A method for preparing functionalized two-dimensional metal-organic framework materials for enriching glycopeptides

Abstract

A method for preparing a functionalized two-dimensional metal-organic framework material for enriching glycopeptides belongs to the field of protein and polypeptide enrichment. The present invention comprises the following steps: first, synthesizing two-dimensional Zr-Fc MOF nanosheets; using an in-situ reduction method to load a large amount of Au nanoparticles on the surface of the nanosheets for subsequent modification; finally, modifying glutathione (GSH) and L-cysteine ​​(L-Cys) on the surface through Au-S interaction to further improve the hydrophilicity, thereby obtaining a two-dimensional metal-organic framework composite material with high hydrophilicity. The present invention overcomes the disadvantage of low glycopeptide enrichment efficiency of traditional MOFs. At the same time, the limited hydrophilicity of the two-dimensional metal-organic framework material is improved by post-synthetic modification of hydrophilic amino acids. The functionalized two-dimensional metal-organic framework material of the present invention is simple to prepare, has excellent sensitivity, selectivity, binding capacity and reusability, and exhibits excellent enrichment ability in both model proteins and actual samples.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein and polypeptide enrichment, and specifically relates to the preparation of a functional two-dimensional metal-organic framework material and its application in glycopeptide enrichment. Background Art

[0002] Proteins are essential components of living organisms. As one of the most common post-translational modifications, protein glycosylation is associated with numerous biological processes, including molecular recognition, signal transduction, and immune activity. Numerous studies have linked abnormal glycosylation to the development and progression of numerous diseases, such as cancer, Alzheimer's disease, and autoimmune diseases. Therefore, the identification of protein glycosylation is crucial for a deeper understanding of disease mechanisms. Currently, mass spectrometry has become a leading tool for comprehensive glycoproteomics analysis due to its rapidity, high throughput, and high sensitivity. There are two main strategies for analyzing protein glycosylation using mass spectrometry: top-down and bottom-up. The top-down approach analyzes intact proteins, but separation and enrichment of glycoproteins is challenging. The bottom-up approach, on the other hand, targets peptides, enzymatically hydrolyzing glycoproteins into peptides for separation and identification, offering greater efficiency and compatibility with mass spectrometry. However, glycopeptides are low in abundance in biological samples and exhibit low ionization efficiency during mass spectrometry analysis. Furthermore, coexisting non-glycopeptides and salts can interfere with detection. Therefore, selective separation and enrichment of glycopeptides in complex biological samples before mass spectrometry analysis is the key to successful identification of glycopeptides.

[0003] Commonly used methods for glycopeptide enrichment include lectin affinity, hydrazide chemistry, boronic acid affinity, and hydrophilic interaction methods. The hydrophilic interaction method has been widely developed due to its advantages, including unbiased glycopeptide enrichment, good compatibility, selectivity, and reproducibility. A large number of hydrophilic materials with ideal enrichment selectivity have been applied, such as carbohydrates, zwitterionic materials, polymeric materials, and organic framework nanomaterials. Metal-organic frameworks (MOFs) offer significant advantages in glycopeptide enrichment due to their large surface area, high porosity, tunable pore structure, and ease of functionalization. Currently, the MOFs commonly used for glycopeptide enrichment are three-dimensional structures, but their large steric hindrance and limited binding sites result in suboptimal glycopeptide enrichment. Two-dimensional MOFs, with their larger surface area and more exposed sites on the outer surface, can avoid these drawbacks. However, the inherent hydrophilicity of two-dimensional MOFs is limited. Therefore, the development of new, more hydrophilic two-dimensional MOFs to improve glycopeptide enrichment efficiency is crucial. Summary of the Invention

[0004] The purpose of the present invention is to prepare a highly hydrophilic two-dimensional metal-organic framework material functionalized with double amino acids, and use it for the separation and enrichment of glycopeptides. The present invention uses a two-dimensional metal-organic framework material to overcome the shortcomings of the low glycopeptide enrichment efficiency of traditional MOFs. At the same time, the limited hydrophilicity of the two-dimensional metal-organic framework material is improved by post-synthetic modification of hydrophilic amino acids. The functionalized two-dimensional metal-organic framework material of the present invention is simple to prepare, with mild conditions, high glycopeptide enrichment efficiency, and excellent selectivity and reusability. The present invention uses horseradish peroxidase (HRP) and human serum immunoglobulin (IgG) as model proteins to demonstrate the excellent performance of the synthesized material for glycopeptide enrichment.

[0005] The invention first synthesizes hydrophilic Zr-Fc MOF nanosheets with Zr-O as metal clusters and ferrocene dicarboxylic acid (Fc(COOH)2) as ligands, then uses an in situ reduction method to load a large number of Au nanoparticles on the surface of the nanosheets to facilitate subsequent modification. Finally, glutathione (GSH) and L-cysteine ​​(L-Cys) are modified on the surface through Au-S interaction to further improve the hydrophilicity, thereby obtaining a functional two-dimensional metal-organic framework material with high hydrophilicity.

[0006] In order to achieve the above object, the present invention is implemented according to the following technical solutions:

[0007] A method for preparing a functionalized two-dimensional metal-organic framework material for enriching glycopeptides, characterized by comprising the following steps:

[0008] (1) Weigh 139.8 mg of ZrCl4 and 164.4 mg of Fc(COOH)2 in a 50 mL beaker, add 20 mL of DMF and 1.7 mL of CH3COOH, ultrasonicate for 30 min to disperse them evenly, then transfer them to a reactor and react at 120 °C for 12 h. After the reaction, the product was collected by centrifugation (3000 rpm, 30 min) and washed three times with DMF (20 mL each time, 3000 rpm, 30 min). Finally, the obtained Zr-FcMOF product was dispersed in 100 mL of deionized water to form 1 mg mL -1 The homogeneous solution was stored at room temperature until use.

[0009] (2) Weigh 10 mL of the Zr-FcMOF solution prepared in (1) and disperse it in 50 mL of deionized water. Ultrasonicate for 30 min to disperse it evenly. Under magnetic stirring, add HAuCl4·3H2O solution (0.05 M, 600 μL) dropwise. After reacting at room temperature for 10 min, transfer to an ice bath, add NaBH4 aqueous solution (0.5 M, 1.5 mL), and continue stirring for 10 min. After the reaction is completed, centrifuge once (8000 rpm, 5 min). The obtained product is washed with deionized water three times (20 mL each time, 8000 rpm, 5 min), and then dried at room temperature to obtain Zr-Fc MOF@Au.

[0010] (3) 10 mg of Zr-Fc MOF@Au, 30 mg of GSH, and 30 mg of L-Cys were weighed and dispersed in 30 mL of ethanol-water solution (v / v, 1 / 3). The mixture was sonicated for 10 min to achieve uniform dispersion and magnetically stirred at room temperature for 12 h. After the reaction, the product was centrifuged once (8000 rpm, 5 min) and washed three times with ethanol-water solution (v / v, 1 / 3) (20 mL each, 8000 rpm, 5 min), then washed once with anhydrous ethanol (8000 rpm, 5 min), and finally dried overnight at room temperature.

[0011] (4) Furthermore, in the above step (1), the molar ratio of ZrCl4 to Fc(COOH)2 is 1:1, and the molar ratio of ZrCl4 to CH3COOH is 1:50.

[0012] (5) Furthermore, the purpose of using 1.7 mL of CH 3 COOH in step (1) is to control the longitudinal growth of Zr-FcMOF and obtain two-dimensional Zr-FcMOF nanosheets with a thin thickness.

[0013] (6) Furthermore, the Au nanoparticles in the above step (2) are loaded to modify the Zr-FcMOF to facilitate subsequent modification.

[0014] (7) Furthermore, in the above step (3), the mass ratio of Zr-Fc MOF@Au, GSH, and L-Cys was 1:3:3.

[0015] (8) The purpose of further adding GSH and L-Cys as mentioned above is to improve the hydrophilicity of Zr-FcMOF.

[0016] (9) Furthermore, the prepared Zr-FcMOF is a two-dimensional rectangular nanosheet with a lateral size of 500 nm and a thickness of 3.6 nm.

[0017] (10) Furthermore, Au nanoparticles in the prepared Zr-FcMOF@Au@GC were distributed on the surface of Zr-FcMOF nanosheets.

[0018] (11) Furthermore, the prepared Zr-Fc MOF@Au@GC has high hydrophilicity, and because the two-dimensional morphology of the carrier itself reduces the steric hindrance with glycopeptides during enrichment and increases more binding sites, its enrichment efficiency is much higher than that of other functionalized materials used for glycopeptide enrichment.

[0019] The advantages of the present invention are:

[0020] (1) This material utilizes the ultra-high hydrophilicity of the carboxyl groups of residual ligands in MOFs and the carboxyl and amino groups of further modified GSH and L-Cys to enrich glycopeptides. This method has high versatility due to its mild reaction conditions and non-destructive properties during glycopeptide enrichment.

[0021] (2) The present invention uses a two-dimensional metal organic framework material, which can reduce the steric hindrance between the material and the glycopeptide when enriching the glycopeptide compared to traditional three-dimensional MOFs, which is beneficial to the enrichment of the glycopeptide.

[0022] (3) This material uses two-dimensional Zr-Fc MOF nanosheets as carriers. The nanosheets have a large specific surface area and can provide more affinity sites for the enrichment of glycopeptides.

[0023] (4) The present invention uses two amino acids, GSH and L-Cys, to enhance the hydrophilicity of Zr-FcMOF. Studies have shown that dual-amino acid functionalized materials exhibit higher enrichment performance compared to composites modified with only GSH or only L-Cys. The synergistic effect of GSH and L-Cys helps improve the material's ability to enrich glycopeptides.

[0024] (5) The present invention can be successfully applied to the separation and enrichment of glycopeptides in model proteins HRP and IgG, as well as the enrichment and identification of glycopeptides in complex biological samples such as human serum. It is a universal glycopeptide enrichment material. DETAILED DESCRIPTION

[0025] The present invention will be described in detail below with reference to the embodiments, but they are not intended to limit the present invention.

[0026] Example 1: Enrichment of glycopeptides from HRP and IgG enzymatic hydrolysates using functionalized two-dimensional metal-organic frameworks

[0027] (1) Preparation process of functionalized two-dimensional metal-organic framework materials

[0028] (a) Preparation process of Zr-Fc MOF: 139.8 mg of ZrCl4 and 164.4 mg of Fc(COOH)2 were weighed and dissolved in 20 mL of DMF, 1.7 mL of CH3COOH was added, and ultrasonication was performed for 30 min to make it uniformly dispersed. The mixture was then transferred to a reactor and reacted at 120°C for 12 h. After the reaction, the product was collected by centrifugation (3000 rpm, 30 min) and washed three times with DMF (20 mL each time, 3000 rpm, 30 min). The obtained product was then dispersed in 100 mL of deionized water to form 1 mg mL -1 The homogeneous solution was stored at room temperature until use.

[0029] (b) Preparation process of Zr-Fc MOF@Au: Zr-Fc MOF solution (1 mg mL -1 , 10 mL) was dispersed in 50 mL of deionized water and sonicated for 30 minutes to achieve uniform dispersion. Under magnetic stirring, HAuCl4·3H2O solution (0.05 M, 600 μL) was added dropwise. After reacting at room temperature for 10 minutes, the mixture was transferred to an ice bath, and NaBH4 aqueous solution (0.5 M, 1.5 mL) was added and stirred for another 10 minutes. After the reaction, the mixture was rapidly centrifuged once (8000 rpm, 5 minutes). The resulting product was washed three times with deionized water (20 mL each, 8000 rpm, 5 minutes each) and then dried at room temperature to obtain Zr-Fc MOF@Au.

[0030] (c) Preparation of Zr-Fc MOF@Au@GC: 10 mg of Zr-Fc MOF@Au, 30 mg of GSH, and 30 mg of L-Cys were dispersed in 30 mL of ethanol-water (v / v, 1 / 3) solution. Ultrasonication was performed for 10 min to achieve uniform dispersion, followed by magnetic stirring at room temperature for 12 h. After the reaction, the product was centrifuged once (8000 rpm, 5 min) and washed three times with ethanol-water (v / v, 1 / 3) solution (20 mL each, 8000 rpm, 5 min), then washed once with anhydrous ethanol (8000 rpm, 5 min), and finally dried overnight at room temperature.

[0031] (2) Preparation of HRP (IgG) enzymatic solution: Weigh 1 mg of HRP or IgG and dissolve it in 625 μL of denaturing buffer solution (50 mM NH4HCO3, 8 M urea, pH = 8.2). Then, reduce it with 5 μL of DTT (200 mM) at 56°C for 45 min. After cooling to room temperature, add 20 μL of IAA (200 mM) for alkylation. React at room temperature in the dark for 45 min. Then, dilute the sample with 50 mM NH4HCO3 buffer solution to a final protein concentration of 0.2 mg mL -1Trypsin (enzyme / protein = 1 / 40, w / w) was added and the reaction was allowed to proceed at 37°C for 18 h. Finally, 5 μL of FA was added to terminate the enzymatic hydrolysis reaction. The resulting enzymatic hydrolysate was frozen and stored at -20°C for further use and analysis.

[0032] (3) For enrichment of glycopeptides in HRP hydrolyzate: 500 μg of the prepared composite material Zr-Fc MOF@Au@GC was weighed and evenly dispersed in 200 μL of loading buffer solution (ACN / H2O / TFA=89 / 10 / 1, v / v / v) containing 20 μL of HRP hydrolyzate, and incubated on a shaker (400 rpm) at room temperature for 35 min. After incubation, centrifuge once (6000 rpm, 3 min) to remove the supernatant, and then wash three times with 200 μL of washing buffer solution (ACN / TFA=99 / 1, v / v) on a shaker (400 rpm, 5 min) to remove the interference of non-glycopeptides. The captured glycopeptides were eluted with 20 μL of elution buffer solution (H2O / TFA=99 / 1, v / v) on a shaker (400 rpm, 15 min), centrifuged once (6000 rpm, 5 min), and the supernatant was collected. The eluate containing glycopeptides was directly subjected to MALDI-TOF-MS analysis (three parallel experiments were performed). Simultaneously, the HRP hydrolyzate was directly analyzed by MALDI-TOF-MS as a comparative experiment. The results showed that when the HRP hydrolyzate was analyzed directly by MALDI-TOF-MS, only three glycopeptides with very low signal intensities were detected. However, after enrichment with Zr-Fc MOF@Au@GC, up to 39 glycopeptides with high-intensity signals were detected, and the signals of non-glycopeptides were also greatly reduced, demonstrating that the prepared Zr-Fc MOF@Au@GC has excellent selective enrichment capabilities for glycopeptides in HRP hydrolyzates.

[0033] (4) For enrichment of glycopeptides in IgG hydrolysate: 500 μg of the prepared composite material Zr-Fc MOF@Au@GC was weighed and evenly dispersed in 200 μL of loading buffer solution (ACN / H2O / TFA=89 / 10 / 1, v / v / v) containing 20 μL of IgG hydrolysate, and incubated on a shaker (400 rpm) at room temperature for 35 min. After incubation, centrifuge once (6000 rpm, 3 min) to remove the supernatant, and then wash three times with 200 μL of washing buffer solution (ACN / TFA=99 / 1, v / v) on a shaker (400 rpm, 5 min) to remove the interference of non-glycopeptides. The captured glycopeptides were eluted with 20 μL of elution buffer solution (H2O / TFA=99 / 1, v / v) on a shaker (400 rpm, 15 min), centrifuged once (6000 rpm, 5 min), and the supernatant was collected. The eluate containing glycopeptides was directly subjected to MALDI-TOF-MS analysis (three parallel experiments were performed). Simultaneously, the IgG enzymatic hydrolysate was directly analyzed by MALDI-TOF-MS as a comparative experiment. The results showed that when the IgG enzymatic hydrolysate was directly analyzed by MALDI-TOF-MS, only four glycopeptide signals with low signal-to-noise ratios were detected. However, after enrichment with Zr-Fc MOF@Au@GC, 44 high-intensity glycopeptide signals were detected, and the signal-to-noise ratio of non-glycopeptides was also greatly reduced, indicating that the prepared Zr-Fc MOF@Au@GC also has excellent glycopeptide enrichment performance for IgG enzymatic hydrolysates.

[0034] (5) Investigation of the sensitivity of functionalized two-dimensional metal-organic framework materials: The HRP hydrolyzate in (2) was diluted with 50 mM NH4HCO3 solution to obtain HRP hydrolyzates with concentrations of 10 fmol / μL, 1 fmol / μL, and 0.1 fmol / μL, respectively. Then, the prepared Zr-Fc MOF@Au@GC (500 μg × 3) was weighed and evenly dispersed in 200 μL of loading buffer solution (ACN / H2O / TFA = 89 / 10 / 1, v / v / v) containing 20 μL of HRP hydrolyzate with concentrations of 10, 1, and 0.1 fmol / μL, respectively. The mixture was incubated on a shaker (400 rpm) at room temperature for 35 min. After incubation, the supernatant was removed and the mixture was washed three times with 200 μL of washing buffer solution (ACN / TFA = 99 / 1, v / v) on a shaker (400 rpm, 5 min) to remove interference from non-glycopeptides. Finally, the resulting Zr-Fc MOF@Au@GC was eluted with 20 μL of elution buffer (H₂O / TFA = 99 / 1, v / v) on a shaker (400 rpm, 15 min). After centrifugation once (6000 rpm, 5 min), the supernatant was collected. The eluate containing glycopeptides was directly subjected to MALDI-TOF-MS analysis (triplicate runs were performed for each HRP hydrolyzate concentration). The results showed that 15 glycopeptides were enriched at an HRP hydrolyzate concentration of 10 fmol / μL, and 8 at 1 fmol / μL. When the concentration was reduced to 0.1 fmol / μL, 2 glycopeptides were still enriched, demonstrating the excellent sensitivity of the Zr-Fc MOF@Au@GC material for glycopeptide detection.

[0035] (6) Investigation of the selectivity of functionalized two-dimensional metal-organic framework materials:

[0036] (a) Preparation of BSA enzymatic hydrolysate: the same as the enzymatic hydrolysis process of HRP and IgG in Example 1 (2).

[0037] (b) HRP and BSA hydrolysates were mixed at a mass ratio of 1:100, 1:1000, and 1:2000, respectively, and then dispersed in 200 μL of loading buffer (ACN / H2O / TFA = 89 / 10 / 1, v / v / v) (wherein the mass of HRP hydrolysate was 4 μg). 500 μg of the prepared Zr-Fc MOF@Au@GC was weighed and evenly dispersed in the mixed solution. The mixture was then incubated on a shaker (400 rpm) at room temperature for 35 min. After incubation, the mixture was centrifuged once (6000 rpm, 3 min) and the supernatant was removed. The mixture was then washed three times (400 rpm, 5 min) with 200 μL of wash buffer (ACN / TFA = 99 / 1, v / v) on a shaker to remove non-glycopeptide interference. The captured glycopeptides were eluted with 20 μL of elution buffer (H₂O / TFA = 99 / 1, v / v) on a shaker (400 rpm, 15 min). After centrifugation once (6000 rpm, 5 min), the supernatant was collected. The glycopeptide-containing eluate was directly subjected to MALDI-TOF-MS analysis (triplicate runs were performed for each concentration ratio). The results showed that 28 glycopeptides were enriched in a mixture of HRP hydrolyzate and BSA hydrolyzate at a ratio of 1:100. A mixture of 18 glycopeptides at a ratio of 1:1000 was enriched. When the ratio was increased to 1:2000, 17 glycopeptides were still enriched, demonstrating the excellent selectivity of the Zr-Fc MOF@Au@GC material.

[0038] (7) Investigation of the binding capacity of functionalized two-dimensional metal-organic framework materials: Different masses of Zr-Fc MOF@Au@GC (10 μg to 100 μg) were weighed and dispersed in 200 μL of loading buffer solution (ACN / H2O / TFA = 89 / 10 / 1, v / v / v) (wherein the mass of HRP enzymatic hydrolyzate was 12 μg), and incubated at room temperature for 35 min on a shaker (400 rpm). After incubation, the mixture was centrifuged once (6000 rpm, 3 min) to remove the supernatant, and then washed three times on a shaker (400 rpm, 5 min) with 200 μL of washing buffer solution (ACN / TFA = 99 / 1, v / v) to remove interference from non-glycopeptides. The captured glycopeptides were eluted with 20 μL of elution buffer (H₂O / TFA = 99 / 1, v / v) on a shaker (400 rpm, 15 min). The supernatant was collected after centrifugation once (6000 rpm, 5 min). The glycopeptide-containing eluate was directly subjected to MALDI-TOF-MS analysis. The results showed that the S / N ratio of the enriched glycopeptides increased with increasing material dosage, reaching a maximum at 60 μg and remaining relatively constant thereafter, demonstrating that the invented Zr-Fc MOF@Au@GC material exhibits excellent glycopeptide enrichment performance.

[0039] (7) Investigation of the reusability of functionalized two-dimensional metal-organic framework materials:

[0040] (a) Glycopeptide enrichment process: the same as the HRP enrichment process in Example 1 (3).

[0041] (b) The Zr-Fc MOF@Au@GC material eluted in (a) was collected and washed three times using 200 μL of elution buffer (H2O / TFA = 99 / 1, v / v) and 200 μL of loading buffer (ACN / H2O / TFA = 89 / 10 / 1, v / v / v) on a shaker (400 rpm, 10 min). The supernatant was removed and used for glycopeptide enrichment in the HRP enzymatic hydrolyzate in the next cycle. This process was repeated five times, and the eluate obtained each time was used for MALDI-TOF-MS analysis. The number of glycopeptides detected after three and five reuses was essentially the same as that after the first reuse, indicating that the Zr-Fc MOF@Au@GC prepared by this invention has good reusability.

[0042] Example 2: Enrichment of glycopeptides from human serum enzymatic hydrolysate using functionalized two-dimensional metal-organic frameworks

[0043] (1) Preparation process of functionalized two-dimensional metal organic framework material Zr-Fc MOF@Au@GC: same as (1) in Example 1.

[0044] (2) Preparation of human serum sample enzymatic hydrolysate: First, 10 μL of human serum was diluted with 10 μL of deionized water and mixed evenly with 90 μL of denaturing buffer solution (50 mM NH4HCO3, 8 M urea, pH = 8.2). Then, 5 μL of DTT (200 mM) was added and reacted at 56°C for 45 min. Then, 20 μL of IAA (400 mM) was added and reacted at room temperature in the dark for 45 min. The sample was then diluted to 1 mL with 50 mM NH4HCO3 buffer solution, and trypsin (enzyme / protein = 1 / 40, w / w) was added and reacted at 37°C for 18 h. Finally, 5 μL of FA was added to terminate the enzymatic hydrolysis reaction. The obtained enzymatic hydrolysate was lyophilized at -20°C for later use.

[0045] (3) The lyophilized human serum sample from (2) of Example 2 was mixed evenly with 1 mL of loading buffer (ACN / H2O / TFA = 89 / 10 / 1, v / v / v) containing 2.5 mg of Zr-Fc MOF@Au@GC and incubated on a shaker (400 rpm) for 35 min at room temperature. After incubation, the mixture was centrifuged once (6000 rpm, 3 min) to remove the supernatant. The obtained product was washed three times (400 rpm, 5 min) with 900 μL of washing buffer (ACN / TFA = 99 / 1, v / v) on a shaker. Finally, the product was eluted twice (400 rpm, 15 min) (15 min each time) with 50 μL of elution buffer (H2O / TFA = 99 / 1, v / v) on a shaker, centrifuged once (6000 rpm, 5 min), and the supernatant was collected.

[0046] (4) The eluate from Example 2(3) was lyophilized and dissolved in 43 μL of deionized water. 4 μL of 10× GlycoBuffer 2 and 2 μL of PNGase F were added, and the mixture was incubated at 37°C in a shaker (400 rpm) for 18 h. Finally, the solution was lyophilized and analyzed by LC-MS / MS.

[0047] (5) The obtained LC-MS / MS data were processed using Proteome Discoverer software. A total of 655 glycopeptides belonging to 366 glycoproteins were detected, indicating that the invented Zr-Fc MOF@Au@GC composite material exhibited excellent enrichment ability in complex biological samples and has broad application prospects in glycoproteomics.

Claims

1. A method for preparing a functionalized two-dimensional metal-organic framework material for enriching glycopeptides, characterized in that: The following steps are involved: (1) Weigh 139.8 mg of ZrCl4 and 164.4 mg of Fc(COOH)2 in a 50 mL beaker, add 20 mL of DMF and 1.7 mL of CH3COOH, and sonicate for 30 min to disperse them uniformly; transfer the mixed solution to a reactor and react at 120 °C for 12 h; after the reaction, wash the obtained Zr-Fc MOF with DMF; finally, disperse the obtained product in 100 mL of deionized water to form a uniform solution and store it at room temperature for use; (2) Disperse 10 mL of Zr-Fc MOF solution in 50 mL of deionized water. The concentration of Zr-Fc MOF solution is 1 mg mL -1 , ultrasonically dispersed for 30 min; under magnetic stirring, 600 μL of 0.05 M HAuCl4·3H2O solution was added dropwise, stirred at room temperature for 10 min, transferred to an ice bath, and 1 mL of 0.5 M NaBH4 aqueous solution was added, and stirring was continued for 10 min; after the reaction was completed, the product was washed with deionized water and then dried at room temperature to obtain Zr-Fc MOF@Au; (3) 10 mg of Zr-Fc MOF@Au, 30 mg of glutathione GSH, and 30 mg of cysteine ​​L-Cys were weighed and dispersed in 30 mL of ethanol-water solution and stirred at room temperature for 12 h. After the reaction, the obtained products were washed with ethanol-water solution and anhydrous ethanol, respectively, and then dried at room temperature overnight to obtain the desired dual amino acid functionalized two-dimensional metal-organic framework material Zr-Fc MOF@Au@GC.

2. The method for preparing a functionalized two-dimensional metal-organic framework material for enriching glycopeptides according to claim 1, characterized in that: When washing with DMF after the reaction in step (1) is completed, the amount of DMF used is 20 mL; the concentration of the uniform solution formed by dispersing the product in 100 mL of deionized water in step (1) is 1 mg·mL -1 .

3. The method for preparing a functionalized two-dimensional metal-organic framework material for enriching glycopeptides according to claim 1, characterized in that: When washing with deionized water after the reaction in step (2) is completed, the amount of deionized water used is 20 mL.

4. The method for preparing a functionalized two-dimensional metal-organic framework material for enriching glycopeptides according to claim 1, characterized in that: The ethanol-water solution in step (3) is a mixed solution of ethanol and water in a volume ratio of 1:3; after the reaction in step (3) is completed, it is first washed three times with the ethanol-water solution and then washed once with anhydrous ethanol, with the amount of each being 20 mL.

5. A material prepared according to the method for preparing a functionalized two-dimensional metal-organic framework material for enriching glycopeptides according to claim 1.