Polyurea-modified macroporous adsorption resin microspheres, preparation and use thereof

Abstract

This invention discloses a polyurea-functionalized macroporous adsorption resin microsphere material and its applications. Specifically, macroporous adsorption resin microspheres are used as the matrix, and terephthalic diisocyanate and tri-(2-aminoethyl)amine are used as functional monomers. Through isocyanate-amine polymerization and epoxy-amine ring-opening reaction, a polyurea-rich crosslinked network is bonded and coated onto the surface of the resin microspheres, resulting in surface polyurea-functionalized macroporous adsorption resin (MAR@PUP) microspheres. The surface of MAR@PUP microspheres is rich in urea groups, which can recognize phosphorylation sites in peptides through multiple hydrogen bonds. Based on the differences in phosphorylation degree and the regulation of enrichment conditions, highly selective enrichment of monophosphorylated peptides and polyphosphorylated peptides can be achieved, especially showing excellent enrichment performance for polyphosphorylated peptides.

Description

Technical Field

[0001] This invention relates to the technical field of polyurea-functionalized macroporous adsorption resins, and discloses a method for preparing polyurea-modified macroporous adsorption resins and their application in the enrichment of phosphorylated peptides. Using macroporous adsorption resin (MAR) microspheres containing epoxy groups as a matrix, and p-phenylene diisocyanate (PDI) and tris(2-aminoethyl)amine (TAEA) as functional monomers, a polymer rich in polyurea groups is grafted onto the resin surface through isocyanate-amine polymerization and epoxy-amine ring-opening reaction. Under different enrichment conditions, the enrichment of monophosphorylated peptides and polyphosphorylated peptides is achieved by controlling the strength of hydrogen bond interactions with different phosphorylated peptides. Background Technology

[0002] Phosphorylation is one of the most widely used and important post-translational modifications of proteins, and it is closely related to human life activities and the occurrence and development of diseases. While mass spectrometry has significant advantages in protein identification, phosphorylated peptides have low ionization efficiency and low signal abundance, making them easily inhibited by high-abundance non-phosphorylated peptides. Therefore, selective enrichment of phosphorylated peptides before mass spectrometry analysis is crucial (Reference 1, Jiang D. et al. “Design of Ce... 3+"Ions functionalized magnetic black phosphorus nanosheets for highly efficient enrichment of phosphopeptides", Analytica Chimica Acta, 2025, 1350, 343878. Currently, researchers have developed various enrichment materials based on strategies such as immunoprecipitation, immobilized metal ion affinity chromatography (IMAC), and metal oxide affinity chromatography (MOAC). However, these materials generally suffer from problems such as cumbersome synthesis, high cost, and difficult separation (Reference 2, Wang D. et al. “Cotton Ti-IMAC: developing phosphorylated cotton as a novel platform for phosphopeptide enrichment”, ACS Applied Materials & Interfaces, 2023, 15, 47893-47901; Reference 3, Wang X. et al. “ZrO2 doped magnetic mesoporous polyimide for the efficient enrichment of phosphopeptides”, Talanta, 2018, 188.). (385-392.) To further improve enrichment selectivity and anti-interference ability, the mild and highly specific multiple hydrogen bond molecular recognition mechanism has gradually attracted attention. Unlike metal coordination, multiple hydrogen bonds can form a multi-point, reversible hydrogen bond network between ligands and phosphate groups, which can accurately identify phosphorylation sites in complex matrices, while effectively avoiding non-specific adsorption of acidic non-phosphorylated peptides (Reference 4, Qing G. et al. “Hydrogen bond based smart polymer for highly selective and tunable capture of multiply phosphorylated peptides”, Nature Communications, 2017, 8, 461.). Novel affinity materials designed based on multiple hydrogen bonds have advantages such as mild interaction, high selectivity, and good biocompatibility, providing a new strategy for the efficient enrichment of phosphorylated peptides. At the same time, this modification method has the advantages of simple operation and mild conditions. Under different enrichment conditions, the strength of multiple hydrogen bond interaction can be tuned, and the separate enrichment of monophosphorylated peptides, polyphosphorylated peptides, and fully phosphorylated peptides can be achieved. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing polyurea-functionalized macroporous adsorption resin microspheres, using polyurea-functionalized macroporous adsorption resin as an adsorbent, which can enrich polyphosphorylated peptides and monophosphorylated peptides through multiple hydrogen bonds.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: (1) Preparation of polyurea-modified macroporous adsorption resin microspheres First, 0.3-0.4 mol TAEA, 0.18-0.32 mol PDI, and 200-1800 mg of macroporous adsorption resin (MAR) containing epoxy groups were weighed and dispersed in 20-120 mL of N,N-dimethylformamide (DMF), and ultrasonically dispersed and mixed. The reaction was carried out at 40-60 °C under nitrogen (N2) atmosphere with continuous mechanical stirring at 100-150 rpm for 10-12 h. Finally, 30-100 mL of 0.8-1.2 M hydrochloric acid (HCl) solution was added to terminate the reaction. The resulting macroporous adsorption resin microspheres, functionalized with polyurea, were named MAR@PUP.

[0005] (2) Application of polyurea-modified macroporous adsorption resin microspheres in the enrichment of phosphorylated peptides Enrichment of polyphosphorylated peptides: Add 10-50 mg of MAR@PUP material to a centrifuge tube and incubate with 100-500 μL of loading solution for 10-40 min. The loading solution is a mixture of acetonitrile (ACN) and water, with ACN comprising 70%-85% by volume, and also contains 0.1%-1% trifluoroacetic acid (TFA) by volume. Incubate at room temperature for 10-40 min, then centrifuge at 3000-9000 rpm for 3-5 min. After removing the supernatant, add 1-20 μL of protein hydrolysate containing peptides to the 100-500 μL loading solution, then add this mixture to the centrifuge tube, mix thoroughly with the material, incubate at room temperature for 10-40 min, and centrifuge at 3000-9000 rpm for 3-5 min to remove the supernatant. The material was then washed 3-5 times with 100-500 μL of loading solution to remove non-specifically adsorbed peptides. Finally, 100-200 μL of elution solution was added to elute the specifically bound phosphorylated peptides. The elution solution was a mixture of acetonitrile (ACN) and water (H₂O), with ACN comprising 45%-55% by volume. Additionally, the elution solution contained 2%-3% by volume of TFA. The elution solution was centrifuged, lyophilized, and then analyzed by MALDI-TOF MS.

[0006] Monophosphorylated peptide enrichment: Add 10-50 mg of MAR@PUP material to a centrifuge tube and incubate with 100-500 μL of loading solution for 10-40 min. The loading solution is a mixture of ACN and water, with ACN comprising 93%-97% by volume, and also contains 0.08%-0.12% acetic acid (HAc) by volume. Incubate at room temperature for 10-40 min, then centrifuge at 3000-9000 rpm for 3-5 min. After removing the supernatant, add 1-20 μL of protein hydrolysate containing the peptide to the 100-500 μL loading solution, then add this mixture to the centrifuge tube, mix thoroughly with the material, incubate at room temperature for 10-40 min, and centrifuge at 3000-9000 rpm for 3-5 min to remove the supernatant. Next, the material was washed 3-5 times with 100-500 μL of a elution solution (a mixture of ACN and water, with ACN comprising 85%-90% by volume) to remove non-specifically adsorbed peptides. Additionally, the elution solution contained 0.08%-0.12% HAc by volume. Finally, an elution solution (a mixture of ACN and water, with ACN comprising 45%-55% by volume) was added to elute the specifically bound monophosphorylated peptides. The elution solution also contained 0.08%-0.12% HAc by volume. The elution solution was centrifuged, lyophilized, and then analyzed by MALDI-TOF MS.

[0007] The present invention has the following advantages:

[0008] 1. The MAR@PUP material preparation method is simple, mild, and inexpensive, making it suitable for large-scale mass production. Compared with traditional Ti-IMAC or Zr-IMAC methods, it eliminates the need to fix expensive Ti or Zr metal ions on the material surface, reduces the number of preparation steps, and minimizes the waste of metal resources.

[0009] 2. MAR@PUP recognizes phosphorylated peptides through multiple hydrogen bonding interactions between abundant urea groups and phosphorylation sites on its surface, rather than through metal ion coordination. This results in better dynamics and stronger specificity, which is fundamentally different from the IMAC method.

[0010] 3. By simply changing the content of ACN, the content and type of organic acids in the enrichment conditions, the selectivity for monophosphorylated peptides and polyphosphorylated peptides can be precisely controlled to achieve bifunctional enrichment, which has better applicability.

[0011] The surface of MAR@PUP microspheres is rich in urea groups, which can recognize phosphorylation sites in peptides through multiple hydrogen bonds. Based on the differences in phosphorylation levels and the regulation of enrichment conditions, highly selective enrichment of monophosphorylated and polyphosphorylated peptides can be achieved, especially showing excellent enrichment performance for polyphosphorylated peptides. The materials involved in this invention have the advantages of simple preparation, mild reaction conditions, and suitability for large-scale preparation, and have good application prospects in phosphorylated proteomics research. Attached Figure Description

[0012] Figure 1 Flowchart of the preparation process of polyurea-modified macroporous adsorption resin microspheres.

[0013] Figure 2 Scanning electron microscope image of MAR@PUP-1 material in Example 1.

[0014] Figure 3 Nitrogen adsorption / desorption curves (a) and pore size distribution diagram (b) of MAR and MAR@PUP-1 in Example 1.

[0015] Figure 4 Infrared spectra of MAR, MAR@PUP-1 and PUP in Example 1.

[0016] Figure 5 The enrichment results of MAR@PUP-1 on polyphosphorylated peptides in β-casein hydrolysate in Example 1: (a) before enrichment, (b) after enrichment of polyphosphorylated peptides. (* indicates polyphosphorylated peptides).

[0017] Figure 6 The enrichment results of MAR@PUP-1 on monophosphorylated peptides in β-casein hydrolysate in Example 1. (▲ indicates monophosphorylated peptides).

[0018] Figure 7 The enrichment results of MAR@PUP-2 on polyphosphorylated peptides in β-casein hydrolysate in Example 2. (* indicates polyphosphorylated peptides).

[0019] Figure 8 The enrichment results of MAR@PUP-2 on monophosphorylated peptides in β-casein hydrolysate in Example 2. (▲ indicates monophosphorylated peptides).

[0020] Figure 9 The enrichment results of MAR@PUP-3 on polyphosphorylated peptides in β-casein hydrolysate in Example 3. (* indicates polyphosphorylated peptides).

[0021] Figure 10 Example 3 shows the enrichment results of MAR@PUP-3 on monophosphorylated peptides in β-casein hydrolysate. (▲ indicates monophosphorylated peptides).

[0022] Figure 11 Comparative Example 4: Enrichment of polyphosphorylated peptides by MAR@PUP-4 in β-casein hydrolysate. (* indicates polyphosphorylated peptides).

[0023] Figure 12 Comparative Example 4: Enrichment of monophosphorylated peptides by MAR@PUP-4 in β-casein hydrolysate. (* indicates polyphosphorylated peptides).

[0024] Figure 13 The enrichment results of MAR@PUP-5 for polyphosphorylated peptides in β-casein hydrolysate in Comparative Example 5. (* indicates polyphosphorylated peptides).

[0025] Figure 14 Comparative Example 5: Enrichment of monophosphorylated peptides by MAR@PUP-5 in β-casein hydrolysate. (* indicates polyphosphorylated peptides).

[0026] Figure 15 Comparative Example 6: Enrichment of polyphosphorylated peptides by MAR-TAEA in β-casein hydrolysate. (▲ indicates monophosphorylated peptides).

[0027] Figure 16 Comparative Example 6: Enrichment of monophosphorylated peptides by MAR-TAEA in β-casein hydrolysate. (▲ indicates monophosphorylated peptides).

[0028] Figure 17 The enrichment results of PUP for polyphosphorylated peptides in β-casein hydrolysate in Comparative Example 7. (* indicates polyphosphorylated peptides).

[0029] Figure 18 Comparative Example 7: Enrichment of monophosphorylated peptides by PUP in β-casein hydrolysate. (▲ indicates monophosphorylated peptides).

[0030] Figure 19 Comparative Example 8 Ti 4+ -IMAC enrichment results of phosphorylated peptides in β-casein hydrolysate. (▲ indicates monophosphorylated peptides, * indicates polyphosphorylated peptides). Detailed Implementation

[0032] Example 1: Preparation of MAR@PUP-1 material:

[0033] Preparation of macroporous adsorption resin microspheres containing epoxy groups

[0034] 1 g of polyvinyl alcohol was dispersed in 120 mL of water; then 18 mL of toluene, 15 mL of ethylene glycol dimethacrylate, and 20 mL of glycidyl methacrylate were mixed thoroughly; 0.12 g of benzoyl peroxide was added to the mixture, and the reaction was carried out at 70 °C under a N2 atmosphere for 12 h. The solid was collected by filtration, and the resulting white powder was washed three times with 50 mL of water and ethanol, respectively, and dried in a vacuum oven at 60 °C for 48 h to obtain macroporous adsorption resin microspheres containing epoxy groups, denoted as MAR.

[0035] Preparation of polyurea-modified macroporous adsorption resin microspheres (MAR@PUP-1) First, 0.24 mol of PDI was weighed and dissolved in 30 mL of DMF. Then, 0.36 mol of TAEA was measured and added to the solution along with 200 mg of MAR. The reaction was then carried out under N2 conditions at 60 °C with continuous mechanical stirring at 150 rpm for 12 h. Finally, 30 mL of 1 M HCl solution was added to terminate the reaction. The product was washed three times each with DMF and ethanol and then dried in a vacuum oven at 60 °C for 48 h. The resulting polyurea-modified macroporous adsorption resin microspheres were named MAR@PUP-1. The preparation process is as follows: Figure 1 As shown.

[0036] Product characterization The morphology of the MAR@PUP-1 material in Example 1 was characterized using scanning electron microscopy. The results are as follows: Figure 2 As shown, MAR@PUP-1 has a smooth surface and a particle size distribution in the range of 100-400 nm. Meanwhile, the surface morphology of the four materials in the examples—MAR@PUP-2, MAR@PUP-3, and amino-modified macroporous adsorption resin microspheres—is not significantly different from MAR@PUP-1, with particle sizes all within the 100-400 nm range and smooth surfaces. This is mainly because we only performed simple modification on the macroporous adsorption resin with epoxy groups, without destroying its surface structure. Figure 3 The nitrogen adsorption / desorption isotherms for MAR and MAR@PUP-1 are shown. The specific surface area of ​​MAR is 45.9 m². 2 / g, while the specific surface area of ​​the modified MAR@PUP-1 decreased to 32.3 m². 2 / g. The pore size of BJH is approximately 27.1 nm. The decrease in the specific surface area of ​​MAR@PUP-1 is due to the polyurea cross-linked network occupying part of the pores, which also proves the successful preparation of the material. Figure 4 Infrared spectra of MAR, MAR@PUP-1, and PUP (Comparative Example 7) are shown. The infrared spectrum of the PUP material is shown at 1571 cm⁻¹. -1 and 3300 cm-1 Two sharp characteristic absorption peaks appear at 1633 cm⁻¹, attributed to the bending and stretching vibrations of the N–H bonds in the polyurea group, respectively. Additionally, there is another absorption peak at 1633 cm⁻¹, attributed to the stretching vibration of the C=O group in the polyurea group. The MAR infrared spectrum shows an absorption peak at 1255 cm⁻¹. -1 The characteristic absorption peak of the C–O bond of the epoxy group appeared at the point; after polyurea modification, the intensity of the peak was significantly reduced, confirming that the epoxy group on the surface of MAR underwent an epoxy-amine ring-opening reaction with the amino group in the polyurea group, proving that the polyurea crosslinked network PUP and MAR@PUP-1 material were successfully prepared.

[0037] Product Application: Using MAR@PUP-1 as an adsorbent, polyphosphorylated peptides in β-casein hydrolysate are enriched.

[0038] Preparation of β-casein hydrolysate samples

[0039] 2 mg of β-casein was dissolved in 10 mL of 0.1 M ammonium bicarbonate solution containing 8 M urea (pH=8.2). 80 µmol of DL-1,4-dithiothreitol was added, and the mixture was kept at 37 °C for 2 h. Then, 40 µmol of 2-iodoacetamide was added, and the mixture was reacted in the dark for 40 min. The urea concentration was diluted to 1 M with 0.1 M ammonium bicarbonate solution. 80 µg of trypsin was added at a protein to trypsin mass ratio of 25:1, and the mixture was kept at 37 °C for 16 h to obtain a protein hydrolysate (β-casein hydrolysate) containing peptides. This hydrolysate was stored at -20 °C for later use.

[0040] Enrichment of polyphosphorylated peptides Using the MAR@PUP-1 material prepared in Example 1 as the adsorbent, 10 mg of the material was weighed into a centrifuge tube and incubated for 30 min with 200 μL of loading solution. The loading solution was a mixture of ACN and H2O, wherein the volume ratio of ACN was 80%, containing 0.1% TFA, and the remainder was water. 1 μg of β-casein hydrolysate was added, and the mixture was incubated at room temperature for 30 min. Subsequently, the mixture was centrifuged at 8000 rpm for 5 min, and the supernatant was removed. The material was then washed and centrifuged three times with 200 μL of eluent (centrifuged at 8000 rpm for 5 min each time, and the supernatant was removed) to remove non-specific adsorbed peptides. The volume ratio of ACN in the eluent was 75%, containing 0.1% TFA, and the remainder was water. Finally, the mixture was eluted with 150 μL of elution buffer for 30 min. The eluent contained 50% ACN by volume, 2.5% TFA by volume, and the remainder was water. After elution, the eluent and adsorbent were separated by centrifugation at 8000 rpm for 5 min. The resulting eluent containing polyphosphorylated peptides could be analyzed directly by MALDI-TOF MS (either lyophilized or freeze-dried).

[0041] MALDI-TOF-MS analysis: Add 1 μL of sample solution (β-casein digest or elution buffer) to the MALDI target and allow it to air dry. Then, cover the sample spot with 1 μL of 2,5-dihydroxybenzoic acid solution (DHB, 25 mg / mL) as a matrix. After it has completely air dried, perform MALDI-TOF MS analysis.

[0042] Enrichment results The analytical results of MAR@PUP-1 on polyphosphorylated peptides in Example 1 are as follows: Figure 5 As shown. Figure 5 a shows the MALDI-TOF MS spectrum of the β-casein hydrolysate. Although three phosphorylated peptide signals were detected (β1, monophosphorylated peptide, mass-to-charge ratio 2061 Da; β2, monophosphorylated peptide, mass-to-charge ratio 2556 Da; β3, polyphosphorylated peptide, mass-to-charge ratio 3121 Da), a large number of non-phosphorylated peptides were also present in the spectrum, interfering with the identification of phosphorylated peptides. Figure 5 b shows the enrichment results of MAR@PUP-1 on β-casein hydrolysate. The results show that MAR@PUP-1 successfully identified one polyphosphorylated peptide (β3), while no other non-phosphorylated peptides or monophosphorylated peptides were detected, indicating that MAR@PUP-1 has excellent selective enrichment performance for polyphosphorylated peptides.

[0043] Using MAR@PUP-1 as an adsorbent, monophosphorylated peptides in β-casein hydrolysate were enriched. The preparation process and conditions for β-casein hydrolysis samples are the same as above.

[0044] Enrichment of monophosphorylated peptides Using the MAR@PUP-1 material prepared in Example 1 as the adsorbent, 10 mg of the material was weighed into a centrifuge tube and incubated for 30 min with 200 μL of loading solution. The loading solution was a mixture of ACN and H2O, wherein the volume ratio of ACN was 95%, containing 0.1% HAc by volume, and the remainder was water. 1 μg of β-casein hydrolysate was added, and the mixture was incubated at room temperature for 30 min. Subsequently, the mixture was centrifuged at 8000 rpm for 5 min, and the supernatant was removed. The material was then washed and centrifuged three times with 200 μL of eluent (centrifuged at 8000 rpm for 5 min each time, and the supernatant was removed) to remove non-specific adsorbed peptides. The volume ratio of ACN in the eluent was 90%, containing 0.1% HAc by volume, and the remainder was water. Finally, the mixture was eluted with 150 μL of elution buffer for 30 min. The eluent contained 50% ACN by volume, including 0.1% HAc by volume, with the remainder being water. After elution, the eluent was centrifuged at 8000 rpm for 5 min to separate the eluent from the adsorbent. The resulting eluent contained monophosphorylated peptides and could be analyzed directly using MALDI-TOF MS (either lyophilized or freeze-dried).

[0045] The MALDI-TOF MS analysis method and conditions are the same as above.

[0046] Enrichment results

[0047] The analytical results of MAR@PUP-1 on monophosphorylated peptides in Example 1 are as follows: Figure 6 As shown in the spectrum, only two monophosphorylated peptides (β1 and β2) were detected, and no non-phosphorylated peptides or polyphosphorylated peptides were found, indicating that MAR@PUP-1 has excellent selective enrichment performance for monophosphorylated peptides under the current conditions.

[0048] Example 2: Preparation of MAR@PUP-2 material The preparation process and conditions for MAR@PUP-2 were the same as in Example 1, except that the amount of TAEA added was 0.3 mol. The resulting material was named MAR@PUP-2.

[0049] Using MAR@PUP-2 as an adsorbent, polyphosphorylated peptides in β-casein hydrolysate were enriched. The preparation process and conditions for the β-casein hydrolysate samples were the same as in Example 1.

[0050] The enrichment process and conditions for polyphosphorylated peptides were the same as those for polyphosphorylated peptides in Example 1, except that the adsorbent used was the MAR@PUP-2 material prepared in Example 2, and the amount used was 10 mg.

[0051] The MALDI-TOF MS analysis methods and conditions are the same as in Example 1.

[0052] Using MAR@PUP-2 as an adsorbent, monophosphorylated peptides in β-casein hydrolysate were enriched. The preparation process and conditions for the β-casein hydrolysate samples were the same as in Example 1.

[0053] The enrichment process and conditions for monophosphorylated peptides were the same as those for monophosphorylated peptides in Example 1, except that the adsorbent used was the MAR@PUP-2 material prepared in Comparative Example 2, and the amount used was 10 mg.

[0054] The MALDI-TOF MS analysis methods and conditions are the same as in Example 1.

[0055] Enrichment results The analytical results of MAR@PUP-2 on polyphosphorylated peptides in Example 2 are as follows: Figure 7 As shown in the image, only one signal of a polyphosphorylated peptide (β3) was detected in the spectrum. No non-phosphorylated peptides or monophosphorylated peptides were found, indicating that MAR@PUP-2 also has excellent selective enrichment performance for polyphosphorylated peptides under the current conditions. The analysis results of MAR@PUP-2 for monophosphorylated peptides in Example 2 are shown below. Figure 8 As shown in the spectrum, only two monophosphorylated peptides (β1 and β2) were detected, and no non-phosphorylated peptides or polyphosphorylated peptides were found, indicating that MAR@PUP-2 also has excellent selective enrichment performance for monophosphorylated peptides under the current conditions.

[0056] Example 3: Preparation of MAR@PUP-3 material The preparation process and conditions for MAR@PUP-2 were the same as in Example 1, except that the amount of TAEA added was 0.4 mol. The resulting material was named MAR@PUP-3.

[0057] Using MAR@PUP-3 as an adsorbent, polyphosphorylated peptides in β-casein hydrolysate were enriched. The preparation process and conditions for the β-casein hydrolysate samples were the same as in Example 1.

[0058] The enrichment process and conditions for polyphosphorylated peptides were the same as those for polyphosphorylated peptides in Example 1, except that the adsorbent used was the MAR@PUP-3 material prepared in Example 3, and the amount used was 10 mg.

[0059] The MALDI-TOF MS analysis methods and conditions are the same as in Example 1.

[0060] Using MAR@PUP-3 as an adsorbent, monophosphorylated peptides in β-casein hydrolysate were enriched. The preparation process and conditions for the β-casein hydrolysate samples were the same as in Example 1.

[0061] The enrichment process and conditions for monophosphorylated peptides were the same as those for monophosphorylated peptides in Example 1, except that the adsorbent used was the MAR@PUP-3 material prepared in Comparative Example 3, and the amount used was 10 mg.

[0062] The MALDI-TOF MS analysis methods and conditions are the same as in Example 1.

[0063] Enrichment results The analytical results of MAR@PUP-3 on polyphosphorylated peptides in Example 3 are as follows: Figure 9 As shown in the image, only one signal of a polyphosphorylated peptide (β3) was detected in the spectrum. No non-phosphorylated peptides or monophosphorylated peptides were found, indicating that MAR@PUP-3 also has excellent selective enrichment performance for polyphosphorylated peptides under the current conditions. The analysis results of MAR@PUP-3 for monophosphorylated peptides in Example 3 are shown below. Figure 10 As shown in the spectrum, only two monophosphorylated peptides (β1 and β2) were detected, and no non-phosphorylated peptides or polyphosphorylated peptides were found, indicating that MAR@PUP-3 also has excellent selective enrichment performance for monophosphorylated peptides under the current conditions.

[0064] Comparative Example 4: Preparation of MAR@PUP-4 material The preparation process and conditions for MAR@PUP-4 are described in Example 1, except that the amount of TAEA added is 0.24 mol. The resulting material is named MAR@PUP-4.

[0065] Using MAR@PUP-4 as an adsorbent, polyphosphorylated peptides in β-casein hydrolysate were enriched. The preparation process and conditions for the β-casein hydrolysate samples were the same as in Example 1.

[0066] The enrichment process and conditions for polyphosphorylated peptides were the same as those for polyphosphorylated peptides in Example 1, except that the adsorbent used was the MAR@PUP-4 material prepared in Example 4, and the amount used was 10 mg.

[0067] The MALDI-TOF MS analysis methods and conditions are the same as in Example 1.

[0068] Using MAR@PUP-4 as an adsorbent, monophosphorylated peptides in β-casein hydrolysate were enriched. The preparation process and conditions for the β-casein hydrolysate samples were the same as in Example 1.

[0069] The enrichment process and conditions for monophosphorylated peptides were the same as those for monophosphorylated peptides in Example 1, except that the adsorbent used was the MAR@PUP-4 material prepared in Comparative Example 4, and the amount used was 10 mg.

[0070] The MALDI-TOF MS analysis methods and conditions are the same as in Example 1.

[0071] Enrichment results The analytical results of MAR@PUP-4 on polyphosphorylated peptides in Comparative Example 4 are as follows: Figure 11 As shown in the MALDI-TOF MS spectrum, only the signal of polyphosphorylated peptide (β3) was detected; no non-phosphorylated peptides or monophosphorylated peptides were found, indicating that MAR@PUP-4 has similar selectivity for polyphosphorylated peptides to MAR@PUP-1. However, the signal value of β3 in the spectrum is lower than that in Example 1, which is due to the lower content of surface urea groups. The signal of MAR@PUP-4 for monophosphorylated peptides in Comparative Example 4 is shown in the image. Figure 12 As shown in the spectrum, while signals of two monophosphorylated peptides (β1 and β2) were clearly detected, signals of the polyphosphorylated peptide β3 and a small amount of interfering signals from non-phosphorylated peptides were also detected. This may be because excess PDI results in unreacted aldehyde groups in the surface polymer network, which could potentially react with the terminal amino groups in the peptide, causing non-specific adsorption. This indicates that MAR@PUP-4 lacks specificity for monophosphorylated peptides.

[0072] Comparative Example 5: Preparation of MAR@PUP-5 material The preparation process and conditions for MAR@PUP-5 were the same as in Example 1, except that the amount of TAEA added was 0.48 mol. The resulting material was named MAR@PUP-5.

[0073] Using MAR@PUP-5 as an adsorbent, polyphosphorylated peptides in β-casein hydrolysate were enriched. The preparation process and conditions for the β-casein hydrolysate samples were the same as in Example 1.

[0074] The enrichment process and conditions for polyphosphorylated peptides were the same as those for polyphosphorylated peptides in Example 1, except that the adsorbent used was the MAR@PUP-5 material prepared in Example 5, and the amount used was 10 mg.

[0075] The MALDI-TOF MS analysis methods and conditions are the same as in Example 1.

[0076] Using MAR@PUP-5 as an adsorbent, monophosphorylated peptides in β-casein hydrolysate were enriched. The preparation process and conditions for the β-casein hydrolysate samples were the same as in Example 1.

[0077] The enrichment process and conditions for monophosphorylated peptides were the same as those for monophosphorylated peptides in Example 1, except that the adsorbent used was the MAR@PUP-5 material prepared in Comparative Example 5, and the amount used was 10 mg.

[0078] The MALDI-TOF MS analysis methods and conditions are the same as in Example 1.

[0079] Enrichment results The analytical results of MAR@PUP-5 on polyphosphorylated peptides in Comparative Example 5 are as follows: Figure 13 As shown in the MALDI-TOF MS spectrum, only the signal of polyphosphorylated peptide (β3) was detected; no non-phosphorylated peptides or monophosphorylated peptides were found, indicating that MAR@PUP-4 has similar selectivity for polyphosphorylated peptides to MAR@PUP-1. However, the signal value of β3 in the spectrum is lower than that in Example 1, which is due to the lower content of surface urea groups. The signal of MAR@PUP-5 for monophosphorylated peptides in Comparative Example 5 is shown in the image. Figure 14 As shown in the spectrum, while signals of two monophosphorylated peptides (β1 and β2) were clearly detected, signals of the polyphosphorylated peptide β3 and a small amount of interfering signals from non-phosphorylated peptides were also detected. This may be because excess PDI results in unreacted aldehyde groups in the surface polymer network, which could potentially react with the terminal amino groups in the peptide, causing non-specific adsorption. This indicates that MAR@PUP-4 lacks specificity for monophosphorylated peptides.

[0080] Comparative Example 6: Preparation of amino-modified macroporous adsorption resin microspheres (MAR-TAEA) The preparation process and conditions of the amino-modified macroporous adsorption resin microspheres are the same as in Example 1, except that PDI is not added, and TAEA is grafted onto the surface only through an epoxy-amine reaction. The resulting material is named MAR-TAEA.

[0081] Using MAR-TAEA as an adsorbent, polyphosphorylated peptides in β-casein hydrolysate were enriched. The preparation process and conditions for the β-casein hydrolysate samples were the same as in Example 1.

[0082] The enrichment process and conditions for polyphosphorylated peptides were the same as those for polyphosphorylated peptides in Example 1, except that the adsorbent used was the MAR-TAEA material prepared in Comparative Example 6, and the amount used was 10 mg.

[0083] The MALDI-TOF MS analysis methods and conditions are the same as in Example 1.

[0084] Using MAR-TAEA as an adsorbent, monophosphorylated peptides in β-casein hydrolysate were enriched. The preparation process and conditions for the β-casein hydrolysate samples were the same as in Example 1.

[0085] The enrichment process and conditions for monophosphorylated peptides were the same as those for monophosphorylated peptides in Example 1, except that the adsorbent used was the MAR-TAEA material prepared in Comparative Example 6, and the amount used was 10 mg.

[0086] The MALDI-TOF MS analysis methods and conditions are the same as in Example 1.

[0087] Enrichment results The analytical results of MAR-TAEA on polyphosphorylated peptides in Comparative Example 6 are as follows: Figure 15 As shown in the spectrum, a clear signal of polyphosphorylated peptide (β3) was detected, while no non-phosphorylated peptides or monophosphorylated peptides were found, indicating that MAR-TAEA has similar selectivity for polyphosphorylated peptides to MAR@PUP-1. However, the signal value of β3 in the spectrum is much lower than that in Example 1. This is because MAR-TAEA only has TAEA molecules bonded to its surface, where the density of amino groups serving as adsorption sites is significantly lower than that of the urea crosslinking network. The analytical results of MAR-TAEA for monophosphorylated peptides in Comparative Example 6 are shown below. Figure 16 As shown in the image, two monophosphorylated peptides (β1 and β2) and one polyphosphorylated peptide (β3) signals were detected. This indicates that under the current conditions, MAR-TAEA cannot distinguish between monophosphorylated and polyphosphorylated peptides.

[0088] Comparative Example 7: Preparation of Polyurea Crosslinked Network Material (PUP) The preparation method of the polyurea crosslinked network material is the same as in Example 1, except that MAR is not added, and only TAEA reacts with PDI. The resulting light pink powder material is named PUP.

[0089] Using PUP as an adsorbent, polyphosphorylated peptides in β-casein hydrolysate were enriched. The preparation process and conditions for the β-casein hydrolysate samples were the same as in Example 1.

[0090] The enrichment process and conditions for polyphosphorylated peptides were the same as those for polyphosphorylated peptides in Example 1, except that the adsorbent used was the PUP material prepared in Comparative Example 7, and the amount used was 10 mg.

[0091] The MALDI-TOF MS analysis methods and conditions are the same as in Example 1.

[0092] Using PUP as an adsorbent, monophosphorylated peptides in β-casein hydrolysate were enriched. The preparation process and conditions for the β-casein hydrolysate samples were the same as in Example 1.

[0093] The enrichment process and conditions for monophosphorylated peptides were the same as those for monophosphorylated peptides in Example 1, except that the adsorbent used was the PUP material prepared in Comparative Example 7, and the amount used was 10 mg.

[0094] The MALDI-TOF MS analysis methods and conditions are the same as in Example 1.

[0095] Enrichment results The analytical results of PUP on polyphosphorylated peptides in Comparative Example 7 are as follows: Figure 17 As shown in the spectrum, although the signal of polyphosphorylated peptide (β3) was detected, the signal of some non-phosphorylated peptides was also detected simultaneously. The analysis results of PUP for monophosphorylated peptides in Comparative Example 7 are shown below. Figure 18 As shown in the spectrum, a very weak signal of monophosphorylated peptide was detected, along with signals of non-phosphorylated peptide. These results indicate that the PUP prepared in Comparative Example 7 has significantly weaker selectivity for both polyphosphorylated and monophosphorylated peptides than MAR@PUP-1 prepared in Example 1. Although the urea group content in PUP is significantly higher than that in MAR@PUP-1, it is an amorphous powder polymer with poor dispersibility and lacks pore structure design, making it unsuitable for enriching phosphorylated peptides.

[0096] Comparative Example 8: Enrichment of phosphorylated peptides in β-casein hydrolysate using commercial IMAC microspheres as adsorbents. The preparation process and conditions for the β-casein hydrolysate samples were the same as in Example 1. Ti 4+ -IMAC method for enriching phosphorylated peptides: With commercial Ti 4+ -IMAC microspheres (purchased from Bailingwei) were used as adsorbents for the enrichment of phosphorylated peptides. 10 mg of Ti was weighed... 4+-IMAC microspheres were placed in centrifuge tubes and equilibrated with 200 μL of loading solution containing 80% ACN, 6% TFA, and the remainder water. Equilibration was repeated three times, 15 min each time. Then, 200 μL of β-casein was added for digestion, and the mixture was incubated at room temperature for 30 min. Following this, the mixture was centrifuged for 5 min at 8000 rpm, and the supernatant was removed. The material was then washed three times with 200 μL of washing buffer A, which contained 50% ACN, 6% TFA, and 200 mM NaCl, with the remainder water, for 15 min each time to remove non-specifically adsorbed peptides. This was followed by washing with washing buffer B, which contained 30% ACN, 0.1% TFA, and the remainder water, for 15 min each time to remove salts. Finally, the material was incubated with 100 μL of 10% (wt%) ammonia at room temperature for 30 min to remove the Ti... 4+ Phosphorylated peptides adsorbed on the IMAC microsphere material were eluted. After elution, the eluent and material were separated, and the resulting eluent was analyzed by MALDI-TOF MS.

[0097] The MALDI-TOF MS analysis methods and conditions are the same as in Example 1.

[0098] Enrichment results The analysis results of Comparative Example 8 are as follows Figure 19 As shown in the spectrum, only three phosphorylated peptide signals were detected, with no interference from non-phosphorylated peptides. This indicates that Ti... 4+ -IMAC materials exhibit ideal selectivity for phosphorylated peptides, but cannot distinguish between polyphosphorylated peptides and monophosphorylated peptides.

Claims

1. A macroporous adsorbent resin with surface polyurea functionalization, characterized in that: Using macroporous adsorption resin microspheres containing epoxy groups as the matrix, p-phenylene diisocyanate (PDI) and tris(2-aminoethyl)amine (TAEA) as functional monomers, a macroporous adsorption resin microsphere material with surface polyurea functionalization was obtained and named MAR@PUP.

2. The polyurea-modified macroporous adsorption resin material according to claim 1, characterized in that, The preparation process of macroporous adsorption resin microspheres containing epoxy groups is as follows: Disperse 1 g of polyvinyl alcohol in 60-180 mL of water, then mix 12-30 mL of toluene, 6-20 mL of ethylene glycol dimethacrylate and 10-30 mL of glycidyl methacrylate evenly; then add 0.08-0.16 g of benzoyl peroxide to the mixture and react for 8-24 h under N2 atmosphere and 60-80 °C. Solid-liquid separation was performed, and the solid was washed with water and ethanol in sequence and dried to obtain macroporous adsorption resin microspheres containing epoxy groups, denoted as MAR. PDI and TAEA are grafted onto the resin surface via isocyanate-amine polymerization and epoxy-amine ring-opening reaction on the matrix. The polymer rich in polyurea groups can be grafted onto the resin surface. Under different enrichment conditions, the hydrogen bond interaction strength between the polymer and different phosphorylated peptides can be controlled to achieve separate enrichment of monophosphorylated peptides and polyphosphorylated peptides.

3. The polyurea-modified macroporous adsorption resin material according to claim 1 or 2, characterized in that, You can follow these steps: First, 0.3-0.4 mol TAEA, 0.24 mol PDI, and 200-1800 mg of macroporous adsorption resin (MAR) containing epoxy groups were weighed and dispersed in 20-120 mL of N,N-dimethylformamide (DMF) and mixed well. The reaction was carried out under nitrogen (N2) at 40-60 °C with continuous mechanical stirring at 100-150 rpm for 10-12 h. Finally, 30-100 mL of 0.8-1.2 M hydrochloric acid (HCl) solution was added to terminate the reaction. The surface-modified polyurea macroporous adsorption resin microsphere material was obtained and named MAR@PUP.

4. An application of a macroporous adsorption resin material modified with polyurea as described in claim 1, 2 or 3 for the selective enrichment of monophosphorylated peptides and / or polyphosphorylated peptides in biological samples of phosphorylated proteomics.

5. Use according to claim 4, characterized in that: This resin utilizes the multiple hydrogen bond interactions between surface polyurea groups and phosphorylated peptides to selectively enrich monophosphorylated peptides and / or polyphosphorylated peptides in complex biological samples under different enrichment conditions.

6. The enrichment of mono- and polyphosphorylated peptides according to claim 4 or 5, characterized in that, You can do it as follows: Enrichment of polyphosphorylated peptides: Add 10-50 mg of MAR@PUP material to a centrifuge tube and incubate with 100-500 μL of loading solution for 10-40 min. The loading solution is a mixture of acetonitrile (ACN) and water, with ACN comprising 70%-85% by volume. Additionally, the loading solution contains 0.1%-1% trifluoroacetic acid (TFA) by volume, with the remainder being water. Add 1-20 μL of protein hydrolysate containing peptides to the centrifuge tube, mix thoroughly with the material, and incubate at room temperature for 10-40 min. Centrifuge at 3000-9000 rpm for 3-5 min to remove the supernatant. Then, wash and centrifuge the material again with 100-500 μL of eluent (each time using 100-500 μL of eluent, centrifuged at 3000-9000 rpm for 3-5 min). After removing the supernatant, rinse 3-5 times to remove non-specifically adsorbed peptides. The elution solution is a mixture of ACN and water, with ACN comprising 70%-80% by volume. In addition, the elution solution contains 0.08%-0.12% TFA by volume, and the remainder is water. Finally, add 100-200 μL of elution solution and incubate for 10-40 min to elute the specifically bound phosphorylated peptides. The elution solution is a mixture of acetonitrile (ACN) and water (H2O), with ACN comprising 45%-55% by volume. In addition, the elution solution contains 2%-3% TFA by volume, and the remainder is water. The eluent and adsorbent were separated by centrifugation, and the eluent containing polyphosphorylated peptides was collected. And / or, enrichment of monophosphorylated peptides: Add 10-50 mg of MAR@PUP material to a centrifuge tube and incubate with 100-500 μL of loading solution for 10-40 min; the loading solution is a mixture of ACN and water, with ACN accounting for 93%-97% by volume, and also contains 0.08%-0.12% acetic acid (HAc) by volume, with the remainder being water; add 1-20 μL of protein hydrolysate containing peptides to the centrifuge tube, mix thoroughly with the material, incubate at room temperature for 10-40 min, centrifuge at 3000-9000 rpm for 3-5 min to remove the supernatant; then, wash and centrifuge the material again with 100-500 μL of rinsing solution (each time using 100-500 μL of rinsing solution, centrifuge at 3000-9000 rpm for 3-5 min). After removing the supernatant, the eluent is rinsed 3-5 times to remove non-specifically adsorbed peptides. The elution solution is a mixture of ACN and water, with ACN comprising 85%-90% by volume and HAc comprising 0.08%-0.12% by volume, with the remainder being water. Finally, 100-200 μL of elution solution is added and incubated for 10-40 min to elute the specifically bound monophosphorylated peptides. The elution solution is a mixture of ACN and water, with ACN comprising 45%-55% by volume and HAc comprising 0.08%-0.12% by volume, with the remainder being water. The elution solution is then centrifuged to separate the eluent from the adsorbent, and the eluent containing the monophosphorylated peptides is collected.

7. The application according to claim 4, 5, or 6, characterized in that: It can be used for the selective enrichment of polyphosphorylated peptides, monophosphorylated peptides, or fully phosphorylated peptides in biological samples (such as one or more of the body fluids, tissues and cells of humans or animals, or tissue or cell enzymatic hydrolysates).

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