification sites, its preparation method, and its application. Background Technology

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

[0003] Traditional research on O-GalNAc glycosylation has focused on glycosylation sites at serine and threonine residues in proteins, with relatively little research on tyrosine O-GalNAc glycosylation sites. Studies have shown that tyrosine O-GalNAc glycosylation on amyloid precursor protein (APP) is associated with Alzheimer's disease (AD), suggesting that the role of tyrosine O-GalNAc glycosylation in the physiological and pathological processes requires further investigation. 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. Commonly used lectins for enriching O-glycans include: VVA (Vicia villosa lectin), which recognizes N-acetylgalactosamine residues at the ends of the sugar chains, especially Tn antigens; PNA (Peanut agglutinin), which preferentially binds to T antigens (DOI: 10.1038 / nmeth.1731); and Jacalin, which recognizes O-linked oligosaccharides, especially T antigen structures (flowchart shown in the diagram). Figure 1(DOI: 10.1038 / s41467-022-33806-8). However, because O-GalNAc glycosylations modifying serine and threonine in proteins are abundant in cells and tissues, this method of using lectins to enrich and identify tyrosine O-GalNAc glycosylation sites is greatly interfered with, resulting in low identification efficiency. That is, lectins have weak affinity, insufficient enrichment efficiency, and are subject to interference from a large number of non-O-glycopeptides. Only 3.7% of the identified glycosylation sites were on tyrosine (DOI: 10.1038 / s41467-022-33806-8), failing to specifically enrich tyrosine glycosylated peptides.

[0005] Therefore, there is an urgent need to develop a biomaterial or method that can specifically enrich tyrosine glycosylated peptides, thereby avoiding the interference and signal masking caused by glycosylation modifications on serine and threonine. Summary of the Invention

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

[0007] A second aspect of the present invention aims to provide the use of the recombinant antibody of the first aspect of the present invention in enriching O-GalNAc glycosylated peptides or in preparing products for enriching O-GalNAc glycosylated peptides.

[0008] The third objective of this invention is to provide an immunomagnetic bead.

[0009] The fourth aspect of this invention aims to provide the application of immunomagnetic beads from the third aspect of this invention in enriching O-GalNAc glycosylated peptides or detecting O-GalNAc glycosylation sites.

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

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] In a first aspect, 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 Fc region of the immunoglobulin.

[0015] In some embodiments of the present invention, the immunoglobulin is 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 Fc region of the immunoglobulin is as shown in SEQ ID NO:3; or an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:3 after substitution, deletion or addition of one or more amino acids.

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

[0019] In some embodiments of the present invention, the linker 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] A 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 in preparing products 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] A third aspect of the present invention provides an immunomagnetic bead comprising a recombinant antibody from 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 conjugate.

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

[0027] The recombinant antibody was 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] Biotin-modified recombinant antibodies are 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) attaches a biotin to a lysine residue of the AviTag tag, thereby achieving biotin labeling of the target antibody.

[0030] A fourth aspect of the present invention provides the application of immunomagnetic beads from 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] The immunomagnetic beads of the third aspect of the present invention were mixed with peptide samples, incubated, and eluted to obtain tyrosine-O-GalNAc glycosylated peptides.

[0034] Mass spectrometry analysis was performed on tyrosine-O-GalNAc glycosylated peptides.

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

[0036] In some embodiments of the present invention, the peptide sample is 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 a method comprising:

[0038] Cell lysis: The cells were mixed with RapiGest and NH4HCO3 solution, sonicated, boiled, and the sonication and boiling were repeated several times to obtain the lysate.

[0039] Reduction / alkylation treatment: Dithiothreitol was added to the lysis buffer and treated at 55-65℃ for 40-50 min. Iodoacetamide was added and incubated in the dark for 25-35 min to 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] Desialic acid removal: Adjust the pH of the supernatant to 5-6, add ceramide enzyme, and shake at 37°C for 5-7 hours;

[0042] Sep-Pak desalting: Desalting was performed using a C18 column to obtain peptide samples.

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

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

[0045] The beneficial effects of this invention are:

[0046] This invention provides a recombinant antibody capable of specifically targeting and enriching tyrosine-O-GalNAc glycosylated peptides. By coupling this recombinant antibody to magnetic beads, the enrichment of tyrosine-O-GalNAc glycosylated peptides can be effectively improved, and interference and signal masking by glycosylation modifications on serine and threonine can be avoided, enabling the identification of more tyrosine glycosylation sites in a single sample. Attached Figure Description

[0047] Figure 1 This 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 a recombinant plasmid map of the recombinant G10C antibody.

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

[0050] Figure 4This is a schematic diagram of the magnetic beads enriched with tyrosine-O-GalNAc prepared according to the present invention and the enrichment of the magnetic beads.

[0051] Figure 5 This is a schematic diagram of the biotin labeling reaction. Detailed Implementation

[0052] The present invention will be further described in detail below through specific embodiments.

[0053] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0054] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0055] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0056] Terminology Explanation:

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

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

[0059] The heavy / light chain amino acid sequence of the antigen-binding region Fab of the G10C antibody (PDB: 7UT3) is shown in SEQ ID NO: 1-2. This sequence was ligated to the homologous mouse antibody IgG2a subtype Fc (SEQ ID NO: 3). The sequence was optimized and the gene synthesized by the company, and then recombined into a dual-promoter expression plasmid. The antibody was further modified by adding an AviTag tag (GLNDIFEAQKIEWHE (SEQ ID NO: 4)) to the C-terminus of the heavy chain for antibody immobilization. Subsequently, the plasmid was electroporated into Chinese hamster ovary cells (CHO cells), and cell lines stably expressing high levels of G10C were screened using enzyme-linked immunosorbent assay (ELISA). After 4 days of cell culture, the culture supernatant was collected and purified using Protein A+G agarose (Beyotime, 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 As shown.

[0060] EVKLVESGGVLVKPGGSLKLSCAASGFTFRNYDMSWVRQTPEKRLEWVASISGGYTTYYPDIMRGRFTISRDNVRNILYLEMRSLRSEDTAMYYCARNYGHDAMDYWGQGT SVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPR(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] The amino acid sequence of the heavy chain of the recombinant G10C antibody:

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

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

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

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

[0067] This embodiment is used to prepare magnetic beads for enriching tyrosine-O-GalNAc. The recombinant G10C antibody purified in Example 1 was subjected to an in vitro reaction, and biotin was labeled with biotin using biotin ligase BirA, which was then further ligated to streptavidin magnetic beads to obtain magnetic beads enriched for tyrosine-O-GalNAc (preparation flowchart shown). 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) attaches a biotin to a lysine residue of the AviTag tag, thereby achieving biotinylation of the target protein (see schematic diagram of the biotinylation reaction). Figure 5 The purified AviTag-tagged recombinant G10C antibody was reacted with biotin (P0630S) using Beyotime Biotin Labeling Kit and incubated overnight at 4°C. The reaction system is shown in Table 1.

[0070] Table 1 Biotin-labeled reaction system

[0071]

[0072]

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

[0074] The non-covalent interaction between biotin and streptavidin is arguably the strongest known protein-ligand interaction, making it an excellent method for antibody immobilization. Biotin-labeled recombinant G10C antibody was incubated with streptavidin magnetic beads (MCE, HY-K0208) at an equivalent ratio of 50 μg to 100 μL, as follows:

[0075] 1) Thoroughly suspend the magnetic beads, and take 200 μL of streptavidin magnetic bead suspension;

[0076] 2) Add 1 mL of phosphate-buffered saline (PBS) and wash twice, inverting and mixing for 5 min each time at 4°C;

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

[0078] 4) Wash 3 times with 1 mL 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 antibody, obtaining magnetic beads for enriching tyrosine-O-GalNAc. These are designated as antibody-coupled streptavidin magnetic beads.

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

[0081] Example 3

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

[0083] (1) Enrichment of tyrosine glycosylation sites

[0084] 1) Cell sample pretreatment

[0085] Collect cell pellet: approximately 100 μL. The cell pellet can be stored at -80°C (using low-protein adsorption centrifuge tubes throughout the process).

[0086] Cell lysis: Dissolve 1 mg of RapiGest (a surfactant) in 500 μL of 50 mM NH4HCO3 to a concentration of 0.2% (w / v), and resuspend in the cell pellet. Sonicate at AMP 25% for 5 seconds on and 5 seconds off, for 3 minutes per sample; then boil at 98°C for 5 minutes. Next, bring the lysis buffer to 1 mL with 50 mM NH4HCO3, bringing the final RapiGest concentration to 0.1%. Repeat the sonication and boiling process. Quantitative analysis using BCA is performed to calculate protein content.

[0087] Reduction / alkylation treatment: Add 10 mM dithiothreitol (DTT) to the above lysis buffer and treat at 60°C for 45 min; after placing at room temperature, add 20 mM iodoacetamide (IAM) and treat at room temperature in the dark for 30 min.

[0088] Enzyme digestion: Add trypsin (enzyme source) according to the protein content, at a ratio of enzyme:protein = 1:100, and incubate overnight at 37°C with shaking. Add 5 μL of TFA to lower the pH to stop the enzyme digestion reaction, centrifuge at 18000g at 4°C for 30 min, and transfer the supernatant to another centrifuge tube.

[0089] Desialic acid: Adjust the pH to 5-6 with 1M sodium hydroxide, add 0.1U / mL ceramide enzyme (Sigma, N3001) to a final concentration, and shake at 37℃ for 6 hours.

[0090] Sep-Pak desalting treatment: Take a 3CC C18 column (Waters, WAT054945), add 8 mL of methanol to activate the column; add 8 mL of 0.1% TFA to equilibrate the column; add the enzyme-digested peptide, repeating the loading twice; add 4 mL of 0.1% TFA and 4 mL of 0.1% FA to wash the salts; finally, elute the peptides with 1.5 mL of 50% methanol + 0.1% FA. Remove the methanol using a rotary evaporator for about 2-3 hours, then freeze-dry overnight.

[0091] 2) Enrichment of tyrosine-glycosylated peptides

[0092] The dried peptide fragments were resuspended in 500 μL PBS, and the pH was adjusted to approximately neutral. The peptide fragments were quantified (13 μg / μL, total 6.5 mg). The peptide fragments were mixed with the antibody-conjugated streptavidin magnetic beads from Example 2 and incubated at 4°C for 4 h. After washing three times with 1 mL PBS, the peptide fragments were washed once with 1 mL of tertiary water. Finally, the peptide fragments were eluted twice with 100 μL of 0.1% TFA. The two eluents were mixed and dried using a rotary evaporator.

[0093] (2) Mass spectrometry analysis

[0094] The mass spectrometer was a Nano LC-Q Exactive Plus (Thermo Fisher). Samples dissolved in 0.1% FA were separated on a C18 column. 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, resolution: 70,000, and automatic gain control (AGC) target value: 5e5. MS2 analysis used data-dependent acquisition with dynamic exclusion set to 60 s. Within each cycle, the most abundant multicharge precursors from the full scan were selected for fragmentation via high-energy collisional dissociation (HCD) in the orbital trap, resolution: 35,000, and AGC target set to 5e4.

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

[0096] Example 4

[0097] The detection and analysis methods of Example 3 were used to detect and analyze tyrosine glycosylation sites in HEK293 wild-type cells and cells in which N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GalT1) was knocked out using CRISPR / Cas9 technology. Cells in which N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GalT1) was knocked out using CRISPR / Cas9 technology resulted in a shortened O-glycan chain, containing both Tn and STn antigen structures; this was termed the SimpleCell cell line. Cells were lysed, sialic acid was removed using ceramidinase, and the cells were desalted before mass spectrometry analysis (the specific procedure is the same as in Example 3).

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

[0099] Table 2 Information on tyrosine glycosylation sites in each cell

[0100]

[0101] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A recombinant antibody, wherein the amino acid sequence of the heavy chain of the recombinant antibody is shown in SEQ ID NO:5, and the amino acid sequence of the light chain is shown in SEQ ID NO:

2.

2. The recombinant antibody of claim 1 can be enriched for purposes other than disease diagnosis or treatment. O -GalNAc glycosylated peptides or preparations for enrichment O Applications of -GalNAc glycosylated peptides in products, the O -GalNAc glycosylated peptides are tyrosine- O -GalNAc glycosylated peptides.

3. An immunomagnetic bead comprising the recombinant antibody of claim 1 and streptavidin magnetic beads, wherein the recombinant antibody is coupled to the surface of the streptavidin magnetic beads via streptavidin-biotin conjugate.

4. The immunomagnetic beads of claim 3 can be used for enrichment of immunomagnetic beads for purposes other than disease diagnosis or treatment. O -GalNAc glycosylated peptides or detection O Application in -GalNAc glycosylation sites, the O -GalNAc glycosylation to tyrosine- O -GalNAc glycosylation.

5. A tyrosine derivative for non-disease diagnostic or therapeutic purposes. O The enrichment and analysis method for GalNAc glycosylation sites includes the following steps: The immunomagnetic beads described in claim 3 were mixed with the peptide sample, incubated, and eluted to obtain tyrosine- O -GalNAc glycosylated peptides; paratyrosine- O Mass spectrometry analysis was performed on the glycosylated peptides of GalNAc.

6. The enrichment and analysis method according to claim 5, characterized in that, The incubation conditions are 3-5℃ for 3-5 hours.

Images