Sulfonyl functionalized covalent organic framework material as well as preparation method and application thereof

Abstract

The invention discloses a sulfonic group functionalized covalent organic framework material as well as a preparation method and application thereof, the sulfonic group functionalized covalent organic framework material (SCOF) is directly obtained by oil bath heating in a Pyrex glass tube, the preparation process is simple and convenient to operate, the synthesis yield is high, and the synthesized nano material has ultrahigh hydrophilicity and good dissolution and dispersion, and can be applied to the field of chemical engineering. The thermal stability is good; the sulfonic acid group functionalized nano material is used for enriching glycosylated peptide fragments, and shows excellent enrichment selectivity, extremely low detection limit and extremely high anti-interference capability, and compared with an unfunctionalized covalent organic framework material, the enrichment efficiency is greatly improved; meanwhile, a good effect is shown on detection of complex samples, and the nano material provided by the invention has excellent performance such as wide application range, good enrichment capacity, high sensitivity and strong anti-interference performance, and is beneficial to large-scale popularization and application.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of functionalized covalent organic framework materials and their glycopeptide enrichment applications, and particularly relates to a sulfonic acid group functionalized covalent organic framework material and a preparation method and application thereof. BACKGROUND

[0002] Covalent organic framework materials (COFs) are a kind of crystalline porous polymer materials composed of organic molecules connected by covalent bonds. Due to their large specific surface area, adjustable pore structure and easy-to-modify surface, COFs have shown good application prospects in gas adsorption and separation, catalysis, energy storage and carbon dioxide capture. In recent years, the research on functionalized COF materials has attracted increasing attention, and they are currently mainly applied in the fields of energy storage, membrane separation and sensing. Due to their high hydrophilicity, large active specific surface area and excellent dispersion performance, these materials show potential for glycopeptide enrichment.

[0003] Protein glycosylation plays an important role in various cellular processes, regulating the activity and function of proteins. Mass spectrometry (MS)-based proteomics technology is an important means for analyzing glycosylated proteins and glycopeptides. However, the abundance of glycopeptides in biological samples is usually very low (less than 1 x 10 -9 M), and their mass spectrometry detection is easily affected by low ionization efficiency, high-abundance non-sugar peptides, signal suppression and other factors, making it difficult to directly detect glycopeptides in complex samples. Given the key role of glycosylation in the regulation of protein function, developing efficient glycopeptide enrichment techniques has become a hot topic in proteomics research.

[0004] Currently, a variety of methods and materials have been used for the selective enrichment of glycopeptides, including hydrazine chemistry, lectin affinity chromatography, boronic acid affinity and hydrophilic interaction liquid chromatography (HILIC). Among them, HILIC has become the most widely used glycopeptide enrichment strategy due to its simple operation, low cost, good reproducibility and high enrichment efficiency. This method relies on the interaction between the hydrophilicity of the material surface and glycosylated peptides, and materials used include graphene oxide, magnetic nanoparticles and metal-organic framework materials. However, some materials still have problems such as complex synthesis steps or limited enrichment performance.

[0005] The patent document CN202110224172.7 discloses a method for specifically separating and enriching endogenous glycosylated peptides. First, a hydrophilic magnetic mesoporous silica material modified with glucosamide is synthesized. This material can specifically enrich glycosylated peptide segments by hydrophilic interaction, while large molecular proteins are rejected outside the mesopore. This method realizes high sensitivity and high repeatability of glycosylated peptide enrichment, and can realize large-scale identification of endogenous glycosylated peptides in combination with MALDI-TOF MS or nano-LC-MS / MS. However, this patent does not disclose sulfonic acid functionalized covalent organic framework materials and their application in glycosylated peptide segment enrichment.

[0006] The patent document CN201610302904.9 discloses a method for enriching glycosylated peptide segments and mass spectrometry analysis. The terminal amino group of dendrimer PAMAM is converted to sulfonic acid group through sulfonation derivatization reaction, and the skeleton nitrogen of the product is converted to quaternary ammonium group by using iodomethane to synthesize amphoteric hydrophilic dendrimer. This material can efficiently and specifically bind glycosylated peptides in serum sample enzymatic hydrolysate, and the non-sugar peptides that do not react with the nanomaterial are removed by ultrafiltration auxiliary technology. Then, the captured glycopeptides are dissociated from the material by deglycosylase, and are sent into liquid chromatography-mass spectrometry for glycosylated peptide analysis, realizing high specificity, high sensitivity, and high selectivity of mass spectrometry analysis of glycosylated peptides. However, this patent does not disclose sulfonic acid functionalized covalent organic framework materials and their application in glycosylated peptide segment enrichment.

[0007] The patent document CN201510534807.8 discloses a preparation method of functionalized modified nanometer mesoporous material, mesoporous material, and application. The nanometer mesoporous material is a nanometer mesoporous material precursor prepared by using tetraethoxysilane as monomer, cetyltrimethylammonium bromide and P123 as pore-forming agent. The outer specific surface of the nanometer material is selectively modified by using the exclusion effect of the template agent and hydrophobic silane coupling agent. Then, the template agent is removed by solvent extraction, and the inner surface of the material is modified with hydrophilic groups (amino group, carboxyl group, monosaccharide, polysaccharide, etc.). The material is further used for selective enrichment of glycosylated peptides. By means of the high specific area and size exclusion effect of the mesoporous material, and the hydrophobic modification of the outer surface, the non-specific adsorption of the matrix material to non-glycosylated peptides is reduced, the selectivity of enrichment is effectively improved, and efficient and highly selective enrichment of glycosylated peptide segments is realized. However, this patent does not disclose sulfonic acid functionalized covalent organic framework materials and their application in glycosylated peptide segment enrichment. SUMMARY

[0008] The present application aims to provide a sulfonic acid group functionalized covalent organic framework material and a preparation method and application thereof. -6 M), high enrichment capacity and extremely low detection limit (0.01fmol mu L -1 ), and is beneficial to popularization and use.

[0009] To achieve the above-mentioned application purposes, the present application adopts the following technical solutions.

[0010] The present application provides a preparation method of a sulfonic acid group functionalized covalent organic framework material, and the specific preparation process is as follows: 1,3,5-triformylphloroglucinol and 2,5-diamino-1,4-benzenedisulfonic acid are dispersed in a mixed solvent of 1,4-dioxane, mesitylene and acetic acid to form a mixed solution, the mixed solution is then subjected to freeze-thaw pump circulation for multiple times in a liquid nitrogen environment, then the mixed solution is sealed and heated for reaction in an oil bath, after the reaction is completed, the solid after reaction is naturally cooled to room temperature, and then washed with N,N-dimethylformamide, pure water and anhydrous tetrahydrofuran, and finally vacuum dried to obtain a dark brown powder of the sulfonic acid group functionalized covalent organic framework material.

[0011] Preferably, the specific preparation steps of the sulfonic acid group functionalized covalent organic framework material are as follows: 1,3,5-triformylphloroglucinol and 2,5-diamino-1,4-benzenedisulfonic acid are dispersed in a mixed solvent of 1,4-dioxane, mesitylene and acetic acid to form a mixed solution, the mixed solution is then subjected to freeze-thaw pump circulation for 3 times in a 77K liquid nitrogen environment, then the mixed solution is sealed and heated for reaction in an oil bath at 110-130 DEG C for 72h, after the reaction is completed, the solid after reaction is naturally cooled to room temperature, and then washed with N,N-dimethylformamide, pure water and anhydrous tetrahydrofuran for 3 times respectively, and finally vacuum dried at 120 DEG C overnight to obtain a dark brown powder of the sulfonic acid group functionalized covalent organic framework material.

[0012] Preferably, the oil bath heating reaction temperature is 120 DEG C.

[0013] Preferably, the molar ratio of 1,3,5-triformylphloroglucinol to 2,5-diamino-1,4-benzenedisulfonic acid is 1:1.2-2.

[0014] Preferably, the water contact angle of the sulfonic group functionalized covalent organic framework material is 35-45°, and the sulfonic group functionalized covalent organic framework material has good solubility and dispersibility and obvious Tyndall phenomenon.

[0015] The application provides the sulfonic group functionalized covalent organic framework material prepared by the method.

[0016] The application also provides application of the sulfonic group functionalized covalent organic framework material in selective enrichment of glycosylated peptide segments.

[0017] Preferably, the sulfonic group functionalized covalent organic framework material is used in enrichment of glycosylated peptide segments, and the application steps are as follows.

[0018] (1) mixing the sulfonic group functionalized covalent organic framework material with an enrichment solution and uniformly ultrasonically dispersing to obtain a suspension;

[0019] (2) adding a sample of glycosylated peptide segments to be separated into the suspension and vortexing for enrichment, so that the hydrophilic groups on the surface of the sulfonic group functionalized covalent organic framework material are combined with hydrophilic peptide segments in the glycosylated peptide segments through hydrophilic interaction;

[0020] (3) centrifuging after enrichment, removing supernatant, adding a washing solution, and washing away non-glycosylated peptide segments;

[0021] (4) adding an elution solution after washing, uniformly ultrasonically dispersing, vortexing for elution, and obtaining enriched glycosylated peptide segments.

[0022] Preferably, the mixing ratio of the sulfonic group functionalized covalent organic framework material to the enrichment solution is 1-1.5 mg:100 μL.

[0023] Preferably, the enrichment solution is a mixed solution prepared from acetonitrile, trifluoroacetic acid and pure water, the volume concentration of the acetonitrile is 80%-95%, and the volume concentration of the trifluoroacetic acid is 0.05%-1.0%; and the washing solution is a mixed solution prepared from acetonitrile, trifluoroacetic acid and pure water, the volume concentration of the acetonitrile is 80%-95%, and the volume concentration of the trifluoroacetic acid is 0.05%-1.0%.

[0024] Preferably, the enrichment solution is a mixed solution prepared from acetonitrile, trifluoroacetic acid and pure water, the volume concentration of the acetonitrile is 90%, and the volume concentration of the trifluoroacetic acid is 0.1%, and the mixing ratio of the enrichment solution makes the enrichment effect optimal; and the washing solution is a mixed solution prepared from acetonitrile, trifluoroacetic acid and pure water, the volume concentration of the acetonitrile is 90%, and the volume concentration of the trifluoroacetic acid is 0.1%.

[0025] Preferably, the elution solution is a mixed solution prepared from acetonitrile, trifluoroacetic acid and pure water, wherein the volume concentration of acetonitrile is 25% to 35% and the volume concentration of trifluoroacetic acid is 0.05% to 1.0%.

[0026] Preferably, the elution solution is a mixed solution prepared from acetonitrile, trifluoroacetic acid and pure water, wherein the volume concentration of acetonitrile is 30% and the volume concentration of trifluoroacetic acid is 0.1%.

[0027] The present invention also provides the application of the above-mentioned sulfonic acid-functionalized covalent organic framework material in the selective detection of glycosylated peptides. This covalent organic framework material exhibits excellent enrichment selectivity, high enrichment efficiency, low detection limit and high anti-interference ability.

[0028] Compared with existing technologies, this invention has the following advantages and beneficial effects: Based on sulfonic acid-functionalized covalent organic framework materials, this invention enables the specific enrichment of glycopeptides. The prepared sulfonic acid-functionalized covalent organic framework materials possess several unique advantages, including abundant hydrophilic binding sites on the surface, a large specific surface area, high porosity, and easy dispersion, thus achieving highly efficient enrichment of glycopeptides. Compared with traditional unfunctionalized HILIC methods for enriching glycosylated peptides, the enrichment method of this invention has superior performance, including wide applicability, good selectivity, strong anti-interference ability, and low detection limit. This method provides a new approach for the efficient enrichment of glycosylated peptides and is expected to be applied in large-scale proteomic analysis of complex biological samples in the future. Attached Figure Description

[0029] Figure 1 Scanning electron microscope (SEM) image (left) and transmission electron microscope (TEM) image (right) of SCOF prepared for Example 1;

[0030] Figure 2 Water contact angle diagram (left) and water contact angle number diagram (right) of SCOF prepared for Example 1;

[0031] Figure 3 High-resolution transmission electron microscope image and elemental distribution map of SCOF prepared in Example 1 (the elemental distribution maps are C, N, O and S elements in order);

[0032] Figure 4 To use MALDI-TOF MS, the mass spectra of the enrichment of glycopeptides from standard glycosylated protein (HRP) digestion by SCOF prepared in Example 1 and unfunctionalized TpPa COF and the direct analysis of glycopeptides from standard glycoprotein digestion were compared.

[0033] Figure 5To detect the mass spectra of glycopeptides after enzymatic digestion of standard glycosylated protein (HRP) at different concentrations prepared by SCOF enrichment in Example 1 using MALDI-TOF MS;

[0034] Figure 6 To use MALDI-TOF MS, the mass spectra of glycopeptides obtained by enzymatic digestion of SCOF-enriched standard glycoproteins (HRP) mixed in different molar ratios prepared in Example 1 and standard non-glycoproteins (BSA) were detected.

[0035] Figure 7 To use nanoLC-MS / MS, we detected glycopeptides, glycoproteins, and identified glycosylation sites in the SCOF-enriched serum samples of human ovarian cancer and normal individuals prepared in Example 1, as well as to obtain quantitative PCA maps and heatmaps. Detailed Implementation

[0036] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0037] The enrichment solution, washing solution, and elution solution used in the experiment were prepared as follows: the enrichment solution was a pure aqueous solution containing 90% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA); the washing solution was a pure aqueous solution containing 90% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA); and the elution solution was a pure aqueous solution containing 30% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA). The glycopeptide samples to be separated were prepared using a standard glycoprotein digestion solution (the standard glycoprotein was horseradish peroxidase (HRP), from Sigma-Aldrich China, dissolved in 50 mM NH4HCO3 solution, and then digested with trypsin).

[0038] Example 1

[0039] 63 mg of 1,3,5-tricarboxymethyl phloroglucinol (0.3 mmol) and 120.6 mg of 2,5-diamino-1,4-benzenedisulfonic acid (0.45 mmol) were added sequentially to a 25 mL Pryex tube. Then, 1.5 mL of 1,4-dioxane, 1.5 mL of mesitylene, and 0.5 mL of 6 M acetic acid solution were added to the mixture. The mixture was sonicated for 20 min to form a homogeneous suspension. The suspension was then circulated three times using a cryogenic pump under liquid nitrogen at 77 K. The suspension was then sealed in an oil bath at 120 °C for 72 h. After the reaction, the Pryex tube was allowed to cool naturally to room temperature. The resulting solid was centrifuged and washed three times each with N,N-dimethylformamide, pure water, and anhydrous tetrahydrofuran. Finally, the solid was vacuum-dried overnight at 120 °C to obtain a dark brown solid powder sulfonic acid-functionalized covalent organic framework material (SCOF).

[0040] 100 μL of enrichment solution was mixed with 1.2 mg SCOF and ultrasonically dispersed for 15 min to obtain a homogeneous suspension. Then, 2 μL of the glycopeptide fragment to be separated (2.5 × 10⁻⁶) was taken. -6 M) The sample was added to the dispersed suspension and vortexed for 30 min for enrichment. The abundant hydrophilic groups on the SCOF surface can bind to the hydrophilic peptides in the glycopeptides through hydrophilic interactions. After enrichment, the sample was centrifuged to remove the supernatant. Then, 100 μL of washing solution was added, vortexed for 6 min, centrifuged again, and the supernatant was removed. This washing process was repeated 3 times. The purpose of this step is to wash away the non-glycosylated peptides adsorbed on the nanosheets. After the washing step, 20 μL of the eluent was added to the washed material, and the sample was sonicated for 8 min and vortexed for 10 min to elute the glycosylated peptides enriched on the SCOF. The resulting eluent was enriched with glycosylated peptides. Finally, 1 μL of the eluent enriched with glycosylated peptides was mixed with 1 μL of matrix solution (10 mg / mL CHCA solution, CHCA dissolved in 30% ACN + 0.1% TFA to prepare a saturated solution) and spotted. The mixture was then analyzed using MALDI-TOF MS.

[0041] Example 2

[0042] Unfunctionalized covalent organic framework material (TpPa COF) was synthesized, and the enrichment process was the same as above. The enrichment effect was compared with that of sulfonic acid-functionalized covalent organic framework material SCOF.

[0043] Example 3

[0044] To verify the technical effect of the present invention, the SCOF prepared in Example 1 and the TpPa COF used in Example 2 were subjected to corresponding detection and analysis.

[0045] Figure 1 The images show the scanning electron microscope (SEM) image (Czech FEI Nova NaoSEM 450) (left) and transmission electron microscope (TEM) image (USA-Thermo Fisher-Talos F200i) (right) of the synthesized SCOF. The SEM image of SCOF shows that it has a distinct layered structure, while the TEM image shows that the synthesized SCOF has a relatively thin thickness.

[0046] Figure 2 The left image shows the water contact angle of the synthesized SCOF (Dataphysics-OCA20, Germany) and the right image shows the water contact angle of the synthesized SCOF. The left image shows that SCOF has excellent hydrophilicity, while the right image shows that the water contact angle of SCOF is 42°.

[0047] Figure 3 The image shows a high-resolution transmission electron microscope (TEM) image of the synthesized SCOF (Thermo Fisher Scientific Talos F200i) and an elemental distribution map (EDAX Octane Supper) (C, N, O, and S elements in order). The elemental distribution map shows that C, N, O, and S are uniformly distributed in the synthesized material, indicating good material homogeneity.

[0048] Figure 4 The mass spectra of SCOF and TpPa COF prepared in Example 1 above are compared for enrichment of glycopeptides from standard glycoprotein digestion and direct analysis of glycopeptides from standard glycoprotein digestion. Figure 4 In the image, A represents the glycopeptide enrichment mass spectrum obtained by SCOF digestion of standard glycoproteins. Figure 4 B is the mass spectrum of glycopeptide enrichment obtained by TpPa COF digestion of standard glycoproteins; Figure 4 C represents the direct analysis mass spectrum of glycopeptides from standard glycoprotein digestion; NL represents the mass spectrum intensity before normalization; the concentration of the standard glycopeptide is 2.5 × 10⁻⁶. -6 M. The experimental procedure follows the process described in the specific implementation method. MALDI-TOF MS detection parameters: positive ion mode, voltage +25kV, laser pulse frequency 200kHz, laser irradiation times 2000 shots, laser energy 70%. Figure 4 As shown in Figure A, after SCOF enrichment, the mass spectrum shows that 28 glycopeptides (#) were enriched in the HRP digest. The peptide signals were high against a clear background, and almost no non-glycopeptides were detected. Therefore, it can be inferred that SCOF exhibits a good enrichment effect on glycopeptides. Figure 4As shown in Figure B, after enrichment by TpPa COF, the mass spectrum shows that only 10 glycopeptides in the HRP glycoprotein digest were enriched, and the signals were weak. Additionally, some non-glycopeptides were also detected in the mass spectrum. Therefore, it can be inferred that TpPa COF has a poor enrichment effect on glycopeptides. Figure 4 As shown in Figure C, after direct spotting analysis, the mass spectrum shows that only 5 glycopeptides in the HRP glycoprotein hydrolysate were detected and the signals were weak. In addition, some non-glycopeptides were also detected in the mass spectrum. Therefore, it can be inferred that the direct analysis of glycopeptides is not very effective.

[0049] Figure 5 The mass spectra of glycopeptides obtained after digestion with standard glycoproteins at different concentrations prepared in Example 1 above are shown; wherein, Figure 5 China A Figure 5 B, Figure 5 C and Figure 5 The concentrations of the standard glycopeptide fragments corresponding to D were 10 fmol / μL. -1 1.0 fmol μL -1 0.1 fmol / μL -1 and 0.01 fmol μL -1 NL: Mass spectrum intensity before normalization. The experimental procedure was as described in the specific implementation instructions. MALDI-TOF MS detection parameters: positive ion mode, voltage +25kV, laser pulse frequency 200kHz, laser irradiation times 2000 shots, laser energy 70%. Figure 5 Experimental data showed that as the concentration of standard glycopeptides gradually decreased, SCOF could still enrich the glycopeptides in the HRP hydrolysate, demonstrating excellent enrichment ability. Even when the concentration of glycopeptides decreased to 0.01 fmol / μL... -1 At times, such as Figure 5 Even with D, two glycopeptide fragments could still be enriched. This suggests that SCOF exhibits highly efficient enrichment capabilities and an extremely low detection limit.

[0050] Figure 6 The mass spectra of glycopeptides obtained from standard glycoprotein digestion and non-glycoprotein digestion at different molar ratios prepared in Example 1 above are shown; wherein, Figure 6 China A Figure 6 B, Figure 6 C and Figure 6The ratios of standard glycopeptides to non-glycopeptides corresponding to D in the formula were 1:100, 1:100, 1:1000, and 1:5000, respectively. NL: mass spectrum intensity before normalization. The experimental procedure was as described in the specific implementation method. MALDI-TOF MS detection parameters: positive ion mode, voltage +25kV, laser pulse frequency 200kHz, laser irradiation times 2000 shots, laser energy 70%. Figure 6 Experimental data showed that as the molar ratio of standard glycopeptides to non-glycopeptides gradually increased, SCOF could still enrich the glycopeptides in the HRP hydrolysate, demonstrating excellent enrichment ability. When the molar ratio increased to 1:5000, as... Figure 6 Even in the middle D region, eight glycopeptide segments can still be enriched. This suggests that SCOF exhibits strong anti-interference capabilities.

[0051] Figure 7 Maps, quantitative PCA maps, and heatmaps of SCOF-enriched glycopeptides, glycoproteins, and identified glycosylation sites in serum samples from human ovarian cancer and normal individuals prepared in Example 1 above. Figure 7 Image A represents enriched glycopeptides, glycoproteins, and identified glycosylation sites in the serum of human ovarian cancer patients and healthy individuals. Figure 7 Figure B shows the PCA plot of glycoproteins enriched in diseased and healthy individuals. Figure 7 Figure C shows a heatmap of glycoproteins identified in diseased and healthy individuals; the experimental procedure was as described in the specific implementation method. The enriched samples were detected using nanoLC-MS / MS. Detection parameters: peptide samples were aspirated using an autosampler and bound to a Trap column (75 μm × 20 mm, 2 μm particle size). The sample was then eluted to an analytical column (75 μm × 250 mm, 1.6 μm particle size, Thermo). Separation was performed using pore size and ion optics. An analytical gradient was established using two mobile phases (mobile phase A: 0.1% formic acid in H₂O and mobile phase B: 0.1% formic acid in ACN). The flow rate of the liquid phase was set to 300 nL / min. Peptides were introduced into the mass spectrometer via a Captive Spray nano-ion source for DDA scanning, with TIMS enabled and PASEF scanning mode used. Each scan cycle time was 1.1 s, consisting of one MS1 scan and 10 PASEF MS / MS scans, with each PASEF MS / MS scan containing 12 MS / MS spectra. Figure 7The experimental data from China A showed that 196 glycopeptides, 82 glycoproteins, and 232 glycosylation sites were identified in serum samples from human ovarian cancer patients and healthy individuals. Figure 7 The results in B show significant differences in the glycoproteins identified in different individual samples. Figure 7 The experimental data from C describe the different expression levels of enriched glycoproteins in diseased and healthy individuals. The above experimental data demonstrate that SCOF also exhibits excellent enrichment effects in complex real-world samples.

[0052] In summary, this invention provides a sulfonic acid-functionalized SCOF for the specific enrichment of glycopeptides, which is then detected using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). This method offers a novel approach for the efficient enrichment of glycopeptides and holds promise for large-scale proteomic analysis of complex biological samples in the future. Compared to the traditional unfunctionalized HILIC method, SCOF possesses several unique advantages, including a large specific surface area, good hydrophilicity, and a surface rich in hydrophilic sulfonic acid groups that provide numerous binding sites for glycopeptides, thus achieving effective glycopeptide enrichment. Experimental results demonstrate its wide applicability, high selectivity, strong anti-interference ability, and low detection limit. Therefore, this invention effectively overcomes the shortcomings of existing technologies and possesses high industrial application value.

[0053] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A method for preparing a sulfonic acid-functionalized covalent organic framework material, characterized in that... The specific preparation process is as follows: 1,3,5-tricarboxymethyl phloroglucinol and 2,5-diamino-1,4-benzenedisulfonic acid are dispersed in a mixed solvent of 1,4-dioxane, mesitylene, and acetic acid to form a mixed solution. The mixed solution is then circulated multiple times under liquid nitrogen using a freeze-thaw pump. Subsequently, the mixed solution is sealed and heated in an oil bath for reaction. After the reaction is completed, it is naturally cooled to room temperature. The solid after the reaction is taken out and washed with N,N-dimethylformamide, pure water, and anhydrous tetrahydrofuran. Finally, it is vacuum dried to obtain a dark brown powdery sulfonic acid-functionalized covalent organic framework material. This covalent organic framework material has ultra-high hydrophilicity, an ordered nanoporous structure, and good solubility and dispersibility.

2. The method for preparing the sulfonic acid-functionalized covalent organic framework material according to claim 1, characterized in that... The specific preparation steps are as follows: 1,3,5-tricarboxymethyl phloroglucinol and 2,5-diamino-1,4-benzenedisulfonic acid are dispersed in a mixed solvent of 1,4-dioxane, mesitylene and acetic acid to form a mixed solution. The mixed solution is then circulated three times by a cryogenic thawing pump under liquid nitrogen at 77 K. Subsequently, the mixed solution is sealed and placed in an oil bath and heated at 110-130 °C for 72 h. After the reaction is completed, it is naturally cooled to room temperature. The solid after the reaction is taken out and washed three times each with N,N-dimethylformamide, pure water and anhydrous tetrahydrofuran. Finally, it is vacuum dried overnight at 120 °C to obtain a dark brown powdery sulfonic acid-functionalized covalent organic framework material.

3. The method for preparing sulfonic acid-functionalized covalent organic framework materials according to claim 1 or 2, characterized in that: The molar ratio of 1,3,5-tricarboxymethyl phloroglucinol to 2,5-diamino-1,4-benzenedisulfonic acid is 1:1.2~2.

4. The method for preparing sulfonic acid-functionalized covalent organic framework materials according to claim 1 or 2, characterized in that: The water contact angle of the sulfonic acid-functionalized covalent organic framework material is 35°. o ~45 o It has good solubility and dispersibility, and exhibits a significant Tyndall effect.

5. A sulfonic acid-functionalized covalent organic framework material prepared by the method according to any one of claims 1 to 4.

6. The application of the sulfonic acid-functionalized covalent organic framework material of claim 5 in the selective enrichment of glycosylated peptides, wherein the covalent organic framework material exhibits excellent enrichment selectivity, high enrichment efficiency, low detection limit and high anti-interference ability.

7. The application according to claim 6, characterized in that... The specific application steps are as follows: (1) The sulfonic acid-functionalized covalent organic framework material was mixed with the enrichment solution and then ultrasonically dispersed to obtain a suspension; (2) The glycosylated peptide sample to be separated is added to the suspension and enriched by vortexing. The abundant hydrophilic sulfonic acid groups on the surface of the sulfonic acid functionalized covalent organic framework material bind to the hydrophilic peptides in the glycosylated peptides through hydrophilic interaction. (3) After enrichment, centrifuge to remove the supernatant, add washing solution to wash away non-glycosylated peptides; (4) After washing, add the elution solution, sonicate to disperse evenly, and then vortex to elute and extract to obtain the enriched glycosylated peptides.

8. The application according to claim 7, characterized in that: The enrichment solution is a mixed solution prepared from acetonitrile, trifluoroacetic acid and pure water, wherein the volume concentration of acetonitrile is 80%~95% and the volume concentration of trifluoroacetic acid is 0.05%~1.0%; the washing solution is a mixed solution prepared from acetonitrile, trifluoroacetic acid and pure water, wherein the volume concentration of acetonitrile is 80%~95% and the volume concentration of trifluoroacetic acid is 0.05%~1.0%.

9. The application according to claim 7, characterized in that: The elution solution is a mixed solution prepared from acetonitrile, trifluoroacetic acid and pure water, wherein the volume concentration of acetonitrile is 25%~35% and the volume concentration of trifluoroacetic acid is 0.05%~1.0%.

10. The application of the sulfonic acid-functionalized covalent organic framework material of claim 5 in the selective detection of glycosylated peptides, wherein the covalent organic framework material exhibits excellent enrichment selectivity, high enrichment efficiency, low detection limit and high anti-interference ability.

Images