A yd monomer-loaded biomimetic material, a preparation method and application thereof

Abstract

The present application belongs to the field of bionic technology, and particularly relates to a kind of bionic material loaded with YD monomer and its preparation method and application.The present application utilizes YD monomer composed of tyrosine (Y) and aspartic acid (D) to synthesize a kind of bionic material through reversible addition-fragmentation chain transfer (RAFT) polymerization. The bionic material can produce spatially adjacent binding pockets through the covalent bond of tyrosine (Y) and aspartic acid (D), and can capture two kinds of PTM (phosphopeptide and O-glycopeptide) simultaneously through synergistic interaction. The results of the present application show that the bionic material of YD monomer optimizes charge balance, the neutral phenolic group of Y (pKa ~ 10) and the negatively charged carboxylate of D (pKa ~ 3.9) together reduce the electrostatic repulsion with phosphopeptide, while maintaining target affinity; self-assembled into nanostructured porous framework can expose multivalent binding sites, and significantly improve capacity and selectivity through synergistic effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomimetic materials, and particularly relates to a biomimetic material loaded with YD monomers and a preparation method and application thereof. BACKGROUND

[0002] Post-translational modifications (PTMs) of proteins, such as phosphorylation and O-glycosylation, play a key role in regulating cell signaling, metabolic pathways, and disease progression. Phosphorylation is dynamically regulated by kinases and phosphatases and is crucial in cancer and neurodegenerative diseases, while O-glycosylation, particularly mucin-type glycosylation, is associated with immune regulation and tumor metastasis. However, the analysis of these low-abundance PTM peptides (<1% of total peptides) remains challenging due to their poor ionization efficiency in mass spectrometry (MS) and the interference of unmodified peptides. For example, the hydrophilic phosphate group (-PO4 3- ) of phosphorylated peptides competes with non-phosphorylated peptides during ionization, resulting in significant signal suppression. Similarly, the heterogeneity of O-glycan chains and their lower ionization efficiency also hinder the detection of O-glycopeptides. Therefore, developing advanced enrichment strategies is essential for achieving accurate PTM analysis in proteomics.

[0003] Currently, the enrichment techniques for phosphorylated and O-glycosylated peptides have problems such as limited selectivity and complex procedures. Metal oxide-based materials (e.g., TiO2) and immobilized metal ion affinity chromatography (IMAC) are widely used for phosphorylated peptide enrichment through phosphate-metal coordination. However, due to the non-specific binding of acidic non-phosphorylated peptides (e.g., glutamic acid / aspartic acid-rich peptides), these methods exhibit a high false positive rate. For O-glycopeptides, hydrophilic interaction chromatography (HILIC) and lectin affinity chromatography are commonly used methods. While HILIC relies on hydrophilic interactions with glycan moieties, it lacks specificity for O-glycopeptides and requires additional steps to remove phosphorylated contaminants. Lectin-based methods, while specific, have low throughput and are costly. Currently, methods for simultaneous enrichment of phosphorylation and N-glycosylation have been reported, using IMAC combined with hydrazide chemistry to enrich phosphorylated peptides and N-glycopeptides, where Ti 4+ -IMAC method can also achieve simultaneous enrichment of phosphorylated peptides and N-glycopeptides. However, there are few reports on the simultaneous enrichment of phosphorylated and O-glycosylated peptides. This is because, although the enrichment method for phosphorylated peptides is relatively mature, it is difficult to enrich and identify O-glycopeptides due to the complex structure of O-linked sugar chains, the relatively short sugar chains, and the lack of conserved O-glycosylation site motifs. Therefore, the simultaneous enrichment and identification of O-glycosylation modification and phosphorylation is extremely challenging.

[0004] Biomimetic materials inspired by natural molecular recognition mechanisms, such as enzyme-substrate or antibody-antigen interactions, offer promising solutions to these limitations. In particular, peptide-based materials have attracted much attention due to their programmable side-chain functionalities and dynamic self-assembly properties, which can adaptively bind to different PTM targets.

[0005] Therefore, it is urgent to develop a biomimetic material that can simultaneously enrich and identify O-glycosylation modification and phosphorylation. SUMMARY

[0006] The purpose of the present application is to provide a biomimetic material, which can not only capture phosphopeptides and O-glycopeptides simultaneously, but also reduce the electrostatic repulsion with phosphopeptides and maintain target affinity.

[0007] In order to achieve the above purpose, the present application provides the following technical solutions:

[0008] The present application provides a YD monomer, which is formed by dehydration condensation of tyrosine and aspartic acid through a peptide bond.

[0009] The present application provides a biomimetic material, which is a YD monomer modified silica microsphere.

[0010] Preferably, the biomimetic material is synthesized by reversible addition-fragmentation chain transfer polymerization of acrylate modified YD monomer; the acrylate modified YD monomer is formed by dehydration condensation of amino group on aspartic acid and carboxyl group on acrylic acid.

[0011] More preferably, the reagents used in the reversible addition-fragmentation chain transfer polymerization are 4-cyanopentanoic acid dithiobenzoate, N-hydroxysuccinimide and N,N'-dicyclohexyl carbodiimide.

[0012] The present application also provides a preparation method of a biomimetic material, which comprises mixing acrylate modified YD monomer with SiO2-CPADB, ethanol and sodium acetate solution, then reacting with azobisisobutyronitrile after oxygen removal, and dispersing the obtained material in water and reacting with hydrogen peroxide to obtain the biomimetic material.

[0013] The SiO2-CPADB is the product of the reaction of a RAFT agent with amino-functionalized silica.

[0014] Preferably, the oxygen removal process is freeze-thaw cycle.

[0015] Preferably, the reaction conditions with azobisisobutyronitrile are 55℃ for 48 h.

[0016] Preferably, the reaction conditions with hydrogen peroxide are 70℃ for 4 h.

[0017] Preferably, the RAFT agent is 4-cyanopentanoic acid dithiobenzoate, N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide.

[0018] The application also provides the use of the biomimetic material prepared by the above preparation method in enriching phosphorylated peptides and O-glycopeptides.

[0019] The beneficial effects of the application are:

[0020] The YD monomers constituting the biomimetic material can exert synergistic interaction and simultaneously capture phosphopeptides and O-glycopeptides.

[0021] The YD monomer is composed of tyrosine (Y) and aspartic acid (D). The covalent bond between Y and D produces a spatially adjacent binding pocket, which can synergistically capture two PTMs (phosphopeptides and O-glycopeptides). This design optimizes the charge balance: the neutral phenolic group of Y (pKa~10) and the negatively charged carboxylate of D (pKa~3.9) together reduce the electrostatic repulsion with phosphopeptides while maintaining target affinity. In addition, the YD self-assembles into a nanostructured porous framework that can expose multivalent binding sites, significantly improving capacity and selectivity through synergistic effects. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0023] Figure 1(A) is SEM characterization of YD material; (B) is FT-IR spectra of Si02 and poly YD; (C) is isothermal titration calorimetry data for the addition of 50 mM Neu5Ac (pH 2.2) into 10 mM YD aqueous solution (pH 2.2) at 20 °C (upper panel), the grey line (lower panel) indicates the non-linear fitting curve based on the sequential binding site model (N = 2); (D) is isothermal titration calorimetry data for the addition of 50 mM phosphate (pH 2.2) into 10 mM YD aqueous solution (pH 2.2) at 20 °C (upper panel), the grey line (lower panel) indicates the non-linear fitting curve based on the sequential binding site model (N = 2); (E) is partial hydrogen nuclear magnetic resonance (1H-NMR) spectra of mixtures of phosphate and YD at a molar ratio of 10:1, 5:1 and 1:1 in D20 at 20 °C, (orange arrows indicate the chemical shift changes of different groups on YD and phosphate); (F) is partial hydrogen nuclear magnetic resonance (1H-NMR) spectra of mixtures of lactose and YD at a molar ratio of 10:1, 5:1 and 1:1 in D20 at 20 °C, (orange arrows indicate the chemical shift changes of different groups on YD and lactose);

[0024] Figure 2 (A) is a diagram of simultaneous identification of phosphorylation and O-glycosylation using poly YD and Ti02 enrichment strategy; (B) is the post-translational modification of key proteins in HepG2 cells, the schematic diagram illustrates the presence of phosphorylation (indicated by "P" in red circle) and O-glycosylation (indicated by "G" in green circle) on three important proteins: NCL (Nucleolin), FAM174C and YB-1 (Y Box Binding Protein 1); each protein is represented by a different part, the corresponding label, the phosphorylated and O-glycosylated amino acid residues are labeled above the protein name;

[0025] Figure 3 (A) is a schematic diagram of the comparison analysis of O-glycopeptide enrichment using poly YD and Ti02 material; (A) is a bar chart of the number of identified glycoproteins, glycosyl groups and intact glycopeptides; (B) is the distribution of O-glycosylation sites on serine (S) and threonine (T); the pie chart represents the percentage of O-glycosylation on serine and threonine using poly YD and Ti02 material; (C) is the number of glycosyl groups of various glycosyl groups identified, the bar chart shows the number of glycosyl groups identified for different glycosyl groups using poly YD and Ti02 material. DETAILED DESCRIPTION

[0026] The present application provides a YD monomer loaded biomimetic material, the design of which is based on the complementary chemical functions and synergistic interactions of tyrosine (Y) and aspartic acid (D) that constitute the YD monomer. Tyrosine (Y) contributes a phenolic side chain that enables the interaction of the pi-anion with the phosphate group (-PO4 3- ) of the phosphopeptide, while its hydrophobic aromatic ring enhances selectivity by targeting the non-polar regions near the phosphorylation sites in complex matrices. In contrast, aspartic acid (D) provides a carboxylate group (-COO⁻) that forms a hydrogen bond network with the hydroxyl (-OH) moieties of O-glycans and facilitates secondary electrostatic interactions with the phosphate group, enabling dual target binding.

[0027] The covalent linkage of Y and D creates spatially adjacent binding pockets that can synergistically capture both PTMs. This design optimizes charge balance: the neutral phenol group of Y (pKa~10) and the negatively charged carboxylate of D (pKa~3.9) collectively mitigate electrostatic repulsion with the phosphopeptide while maintaining target affinity. Furthermore, the self-assembly of YD into nanostructured porous frameworks can expose multivalent binding sites, significantly improving capacity and selectivity through synergistic effects.

[0028] The present application biomimetic material is synthesized by addition-fragmentation chain transfer (RAFT) polymerization using YD monomers. The addition-fragmentation chain transfer (RAFT) reagent used is 4-cyanopentanoic acid dithiobenzoate, N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide.

[0029] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0030] The amino-functionalized silica (NH2-SiO2) microspheres used in the present application are commercially available.

[0031] The production process, experimental method or detection method involved in the embodiments of the present application, if not specifically stated, are all conventional methods in the prior art, and their names and / or abbreviations belong to conventional names in the art, which are very clear and explicit in the related application field. The skilled person in the art can understand the conventional process steps and apply the corresponding equipment according to the conventional conditions or the manufacturer's recommended conditions.

[0032] The various instruments, equipment, raw materials or reagents used in the embodiments of the present application do not have special restrictions on the source, and are all conventional products that can be purchased through normal commercial channels, or can be prepared according to the conventional methods well known to those skilled in the art.

[0033] Example 1 Synthesis of YD modified silica microspheres

[0034] The RAFT agent, 4-cyanopentanoic acid dithiobenzoate (CPADB) (200 mg), N- hydroxysuccinimide (NHS) (647 mg) and N,N'-dicyclohexylcarbodiimide (DCC) (218 mg) were added to a flask, purged with nitrogen, 5 mL of anhydrous dichloromethane and 100 μL of triethylamine (TEA) were added and the reaction stirred at room temperature under nitrogen for 42 hours, protected from light for 42 hours to give NHS-CPADB.

[0035] The amino-functionalised silica (NH2-Si02) microspheres were added to a flask, purged with nitrogen and reacted with NHS-CPADB and 8 mL of tetrahydrofuran (THF) at room temperature, protected from light for 48 hours to give Si02-CPADB.

[0036] The acrylate modified YD monomer was prepared by dehydration condensation of the amino group on aspartic acid and the carboxyl group of acrylic acid.

[0037] The acrylate modified YD monomer (AA-YD) (62 mg), Si02-CPADB (53 mg), 1 mL of ethanol, 1 mL of a sodium acetate solution (7.2 mM) were added to a flask, which was purged of oxygen by three freeze-thaw cycles. Azobisisobutyronitrile (AIBA) (5.1 mg) was added slowly and the reaction was left to proceed at 55 °C for 48 hours. The resulting material was dispersed in 1 mL of water in a flask, then 20 μL of hydrogen peroxide was added and the reaction was left to proceed at 70 °C for 4 hours to give the product YD-Si02. The resulting YD-Si02 was washed three times with water, centrifuged at 5000 rpm for 3 minutes and stored after drying.

[0038] Example 2 Characterisation of YD material

[0039] 2.1 Image characterisation

[0040] The YD material was characterised using infrared spectroscopy and scanning electron microscope (SEM) images.

[0041] The results are shown in Figures Figure 1 (A-B) and the FT-IR spectrum of the YD material indicates the presence of absorption peaks at 2935 cm -1 corresponding to C-H stretching vibrations, indicating the presence of organic or hydrocarbon compounds in the sample; 2364 cm -1 associated with CO2or other carbon-containing compounds; 1548 cm -1 associated with C=0 stretching vibrations or other functional groups; 1448 cm -1The absorption peak is related to Si-O-Si stretching vibration, which is one of the characteristic absorption peaks of silicon dioxide; it shows that the absorption peak at each position is enhanced compared with SiO2 and YD.

[0042] 2.2 Thermodynamic information characterization

[0043] ITC measurements were performed using a MicroCal VP-ITC 200 calorimeter (MicroCal, Northampton, MA). ITC (Isothermal Titration Calorimetry, Isothermal Titration Calorimetry ) is a technique used to study biomolecular interactions. It provides detailed thermodynamic information about these interactions by measuring the heat released or absorbed during the binding process of biomolecules.

[0044] To avoid bubbles, all solutions were degassed under vacuum before use.

[0045] Neu5Ac and phosphate titrations were performed by injecting 10 μL (50 mM, pH 2.2) into the calorimeter cell containing YD monomer (10 mM, pH 2.2) with a 2-minute interval. A control titration of Neu5Ac and phosphate into buffer solution (0.1% FA, pH 2.2) was used to correct for dilution heat. Raw data were processed using Origin plotting software accompanying the instrument.

[0046] The results are shown in Figure 1 (C-D), which show that YD monomer has strong binding force with Neu5Ac and phosphate. 2.3

[0048] N-acetylneuraminic acid (Neu5Ac), also known as sialic acid, is a carbohydrate widely distributed in nature, mainly composed of glycoprotein, glycolipid or bacterial capsule material in animal cell membrane or secretions. N-acetylneuraminic acid YD monomer (20 mM) was added to Neu5Ac to form YD mixture, and YDhunheye was added to host solution of lactose he phosphate (10 mM). After equilibration at room temperature overnight, the chemical shift of C-H proton was recorded and analyzed on a Bruker NEO 400 MHz spectrometer.

[0049] NMR (nuclear magnetic resonance spectroscopy) was used to test the nuclear magnetic resonance spectrum of phosphate and YD mixture with molar ratios of 10:1, 5:1 and 1:1 in D2O at 20°C; and the nuclear magnetic resonance spectrum of lactose and YD mixture with molar ratios of 10:1, 5:1 and 1:1 in D2O at 20°C.

[0050] The results are shown in

[0051] Figure 1 ​As shown in (EF), the peak values ​​corresponding to the groups in the spectrum increase with the change in the mixture ratio. This further indicates that the ability of the YD material to capture phosphopeptides and O-glycopeptides is enhanced with the increase of the concentration of the YD material in this application.

[0052] Example 3: Selectivity Evaluation of YD Materials

[0053] Weigh approximately 3 mg of poly(YD) material and dissolve it in 500 μL of 50% acetonitrile (ACN) / 1% formic acid (FA). Shake well to mix, centrifuge at 10000 g for 2 minutes, and discard the supernatant.

[0054] Equilibrate the material with 500 μL of 80% ACN / 1% FA; dissolve 100 μg of digested HepG2 protein (human hepatocellular carcinoma cells) in 80% ACN / 1% FA and mix with the equilibrated material. Shake for 30 min, centrifuge at 10000 g for 2 min. Separate the supernatant (retain for further enrichment with commercial TiO2 material). Discard the supernatant, continue washing with 70% ACN / 1% FA (1 mL × 2 times), mix, shake, and centrifuge. Load the sample onto a GELoader microcolumn packed with a 3M membrane. Wash with 70% ACN / 1% FA (30 μL × 2 times) and 65% ACN / 1% FA (30 μL × 2 times). Finally, elute with 10% NH3H2O ​​(20 μL × 2 times). Collect the eluent, dry and reconstitute. Perform further LC-MS / MS analysis.

[0055] Phosphorylated and O-glycosylated peptides enriched from HepG2 protein were isolated and identified using Q-Exactive mass spectrometry.

[0056] The samples were separated using reversed-phase liquid chromatography. Solvent A consisted of 0.1% formic acid (FA, pH 2.59), and solvent B was acetonitrile (ACN) containing 0.1% FA. The analytical column had an inner diameter of 75 μm, and the chromatographic packing material was C18AQ particles (3 μm, 120 A) packed to a length of 12 cm. The flow rate was set to 600 nL / min.

[0057] The LC gradients used for sample analysis were as follows: 2–8% B for 0.2 min, 8–50% B for 45 min, 50–90% B for 0.5 min, and 90% B for 5 min. Full-scan mass spectrometry was performed in Orbitrap, with a mass range of 500 m / z to 1500 m / z. High-energy collisional dissociation (HCD) was used for fragmentation, with the fractionation collision energies set to 20–30%. The top 20 ions with the highest intensities from the full scan were selected for fragmentation.

[0058] The results of simultaneous identification of phosphorylation and O-glycosylation using poly(YD) and TiO2 enrichment strategies are as follows: Figure 2The post-translational modifications of key proteins in HepG2 cells were shown, indicating the presence of phosphorylation and O-glycosylation on three important proteins: NCL (Nucleolin), FAM174C, and YB-1 (Y Box Binding Protein 1), further confirming that the YD material of the application can simultaneously capture phosphopeptides and O-glycopeptides.

[0059] Example 4 Data analysis and identification experiments

[0060] The raw data of O-glycopeptides were searched from the human protein database using Byonic software (ProteinMetrics, San Carlos, CA).

[0061] The parent mass tolerance and fragment ion were set at 10 ppm and 20 ppm, respectively. The fixed modification was set as carbamidomethylation (C), and the variable modifications included oxidation (M), deamidation (N), and acetylation (protein N-terminus). The O-linked glycan database contained nine common O-linked oligosaccharides: HexNAc(l), HexNAc(l)Hex(l), HexNAc(l)Hex(l)NeuAc(l), HexNAc(l)Hex(l)NeuAc(2), HexNAc(2)Hex(l), HexNAc(2)Hex(2)NeuAc(l), HexNAc(2)Hex(2)NeuAc(2), HexNAc(2)Hex(l)NeuAc(l), HexNAc(2)Hex(2)NeuAc(2). Trypsin and GluC were set as specific proteases, allowing up to two missed cleavages. Peptides with charge states of 2, 3, and 4 were selected for further fragmentation. Peptides with a false discovery rate (FDR) < 1% and a Byonic score > 30 were filtered out. The data were also searched against reverse sequences and contaminant sequences for comparison.

[0062] The enriched phosphopeptides were searched in the human protein database downloaded from UniProt using MaxQuant software (version 1.5.8.3). Phosphorylation of serine (S), threonine (T), and tyrosine (Y), as well as oxidation of methionine (M), deamidation of asparagine (N), and acetylation of protein N-terminus were set as variable modifications. Carbamidomethylation of cysteine (C) was set as a fixed modification. The parent ion mass tolerance was set at 10 ppm, and the same tolerance was applied to fragment ion mass. Phosphorylated peptides with a false discovery rate (FDR) of 1% and a minimum score of 40 were considered as authentic identifications.

[0063] The parent ion mass tolerance ( Parent Ion Mass Tolerance ) refers to the range of allowed deviation between the experimentally measured parent ion mass and the theoretically calculated parent ion mass during mass spectrometry analysis.

[0064] The results, as shown in Figure 3 Figure 6, indicate that the number of glycoproteins, glycosyl and intact glycopeptides identified using the YD material of the present application is much higher than that of Ti02; the distribution of O-glycosylation sites on serine (S) and threonine (T) indicates the percentage of O-glycosylation on serine and threonine using poly YD and Ti02 material, Ti02: 40% of O-glycosylation occurs on sulfur (S), and 60% of O-glycosylation occurs on tyrosine (T).

[0065] YD: 34% of O-glycosylation occurs on sulfur (S), and 66% of O-glycosylation occurs on tyrosine (T); overall, O-glycosylation of these two materials mainly occurs on tyrosine (T), but the proportion of glycosylation on sulfur (S) of Ti02 is slightly higher than that of YD. The results of the number of glycosyl identified for different glycosyl using poly YD and Ti02 material indicate that the degree of glycosylation of YD is generally higher than that of Ti02 at each site, especially at HexNAc(2)Hex(l)NeuAc(l) and HexNAc(2)Hex(2)NeuAc(l) sites, the degree of glycosylation of YD is significantly higher. This indicates that YD has a more complex glycosylation structure and a higher level of glycosylation.

[0066] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which are within the protection scope of the present application.

Claims

1. A method for preparing a biomimetic material, characterized in that, The preparation method includes mixing acrylate-modified YD monomer with SiO2-CPADB, ethanol and sodium acetate solution, deoxygenating and reacting with azobisisobutyronitrile, and dispersing the resulting material in water and reacting with hydrogen peroxide to obtain a biomimetic material. The SiO2-CPADB is the product of NHS-CPADB obtained by reacting 4-cyanopentanoic acid dithiobenzoate, N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide with amino-functionalized silica. The YD monomer is formed by the dehydration condensation of tyrosine and aspartic acid through peptide bonds. The acrylate-modified YD monomer is formed by the dehydration condensation of the amino group on aspartic acid and the carboxyl group on acrylic acid.

2. The preparation method according to claim 1, characterized in that, The deoxygenation process is a freeze-thaw cycle.

3. The preparation method according to claim 1, characterized in that, The reaction conditions with azobisisobutyronitrile were 55°C for 48 h.

4. The preparation method according to claim 1, characterized in that, The reaction conditions with hydrogen peroxide were 70°C for 4 hours.

5. A biomimetic material, characterized in that, The biomimetic material is prepared by the preparation method described in any one of claims 1-4.

6. The application of the biomimetic material prepared by the method of claim 1 or the biomimetic material of claim 5 in the enrichment of phosphorylated peptides and O-glycopeptides.

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