Preparation method and application of magnetic core-porous hydrophilic MOFs composite for serum glycopeptide analysis

Abstract

The application discloses a preparation method and application of a novel composite material of magnetic core-porous hydrophilic MOFs for serum glycopeptide analysis. First, based on the in-situ MOF growth strategy, the composite material with a magnetic inner core and a multistage-pore MOFs shell is synthesized. Then, the composite material is used for glycopeptide separation and enrichment of a target sample, and then MALDI-TOF MS analysis and LC-MS / MS analysis are carried out. The combination of the composite material and the robust enrichment process can realize ultra-low detection limit (1 amol / μL) and ultra-high repeatability (CV<15%) of glycopeptides, and the finally detected serum glycopeptides can be further used for screening of disease-related differential expression markers.

Description

Technical Field

[0001] This invention relates to a method for preparing a magnetic core-porous hydrophilic MOF composite material for serum glycopeptide analysis and its application, particularly to a method for preparing a composite material with a magnetic core and a hierarchical porous hydrophilic MOF shell and its application in the separation and enrichment of glycopeptides. The glycopeptide separation and enrichment process based on this composite material exhibits excellent technical reproducibility and detection limits, and successfully characterized significant differences between healthy individuals and patients with urinary system cancers. Background Technology

[0002] As one of the most common post-translational modifications of human proteins, glycosylation contributes to the heterogeneity of cellular molecules and the diversity of biological functions. Therefore, glycosylation processes in mammals contain rich biological information and are closely intertwined with key biological processes such as substance transport, cell recognition, signal transduction, and cell adhesion. Particularly in tumorigenesis and progression, glycosylation plays a crucial role in the growth, proliferation, adhesion, and invasion of cancer cells. Cancer development is often accompanied by changes in the glycosylation of related proteins, such as prostate-specific antigen (PSA), a widely used biomarker for prostate cancer monitoring and diagnosis. Studies have shown that changes in sialylation and core fucosylation levels in serum PSA can distinguish between benign and malignant prostate cancer. To further establish the association between disease and glycosylation, more information about glycosylated peptides, glycosylation sites, and glycan structures is needed.

[0003] Despite the wealth of information contained in glycopeptides, the direct detection of glycopeptides in blood faces significant challenges due to factors such as low abundance of individual glycopeptides, interference from non-glycopeptides, the complexity of serum proteins, and the technical limitations of mass spectrometry platforms. Therefore, glycopeptide analysis techniques based on nanomaterials offer a promising solution to overcome these limitations. Ideally, nanomaterial-based enrichment techniques could serve as a standardized platform to concentrate low-abundance glycopeptides in complex samples, thereby increasing their relative concentration. Currently, various glycopeptide enrichment strategies exist, including boric acid affinity, lectin affinity, hydrophilic interactions, and hydrazine chemistry methods. Among these, the widely adopted hydrophilic interaction strategy utilizes a relatively hydrophobic organic mobile phase or solution to retain glycopeptides on a hydrophilic material, thus separating them from other components in the mixture. Given the high selectivity, high retention capacity, and unbiased enrichment properties of hydrophilic materials for glycopeptides, they have become the preferred choice for separating glycopeptides from complex samples. Common hydrophilic materials include silica-coated materials, carbohydrate-modified materials, zwitterionic functionalized materials, amide-functionalized materials, and metal-organic framework (MOF)-based materials.

[0004] In recent years, due to their large specific surface area, strong adsorption capacity, tunable pore size, chemical modification capabilities, and excellent stability, certain hydrophilic MOFs have shown great application potential in the field of glycopeptide enrichment. Currently, MOFs used for glycopeptide research can be divided into three categories: original MOFs, surface-modified MOFs, and MOF-derived composites. Considering the portability of the glycopeptide enrichment process, Fe3O4-based MOF composites have significant advantages due to their inherent magnetic responsiveness, enabling rapid material separation and repeated use. Furthermore, hierarchical porous MOFs have also attracted widespread attention, not only because their micro-mesoporous channels provide a high specific surface area, but also because their macroporous energy promotes molecular diffusion and improves mass transfer efficiency. However, as an emerging material, Fe3O4-based MOF composites reported for glycopeptide enrichment are still relatively rare, especially those with open macro-mesoporous MOF structures. Li et al. proposed an in-situ MOF growth strategy as a reference, in which Mg²⁺ ions are immobilized through interaction with surface carboxyl groups, and then organic ligands are introduced to form MOFs (Fe3O4@Mg-MOF-74).

[0005] Therefore, in order to improve the efficiency of glycopeptide enrichment, this invention is dedicated to developing a MOF composite material that combines the core structural features of Fe3O4 with a hierarchical porous MOF shell, as well as its preparation method and application. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a magnetic core-porous hydrophilic MOF composite material for serum glycopeptide analysis and its application.

[0007] This invention proposes a method for preparing a novel magnetic core-porous hydrophilic MOF composite material (FeM@PMOFs) for serum glycopeptide analysis. The method employs an in-situ MOF growth strategy, using Ce³⁺ / Ce 4 ⁺ Anchored on the Fe3O4 surface modified with mercaptoacetic acid (MAA), a template agent and ligand are introduced via a microemulsion-mediated self-assembly method to guide the epitaxial growth of MOFs on the Fe3O4@MAA surface; the specific steps are as follows:

[0008] (1): Dissolve 1-2 g of ferric chloride hexahydrate completely in 50-100 mL of ethylene glycol. Stir until the solution is clear and transparent, then add 2-5 g of sodium acetate. After stirring and sonicating with magnetic force, transfer the solution to a polytetrafluoroethylene reactor and heat at 200°C for 12-16 hours. After the reaction is complete, let the reactor cool to room temperature and collect the supernatant by magnetic force. Wash the solid residue repeatedly with ethanol / deionized water until the supernatant becomes clear, and then dry it overnight.

[0009] (2): Add 50-100 mL of ethanol solution containing 2-4 mM mercaptoacetic acid (MAA) to 100-200 mg of the product obtained in step (1) and continue sonication for 5 minutes; then mechanically stir the reaction system overnight at room temperature under a nitrogen atmosphere; remove the supernatant by magnetic collection and wash the magnetic solid several times with ethanol; and vacuum dry for 8-10 hours.

[0010] (3): Add 50-100 mL of an aqueous solution containing 10 mM cerium ammonium nitrate to 50-100 mg of the product obtained in step (2); stir at 40 °C for 1-2 hours, and wash the mixture three times with alternating ethanol / deionized water; dissolve 500-1000 mg of P123 in 20-50 mL of deionized water, and then add 500-1000 μL of acetic acid, 5-10 mL of mesitylene and 500-1200 mg of terephthalic acid in sequence to prepare a ligand solution; after stirring for 5-10 minutes, mix the ligand solution thoroughly with the magnetic particles anchored with cerium ions on the surface; stir the system at 40-50 °C for 30-60 minutes, and then wash several times with DMF / ethanol by magnetic separation; finally, stir and reflux the product in ethanol at 60 °C for 8-10 hours; after vacuum drying, a magnetic core-porous hydrophilic composite material is obtained, named FeM@PMOFs.

[0011] The application of FeM@PMOFs prepared using the method of this invention in the field of glycopeptide separation and enrichment is described in the following steps:

[0012] (1) FeM@PMOFs were prepared into a nanoparticle suspension with a concentration of 1-10 μg / μL and dissolved in buffer A to obtain a material suspension; 10-20 μL of the material suspension was thoroughly mixed with 10-30 μL of biological sample and the volume was fixed to 100-200 μL with buffer A to obtain a mixture; the mixture was incubated at 37 ℃ for 30-60 minutes, and then washed several times with 100-200 μL of buffer A by magnetic separation; 10-30 μL of buffer B was added and glycopeptides were eluted at 37 ℃ for 30-60 minutes; for serum samples, the elution steps were repeated twice; the eluent was extracted by magnetic separation.

[0013] (2) Take 1-2 μL of the eluent obtained in step (1) and mix it with 1 μL of 2,5-dihydroxybenzoic acid solution (15 mg / mL, trifluoroacetic acid / acetonitrile / water = 0.1 / 50 / 49.9, v / v / v) and spot it onto a MALDI-TOF MS target plate. After drying with cold air, perform MALDI-TOF MS detection to obtain a mass spectrum. The organic matrix used for protein analysis is sinapic acid solution (15 mg / mL, trifluoroacetic acid / acetonitrile / water = 0.1 / 50 / 49.9). Take 1-2 μL of the eluent obtained in step (1) and mix it with 1 μL of sinapic acid solution, spot it onto a MALDI-TOF MS target plate, and after drying with cold air, perform MALDI-TOF MS detection to obtain a mass spectrum. The specific conditions for MALDI-TOF MS analysis are: Bruker UltrafleXtreme MALDI-TOF / TOF MS was used, and a 355 nm wavelength range was prepared. The Nd:YAG laser source was used with a laser intensity of 95% and a frequency of 2000 Hz, and the analysis mode was set to accumulate 1000 laser pulses. The MALDI-TOF MS spectra of glycopeptides were acquired in reflector positive ion mode, while the protein spectra were acquired in linear positive ion mode. The raw mass spectrometry data of each sample obtained by MALDI-TOF MS were processed on Bruker Compass flexAnalysis 3.4 software.

[0014] (3) After desalting the eluent obtained in step (2), the eluent was vacuum-dried and then analyzed by LC-MS / MS. The specific conditions for LC-MS / MS analysis were as follows: analysis was performed using a TIMS-TOF Pro2 mass spectrometer (Bruker Daltonics) coupled with an UltiMate 3000 UHPLC system (Thermo Fisher Scientific); 1 µL of peptide sample (200 ng) was injected, and separation was performed using a 15 cm analytical column with an inner diameter of 75 μm and filled with 1.7 μm C18 particles (IonOpticks, Aurora series); the column temperature was 60 °C and the flow rate was 300 nL / min; the HRP sample was separated by multi-step gradient elution for 30 minutes. 0 to 3 minutes: 2.2%–8% Buffer B; 3 to 16 minutes: 8%–28% Buffer B; 16 to 24 minutes: 28%–38% Buffer B; 24 to 27 minutes: 38%–90% Buffer B; 27 to 30 minutes: 90% Buffer B; Serum sample separation uses a 60-minute multi-stage gradient: 0 to 3 minutes: 2.2%–8% Buffer B; 3 to 46 minutes: 8%–28% Buffer B; 46 to 54 minutes: 28%–38% Buffer B; 54 to 57 minutes: 38%–90% Buffer B; 57 to 60 minutes: 90% Buffer B; Mobile phase A consists of an aqueous solution containing 0.1% formic acid, and mobile phase B contains 80% acetonitrile and 0.1% formic acid; Ion source operating parameters are: capillary voltage 1500 V, drying gas flow rate 3.0 L / min, drying gas temperature 150 °C;

[0015] Data were acquired using the dda-PASEF (data-dependent acquisition-parallel accumulation-serial fragmentation) method; the operating mass range of the mass spectrometer was 100–2000 m / z; for precursor ions, the PASEF ramp number was 7, and the total cycle time was 1.59 s; the TIMS (trapped ion mobility spectrometry) scan range was set to 1 / K0 = 0.60 V·s / cm² to 1.60 V·s / cm²; during fragmentation, a step-like collision energy linearly related to ion mobility was used, ranging from 35 eV at 1 / K0 = 0.5 V·s / cm² to 65 eV at 1 / K0 = 1.6 V·s / cm².

[0016] In this invention, the glycopeptide mentioned in step (1) is any one of horseradish peroxidase (HRP) glycopeptide hydrolysate or serum glycopeptide hydrolysate.

[0017] In this invention, the preparation of horseradish peroxidase (HRP) glycopeptide hydrolysate is as follows: A fixed concentration of HRP protein solution is prepared using 20-50 mM ammonium bicarbonate buffer as the solvent; the HRP solution is shaken at 95-100 °C for 5-10 minutes to induce protein denaturation, and then cooled to room temperature; trypsin is added at an enzyme-to-protein mass ratio of 1:50-1:100; the mixture is shaken overnight at 37 °C to prepare the HRP hydrolysate, which is then stored at -20 °C for subsequent use.

[0018] In this invention, the preparation of serum glycopeptide digest is as follows: Serum samples are removed from -80 °C and thawed at 4 °C; serum containing 60 μg of protein is diluted to 50-100 μL with ammonium bicarbonate buffer according to the BCA method; the serum solution is shaken at 95-100 °C for 8-15 minutes to induce protein denaturation, and then cooled to room temperature; 0.5-5 μL of a mixture containing 20-100 mM tris(2-carboxyethyl)phosphine and 50-200 mM iodoacetamide is added to the denatured protein solution, and the mixture is shaken in the dark for 30-50 minutes; subsequently, trypsin is added at an enzyme-to-protein mass ratio of 1:50-1:100, and the mixture is shaken overnight at 37 °C; finally, a serum digest solution with a final volume of 100-500 μL is prepared and stored at -20 °C for subsequent use.

[0019] In this invention, the raw data files generated by TIMS-TOF Pro2 were analyzed and identified using FragPipe (v23.1). The database used contained 708 human N-glycans and the UniProt Homo sapiens FASTA sequence database (2025.12.04). In the workflow section, "Glyco-N-LFQ" was selected as the workflow, and "IM-MS" was selected as the mass spectrometry data type. In the MSFragger section, trypsin digestion was selected, allowing two uncut sites. The peptide mass range was 400-5000 Da. At the same time, methionine oxidation (M) and protein N-terminal acetylation were set as variable modifications, and cysteine ​​residues and lysine residues were set as fixed modifications. A false discovery rate (FDR) threshold of 1% was applied to glycan filtering, peptide matching (PSMs), and peptide and protein levels in Philosopher in PTM-Shepherd.

[0020] The application of FeM@PMOFs prepared using the method of this invention in the screening of glycopeptide biomarkers related to urinary system cancers is described in the following steps:

[0021] (1) Integrate all N-glycopeptides related to healthy individuals, prostate cancer patients, bladder cancer patients, and kidney cancer patients extracted by FeM@PMOFs as a total set;

[0022] (2) Collect common N-glycopeptides related to healthy individuals, prostate cancer patients, bladder cancer patients, and kidney cancer patients;

[0023] (3) Screen out N-glycopeptides that were detected in all samples with a detection rate of 100%;

[0024] (4) Using FC>1.5 or FC<0.67 and p<0.05 as screening criteria, N-glycopeptides with significant expression differences among the four groups of healthy people, prostate cancer patients, bladder cancer patients and kidney cancer patients were screened.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] 1. This invention designs and synthesizes a novel hydrophilic composite material, FeM@PMOFs, combining a Fe3O4 core structure with a hierarchical porous MOF shell. FeM@PMOFs, with its high magnetic responsiveness, can accelerate the glycopeptide enrichment process. Furthermore, compared to Fe3O4, the porous MOF shell endows FeM@PMOFs with a larger specific surface area, enhanced hydrophilicity, and additional coordination effects based on Ce-OH active sites. Therefore, in glycopeptide extraction, FeM@PMOFs exhibits extremely low detection limits (~1 amol / μL), high selectivity, high enrichment capacity, and excellent anti-interference properties.

[0027] 2. This invention exhibits satisfactory reproducibility in both HRP digests and serum samples, maintaining accurate glycopeptide enrichment even in biomimetic and serum samples. In four parallel serum glycopeptide enrichment trials, FeM@PMOFs maintained stable performance and identified 324 N-glycopeptides from 185 glycoproteins in four serum samples, covering 342 glycosylation sites, thus enabling the screening of cancer-related N-glycopeptide biomarkers.

[0028] 3. This invention successfully identified 13 differentially expressed N-glycopeptides in healthy individuals and patients with urinary system cancers through a stepwise screening process, and screened out 7 differentially expressed N-glycopeptides in the prostate cancer / bladder cancer group, prostate cancer / kidney cancer group, and bladder cancer / kidney cancer group, and conducted in-depth analysis of their biological functions. Attached Figure Description

[0029] Figure 1 This is a scanning electron microscope image of the magnetic core-porous hydrophilic composite material of Example 1 of the present invention.

[0030] Figure 2 This is a transmission electron microscope image of the magnetic core-porous hydrophilic composite material of Example 1 of the present invention.

[0031] Figure 3This is an elemental distribution diagram of the magnetic core-porous hydrophilic composite material of Example 1 of the present invention.

[0032] Figure 4 This is the Fourier transform-infrared spectrum of the magnetic core-porous hydrophilic composite material of Example 1 of the present invention.

[0033] Figure 5 XPS images of the magnetic core-porous hydrophilic composite material of Example 1 of the present invention are shown. Among them: (a) is Ce 3d XPS analysis of Fe3O4@MAA and the magnetic core-porous hydrophilic composite material FeM@PMOFs, and (b) is O 1s XPS analysis of Fe3O4, Fe3O4@MAA and FeM@PMOFs.

[0034] Figure 6 The figures show the nitrogen adsorption / desorption isotherms and pore size distribution curves of the magnetic core-porous hydrophilic composite material of Example 1 of the present invention. Specifically: (a) is the nitrogen adsorption / desorption isotherm of FeM@PMOFs, and (b) is the pore size distribution curve of FeM@PMOFs.

[0035] Figure 7 This is a hydrophilic contact angle diagram of the magnetic core-porous hydrophilic composite material of Embodiment 1 of the present invention.

[0036] Figure 8 The image shows representative MALDI-TOF MS images of the magnetic core-porous hydrophilic composite material of Example 2 of this invention before and after enrichment of HRP glycopeptides.

[0037] Figure 9 The values ​​are the CV values ​​of HRP glycopeptides obtained from three parallel enrichments within and between groups based on MALDI mass spectrometry in Example 2 of this invention.

[0038] Figure 10 This shows the CV distribution of all peptides and N-glycopeptides in the LFQ signal intensity during four replicate experiments based on LC-MS / MS in Example 2 of this invention.

[0039] Figure 11 The number of glycopeptides detected in serum samples from different groups in Example 3.

[0040] Figure 12 This is a heatmap of glycopeptide levels after screening all samples in Example 3. Detailed Implementation

[0041] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0042] Example 1

[0043] Synthesis of magnetic core-porous hydrophilic composite materials:

[0044] (1): 1.35 g of ferric chloride hexahydrate was completely dissolved in 75 mL of ethylene glycol. After stirring until the solution was clear and transparent, 3.6 g of sodium acetate was added, and the mixture was magnetically stirred for 1 hour, followed by ultrasonic treatment for 10 minutes. The mixture was then transferred to a polytetrafluoroethylene reactor and heated at 200 °C for 16 hours. After the reaction was complete, the reactor was cooled to room temperature, and the supernatant was collected and removed by magnetic force. The solid residue was repeatedly washed with ethanol / deionized water until the supernatant became clear, and then dried overnight.

[0045] (2): Add 100 mL of an ethanol solution containing 3.2 mM mercaptoacetic acid to 200 mg of the product obtained in step (1) and sonicate continuously for 5 minutes. Mechanically stir the reaction system for 24 hours at room temperature under a nitrogen atmosphere. Collect the supernatant by magnetic force and wash the magnetic solid 5 times with ethanol. Dry under vacuum overnight.

[0046] (3): Add 60 mL of an aqueous solution containing 10 mM cerium ammonium nitrate to 100 mg of the product obtained in step (2). After stirring at 40 °C for 1 hour, wash the mixture three times with alternating ethanol / deionized water. Dissolve 666 mg of P123 in 50 mL of deionized water, and then add 666 μL of acetic acid, 8 mL of mesitylene, and 1106.4 mg of terephthalic acid sequentially to prepare a ligand solution. After stirring for 10 minutes, thoroughly mix the ligand solution with magnetic particles anchored with cerium ions on the surface. Stir the system at 45 °C for 50 minutes, and then wash several times with DMF / ethanol by magnetic separation. Finally, reflux the product in ethanol at 60 °C overnight. After vacuum drying, a magnetic core-porous hydrophilic composite material is obtained.

[0047] Analysis results: Figure 1-7 The successful synthesis of the magnetic core-porous hydrophilic composite material was confirmed. Figure 1 , 2 The overall morphology of the magnetic core-porous hydrophilic composite material was characterized, and it was confirmed that the magnetic core-porous hydrophilic composite material contains four key elements: O, Fe, S, and Ce. Figure 4 Infrared images confirmed the group changes from Fe3O4 to Fe3O4@MAA, and finally to the magnetic core-porous hydrophilic composite material, confirming the successful synthesis of the MOF shell; Figure 5 The XPS plots, comparing the stages of the two important elements, Ce source and O source, confirmed the successful synthesis of the MOF shell. Figure 6 The nitrogen adsorption / desorption isotherms and pore size distribution curves confirm the hierarchical porous MOF shell characteristics of the magnetic core-porous hydrophilic composite material. Figure 7 Contact angle analysis confirmed the hydrophilicity of the magnetic core-porous hydrophilic composite material.

[0048] Example 2

[0049] The magnetic core-porous hydrophilic composite material obtained in Example 1 was used for enrichment evaluation of HRP / human serum glycopeptide samples:

[0050] (1) Preparation of horseradish peroxidase (HRP) glycopeptide hydrolysate: A 2 mg / mL HRP protein solution was prepared using 25 mM ammonium bicarbonate buffer as the solvent. The HRP solution was shaken at 100 °C for 10 minutes to induce protein denaturation, and then cooled to room temperature. Trypsin was added at an enzyme-to-protein mass ratio of 1:50. The mixture was shaken at 37 °C for 16 hours to prepare the HRP hydrolysate, which was then stored at -20 °C for subsequent use.

[0051] (2) Preparation of serum glycopeptide digest: Serum samples were removed from -80 °C and thawed at 4 °C. Serum containing 60 μg of protein was diluted to 50 μL with ammonium bicarbonate buffer according to the BCA method. The serum solution was shaken at 100 °C for 15 minutes to induce protein denaturation, and then cooled to room temperature. 2 μL of a mixture containing 100 mM tris(2-carboxyethyl)phosphine and 200 mM iodoacetamide was added to the denatured protein solution, and the mixture was shaken in the dark for 30 minutes. Subsequently, trypsin was added at an enzyme-to-protein mass ratio of 1:50, and the mixture was shaken at 37 °C for 16 hours. Finally, a serum digest solution with a final volume of 100 μL was prepared and stored at -20 °C for subsequent use.

[0052] (3) Glycopeptide enrichment scheme based on magnetic core-porous hydrophilic composite material: The magnetic core-porous hydrophilic composite material was prepared into a nanoparticle suspension with a concentration of 10 μg / μL and dissolved in buffer A (acetonitrile / water / trifluoroacetic acid = 90 / 9 / 1, v / v / v). 20 μL of the material suspension was thoroughly mixed with a specific volume of the enzymatic hydrolysate obtained in step (1) or step (2) and the volume was fixed to 100 μL with buffer A. The mixture was incubated at 37 °C for 30 min and then washed three times with 100 μL of buffer A by magnetic separation. 20 μL of buffer B (acetonitrile / water / trifluoroacetic acid = 30 / 69.9 / 0.1, v / v / v) was added and the glycopeptides were eluted at 37 °C for 30 min. For serum samples, the elution steps were repeated twice. After magnetic separation and extraction, the eluent is directly sent to a MALDI-TOF mass spectrometer for detection; or it is desalted, or desalted and vacuum centrifuged and dried before LC-MS / MS analysis.

[0053] Analysis results: Figure 8 , Figure 9 , Figure 10As shown in Table 1, glycopeptides derived from HRP or serum can be captured by this material. The detection limit is as low as 1 amol / μL, and the CV value for glycopeptide detection is <15%.

[0054] Table 1

[0055]

[0056] Where: N# represents the N-glycosylation site.

[0057] Example 3

[0058] The magnetic core-porous hydrophilic composite material obtained in Example 1 was used for the enrichment analysis of serum glycopeptides and the screening of related glycopeptide biomarkers in 3 healthy individuals, 3 prostate cancer patients, 3 bladder cancer patients, and 3 kidney cancer patients.

[0059] (1) Integrate all N-glycopeptides related to healthy individuals, prostate cancer patients, bladder cancer patients, and kidney cancer patients extracted by FeM@PMOFs as a total set;

[0060] (2) Collect common N-glycopeptides related to healthy individuals, prostate cancer patients, bladder cancer patients, and kidney cancer patients;

[0061] (3) Screen out N-glycopeptides that were detected in all samples with a detection rate of 100%;

[0062] (4) Using FC>1.5 or FC<0.67 and p<0.05 as screening criteria, N-glycopeptides with significant expression differences among the four groups of healthy people, prostate cancer patients, bladder cancer patients and kidney cancer patients were screened.

[0063] Analysis results: Figure 11 As can be seen, the enrichment process evaluated in Example 2, when applied to serum samples from healthy individuals, those with prostate cancer, those with bladder cancer, and those with kidney cancer, can effectively detect differentially expressed glycopeptides. Furthermore, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. Figure 12 It can be seen that the glycopeptides identified after the screening process showed significant differences among the four groups of serum samples.

[0064] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a magnetic core-porous hydrophilic MOF composite material (FeM@PMOFs) for serum glycopeptide analysis, characterized in that... Using an in-situ MOF growth strategy, Ce³⁺ / Ce 4 ⁺ Anchored on the Fe3O4 surface modified with mercaptoacetic acid (MAA), a template agent and ligand are introduced via a microemulsion-mediated self-assembly method to guide the epitaxial growth of MOFs on the Fe3O4@MAA surface; the specific steps are as follows: (1): Dissolve 1-2 g of ferric chloride hexahydrate completely in 50-100 mL of ethylene glycol. Stir until the solution is clear and transparent, then add 2-5 g of sodium acetate. After stirring with magnetic force and sonicating, transfer the solution to a polytetrafluoroethylene reactor and heat at 200℃ for 12-16 hours. After the reaction is complete, let the reactor cool to room temperature and collect the supernatant by magnetic force. Wash the solid residue repeatedly with ethanol / deionized water until the supernatant becomes clear, and then vacuum dry for 12-16 hours. (2): Add 50-100 mL of ethanol solution containing 2-4 mM mercaptoacetic acid (MAA) to 100-200 mg of the product obtained in step (1) and continue sonication for 5 minutes; then mechanically stir the reaction system for 12 hours at room temperature under a nitrogen atmosphere; remove the supernatant by magnetic collection and wash the magnetic solid several times with ethanol; and vacuum dry for 12-16 hours. (3): Add 50-100 mL of an aqueous solution containing 10 mM cerium ammonium nitrate to 50-100 mg of the product obtained in step (2); stir at 40 °C for 1-2 hours, and wash the mixture three times with alternating ethanol / deionized water; dissolve 500-1000 mg of P123 in 20-50 mL of deionized water, and then add 500-1000 μL of acetic acid, 5-10 mL of mesitylene and 500-1200 mg of terephthalic acid in sequence to prepare a ligand solution; after stirring for 5-10 minutes, mix the ligand solution thoroughly with the magnetic particles anchored with cerium ions on the surface; stir the system at 40-50 °C for 30-60 minutes, and then wash several times with DMF / ethanol by magnetic separation; finally, stir and reflux the product in ethanol at 60 °C for 12-16 hours; after vacuum drying, a magnetic core-porous hydrophilic composite material is obtained, named FeM@PMOFs.

2. An application of FeM@PMOFs obtained by the preparation method as described in claim in the field of glycopeptide separation and enrichment, characterized in that... The specific steps are as follows: (1) FeM@PMOFs were prepared into a nanoparticle suspension with a concentration of 1-10 μg / μL and dissolved in buffer A to obtain a material suspension; 10-20 μL of the material suspension was thoroughly mixed with 10-30 μL of biological sample and the volume was fixed to 100-200 μL with buffer A to obtain a mixture; the mixture was incubated at 37 ℃ for 30-60 minutes, and then washed several times with 100-200 μL of buffer A by magnetic separation; 10-30 μL of buffer B was added and glycopeptides were eluted at 37 ℃ for 30-60 minutes; for serum samples, the elution steps were repeated twice; the eluent was extracted by magnetic separation. (2) Take 1-2 μL of the eluent obtained in step (1) and mix it with 1 μL of 2,5-dihydroxybenzoic acid solution (15 mg / mL, trifluoroacetic acid / acetonitrile / water = 0.1 / 50 / 49.9, v / v / v) and spot it onto a MALDI-TOF MS target plate. After drying with cold air, perform MALDI-TOF MS detection to obtain a mass spectrum. The organic matrix used for protein analysis is sinapic acid solution (15 mg / mL, trifluoroacetic acid / acetonitrile / water = 0.1 / 50 / 49.9). Take 1-2 μL of the eluent obtained in step (1) and mix it with 1 μL of sinapic acid solution, spot it onto a MALDI-TOF MS target plate, and after drying with cold air, perform MALDI-TOF MS detection to obtain a mass spectrum. The specific conditions for MALDI-TOF MS analysis are: Bruker UltrafleXtreme MALDI-TOF / TOF MS was used, and a 355 nm Nd:YAG medium was prepared. The laser source was set to 95% intensity and 2000 Hz frequency, and the analysis mode was set to accumulate 1000 laser pulses. The MALDI-TOF MS spectra of glycopeptides were acquired in reflector positive ion mode, while the protein spectra were acquired in linear positive ion mode. The raw mass spectrometry data of each sample obtained by MALDI-TOF MS were processed on Bruker Compass flexAnalysis 3.4 software. (3) After desalting the eluent obtained in step (2), the eluent was vacuum-dried and then analyzed by LC-MS / MS. The specific conditions for LC-MS / MS analysis were as follows: analysis was performed using a TIMS-TOF Pro2 mass spectrometer coupled with an UltiMate 3000 UHPLC system; 1 µL of peptide sample (200 ng) was injected, and separation was performed using a 15 cm analytical column of IonOpticks, Aurora series with an inner diameter of 75 μm and filled with 1.7 μm C18 particles. The column temperature was 60 °C and the flow rate was 300 nL / min. The separation of HRP samples was performed using a 30-minute multi-step gradient elution: 0 to 3 minutes: 2.2%–8% buffer B; 3 to 16 minutes: 8%–28% buffer B; 16 to 24 minutes: 28%–38% buffer B; 24 to 27 minutes: 38%–90% buffer B; 27 to 30 minutes: 90% buffer B. The separation of serum samples was performed using a 60-minute multi-step gradient elution. 0 to 3 minutes: 2.2%–8% Buffer B; 3 to 46 minutes: 8%–28% Buffer B; 46 to 54 minutes: 28%–38% Buffer B; 54 to 57 minutes: 38%–90% Buffer B; 57 to 60 minutes: maintain 90% Buffer B; Mobile phase A consists of an aqueous solution containing 0.1% formic acid, and mobile phase B contains 80% acetonitrile and 0.1% formic acid; Ion source operating parameters are: capillary voltage 1500 V, dry gas flow rate 3.0 L / min, dry gas temperature 150 °C; Data were acquired using the dda-PASEF method; the operating mass range of the mass spectrometer was 100–2000 m / z; for precursor ions, the PASEF ramp number was 7, and the total cycle time was 1.59 s; the capture ion mobility spectrum scan range was set from 1 / K0 = 0.60 V·s / cm² to 1.60 V·s / cm²; during fragmentation, a step-wise collision energy linearly related to ion mobility was used, ranging from 35 eV at 1 / K0 = 0.5 V·s / cm² to 65 eV at 1 / K0 = 1.6 V·s / cm².

3. The application according to claim 2, characterized in that... The glycopeptide mentioned in step (1) is either horseradish peroxidase glycopeptide hydrolysate or serum glycopeptide hydrolysate.

4. The application according to claim 3, characterized in that... Preparation of horseradish peroxidase glycopeptide hydrolysate: Prepare a fixed concentration of HRP protein solution using 20-50 mM ammonium bicarbonate buffer as solvent; inducing protein denaturation by shaking the HRP solution at 95-100 °C for 5-10 minutes, then cooling to room temperature; add trypsin at an enzyme-to-protein mass ratio of 1:50-1:100; shake the mixture overnight at 37 °C to prepare the HRP hydrolysate, and store it at -20 °C for subsequent use.

5. The application according to claim 3, characterized in that... Preparation of serum glycopeptide digest: Serum samples were removed from -80°C and thawed at 4°C. Serum containing 60 μg of protein was diluted to 50-100 μL with ammonium bicarbonate buffer according to the BCA method. The serum solution was shaken at 95-100°C for 8-15 minutes to induce protein denaturation, followed by cooling to room temperature. 0.5-5 μL of a mixture containing 20-100 mM tris(2-carboxyethyl)phosphine and 50-200 mM iodoacetamide was added to the denatured protein solution, and the mixture was shaken in the dark for 30-50 minutes. Subsequently, trypsin was added at an enzyme-to-protein mass ratio of 1:50-1:100, and the mixture was shaken overnight at 37°C. Finally, a serum digest solution with a final volume of 100-500 μL was prepared and stored at -20°C for subsequent use.

6. The application according to claim 2, characterized in that... The raw data files generated by TIMS-TOF Pro2 were analyzed and identified using FragPipe (v23.1). The database used included 708 human N-glycans and the UniProtHomo sapiens FASTA sequence database (2025.12.04). In the workflow section, "Glyco-N-LFQ" was selected as the workflow, and "IM-MS" was selected as the mass spectrometry data type. In the MSFragger section, trypsin digestion was selected, allowing two uncut sites. The peptide mass range was 400-5000 Da. Methionine oxidation (M) and protein N-terminal acetylation were set as variable modifications, while cysteine ​​and lysine residues were set as fixed modifications. A 1% false discovery threshold was applied to glycan filtering, peptide matching, and peptide and protein levels in Philosopher in PTM-Shepherd.

7. The application of FeM@PMOFs obtained by the preparation method as described in claim 1 in the screening of glycopeptide biomarkers related to urinary system cancers, characterized in that... The specific steps are as follows: (1) Integrate all N-glycopeptides related to healthy individuals, prostate cancer patients, bladder cancer patients, and kidney cancer patients extracted by FeM@PMOFs as a total set; (2) Collect common N-glycopeptides related to healthy individuals, prostate cancer patients, bladder cancer patients, and kidney cancer patients; (3) Screen out N-glycopeptides that were detected in all samples with a detection rate of 100%; (4) Using FC>1.5 or FC<0.67 and p<0.05 as screening criteria, N-glycopeptides with significant expression differences among the four groups of healthy people, prostate cancer patients, bladder cancer patients and kidney cancer patients were screened.

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