Ti 4+ Modified tannic acid-chitosan composite film and its preparation and application

Abstract

The present invention relates to a reusable Ti<supgt;4+< / supgt>-modified tannic acid-chitosan composite membrane and its preparation and application. Specifically, a "one-pot" strategy is adopted, using chitosan and tannic acid as functional monomers and polyethylene glycol diglycidyl ether as a cross-linking agent. The three reaction monomers are dissolved in acetic acid solution and freeze-dried to form a solid membrane, and then it is heated for epoxy amine ring-opening reaction to obtain a tannic acid-chitosan composite membrane. Since the structure of tannic acid contains pyrogallol groups, which can chelate with titanium ions (Ti<supgt;4+< / supgt;) as ligands, a Ti<supgt;4+< / supgt>-modified tannic acid-chitosan composite membrane is further obtained. This composite membrane can be used as an adsorbent for immobilized metal ion affinity chromatography for the efficient enrichment of phosphopeptides in biological samples. The Ti<supgt;4+< / supgt>-modified tannic acid-chitosan composite membrane has a three-dimensional pore structure. Because Ti<supgt;4+< / supgt> and the pyrogallol ligand can form stable chelation bonds, after the phosphopeptides are eluted from the composite membrane, Ti<supgt;4+< / supgt> will not dissociate from the composite membrane, so it can be reused.

Description

Technical Field

[0001] The present invention relates to a reusable Ti 4+ modified tannic acid-chitosan composite membrane and its preparation and application. Specifically, a "one-pot" strategy is adopted, using chitosan and tannic acid as functional monomers and polyethylene glycol diglycidyl ether as a cross-linking agent. The three reaction monomers are dissolved in acetic acid solution, freeze-dried into a solid membrane, and then heated for epoxyamine ring-opening reaction to obtain a highly cross-linked chitosan composite membrane. Since it contains pyrogallol ligand groups, it can further chelate with titanium ions to obtain a Ti 4+ modified tannic acid-chitosan composite membrane. This composite membrane can be used as an adsorbent for immobilized metal ion affinity chromatography for efficient enrichment of phosphopeptides in biological samples. At the same time, it has a three-dimensional pore structure to improve the mass transfer efficiency; because the pyrogallol ligand can form a stable chelation bond with Ti 4+ During the elution process of phosphopeptides, Ti 4+ will not dissociate from the composite membrane, so the composite membrane can be reused. Background Art

[0002] With the development of genomics, proteomics has become one of the current research focuses in the field of biology. Proteomics conducts systematic qualitative and quantitative analysis of proteins or polypeptides in organisms, and provides material and theoretical basis for the physiological changes, pathological research and target drug development of organisms through the identification of protein structure and function, etc. (Reference 1. I-Hsuan C., et al. “Phosphoproteins in extracellular vesicles as candidate markers for breast cancer” Proceedings of the National Academy of Sciences, 2017, 114, 3175-3180). Protein phosphorylation is a common reversible process in post-translational modification of proteins in organisms. Irregular phosphorylation can lead to cell metabolic disorders and even diseases. However, the research of phosphoproteomics still faces many challenges. Limited by the few phosphorylation sites and low abundance of phosphorylated proteins, phosphoproteins are often masked by high-abundance non-phosphorylated proteins in the analysis of complex biological samples. Therefore, in the research of mass spectrometry-based phosphoproteomics, the selective separation of phosphopeptides becomes the premise and key of the analysis. At present, a variety of methods for enriching phosphopeptides have been developed by using the interaction between phosphoamino acids and materials, mainly including affinity chromatography, ion exchange, antigen-antibody binding and chemical reactions, etc. Among these methods, immobilized metal ion affinity chromatography (IMAC) is widely used due to its advantages such as simple experimental process, high specificity and good reproducibility. However, the existing IMAC adsorbents are generally disposable, cannot be reused, and some toxic reagents are used in the preparation process, bringing a series of problems such as resource waste and environmental pollution. Therefore, it is necessary to develop green and reusable IMAC materials and promote their commercialization.

[0003] In recent years, membrane science and technology have developed rapidly and play an increasingly important role in fields such as chemical analysis, food production, gas separation, wastewater treatment, resource collection, and medical diagnosis and treatment. Chitosan is abundant in nature in terms of storage and sources, and has good antibacterial properties, biocompatibility, biodegradability, and easy film-forming properties. Tannic acid is widely present in various plant tissues in nature such as pine trees, oak trees, and mimosa, and is also contained in common tea and wine in life. At present, many new bio-based polymers have been developed using the different properties of phenolic hydroxyl groups in tannic acid and are widely used in industries such as medicine, food, cosmetics, and water treatment (Literature 2. Chen C., et al. “Tannic acid: a crosslinker leading to versatile functional polymeric networks: a review” RSC Advances, 2022, 12, 7689 - 7711; Literature 3. Ibrahim A., et al. “Synthesising tannin-based coagulants for water and wastewater application: A review” Journal of Environmental Chemical Engineering, 2021, 9(1): 105007). Among them, the strong chelation between the trihydroxybenzene group contained in tannic acid and metal ions enables tannic acid to exhibit excellent adsorption performance in the adsorption of heavy metal ions. Therefore, in this invention, the easy film-forming property of chitosan and the strong chelation between tannic acid and metal ions are utilized to prepare a green and environmentally friendly Ti 4+ -modified tannic acid-chitosan composite membrane, which has reusability in the application of phosphopeptide enrichment due to the modification with tannic acid. Summary of the Invention

[0004] The purpose of this invention is to provide a reusable Ti 4+ -modified tannic acid-chitosan composite membrane, which can be used as an adsorbent for immobilized metal ion affinity chromatography for the separation and enrichment of phosphopeptides in complex biological samples.

[0005] To achieve the above purpose, the following steps can be taken:

[0006] (1) Preparation of tannic acid-chitosan composite film: Disperse 50 - 1000 mg of chitosan in 10 - 100 mL of water, and under the rotation speed of 50 - 150 r / min, carry out mechanical stirring for 1 - 10 min; then add 100 - 500 μL of acetic acid and continue stirring to form a clear and homogeneous solution; then add 50 - 200 mg of polyethylene glycol diglycidyl ether and 50 - 500 mg of tannic acid, and continue stirring for 3 - 15 min until completely dissolved; then transfer the solution to a flat-bottom metal mold or a metal plate, freeze it in liquid nitrogen for 5 - 25 min until the solution is completely frozen into a solid film, then place it in a freeze dryer and dry for 12 - 24 h, and finally place it at 50 - 80 °C and react for 2 - 6 h to obtain the tannic acid-chitosan composite film;

[0007] (2) Ti 4+ Preparation of Ti 4+ -modified tannic acid-chitosan composite film: Immerse the tannic acid-chitosan composite film obtained in (1) in 15 - 100 mL of an aqueous solution of titanium sulfate at 40 - 200 mg / mL, incubate at room temperature for 4 - 12 h, and then wash the material with 15 - 100 mL of water each time for 3 - 5 times to remove excess titanium ions; finally, freeze the washed material in liquid nitrogen for 5 - 25 min again and then place it in a freeze dryer and dry for 12 - 24 h to obtain the Ti

[0008] The present invention adopts a "one-pot" strategy, uses chitosan and tannic acid as functional monomers, and polyethylene glycol diglycidyl ether as a cross-linking agent. Dissolve the three reaction monomers in an acetic acid solution, and after freeze-drying, it becomes a solid film. Then heat it to carry out an epoxy amine ring-opening reaction to obtain the tannic acid-chitosan composite film. Because the structure of tannic acid contains pyrogallol groups, which can chelate with titanium ions (Ti 4+ ) as ligands, and further obtain the Ti 4+ -modified tannic acid-chitosan composite film. This composite film can be used as an adsorbent for immobilized metal ion affinity chromatography for the efficient enrichment of phosphopeptides in biological samples.

[0009] The prepared Ti 4+ -modified tannic acid-chitosan composite film can be used as an IMAC adsorbent for the separation and enrichment of phosphopeptides in complex biological samples. This composite film has a three-dimensional pore structure. Because Ti 4+ can form a stable chelation bond with the pyrogallol ligand, after the phosphopeptides are eluted from the composite film, Ti 4+ will not dissociate from the composite film, so it can be reused.

[0010] The present invention has the following advantages:

[0011] (1) The reagents used in the preparation process of this method have low cost, low toxicity, and are green and environmentally friendly;

[0012] (2) The prepared composite membrane material has a three-dimensional pore structure that is conducive to rapid mass transfer;

[0013] (3) The obtained Ti 4+ -modified tannic acid-chitosan composite membrane as an IMAC adsorbent has excellent specificity and enrichment efficiency for phosphopeptides;

[0014] (4) Since Ti 4+ can form stable coordination bonds with pyrogallol ligands, Ti 4+ will not fall off from the composite membrane during the phosphopeptide enrichment process, and the Ti 4+ -modified tannic acid-chitosan composite membrane as an IMAC adsorbent can be reused. Brief Description of the Drawings

[0015] Figure 1 Ti 4+ Schematic diagram of the preparation of the -modified tannic acid-chitosan composite membrane.

[0016] Figure 2 Helium ion scanning electron micrograph of the cross-section of the tannic acid-chitosan composite membrane (I-III). Figure 2 a-c are the tannic acid-chitosan composite membrane (I) prepared in Example 1; Figure 2 d-f: The tannic acid-chitosan composite membrane (II) prepared in Example 2; Figure 2 g-i: The tannic acid-chitosan composite membrane (III) prepared in Example 3.

[0017] Figure 3 Fourier transform-infrared spectroscopy comparison chart of the tannic acid-chitosan composite membrane (I) and three preparation monomers (chitosan, polyethylene glycol diglycidyl ether, and tannic acid).

[0018] Figure 4 Mass spectrometry comparison chart before and after the enrichment of β-casein hydrolysate. Figure a: Before enrichment; Figure b: After enrichment with the Ti 4+ -modified tannic acid-chitosan composite membrane (I) prepared in Example 1, Figure c: After enrichment with the Ti 4+ -modified tannic acid-chitosan composite membrane (II) prepared in Example 2, Figure d: After enrichment with the Ti 4+ -modified tannic acid-chitosan composite membrane (III) prepared in Example 3. In the figure, (*) represents the identified phosphopeptide signal peak, and (#) represents the identified dephosphorylated fragment signal peak.

[0019] Figure 5 In Examples 1-3, Ti 4+Comparison charts of signal intensities of three characteristic phosphopeptides repeatedly identified from β-casein hydrolysate by the modified tannic acid-chitosan composite membranes (a)-I, (b)-II, and (c)-III as adsorbents.

[0020] Figure 6 Mass spectrometry detection charts of the materials in Comparative Examples 1-3 after enriching phosphopeptides in β-casein hydrolysate. Figure a shows the chitosan membrane in Comparative Example 1; Figures b and c show Ti in Comparative Example 2 4+ Mass spectrometry charts of the first enrichment and the second enrichment of the modified tannic acid-chitosan composite membrane (IV); Figures d and e show the commercial Ti in Comparative Example 3 4+ Mass spectrometry charts of the first enrichment and the second enrichment of the -IMAC material. In the figure, (*) represents the phosphopeptide signal, and (#) represents the dephosphorylated fragment signal Specific implementation manners

[0021] Example 1: Ti 4+ Preparation and application of the modified tannic acid-chitosan composite membrane (I):

[0022] Ti 4+ Preparation of the modified tannic acid-chitosan composite membrane (I):

[0023] (1) Preparation of the tannic acid-chitosan composite membrane (I): Disperse 500 mg of chitosan in 25 mL of water, and under a rotation speed of 60 r / min, carry out mechanical stirring for 5 min; then add 200 μL of acetic acid and continue stirring until it is completely dissolved to form a clear and homogeneous solution; then add 50 mg of polyethylene glycol diglycidyl ether and 100 mg of tannic acid, and continue stirring for 5 min until it is completely dissolved into a homogeneous solution; then transfer the solution to a flat-bottomed (the inner bottom surface is flat) metal (such as copper) mold, freeze it in liquid nitrogen for 10 min until the solution is completely frozen into a solid membrane, then place it in a freeze dryer (vacuum degree 18 Pa) and freeze-dry it at -50 °C for 16 h, and finally place it at 60 °C for reaction for 3 h to obtain the tannic acid-chitosan composite membrane (I) with a thickness of 5-8 mm.

[0024] (2) Preparation of the modified tannic acid-chitosan composite membrane (I): Immerse the tannic acid-chitosan composite membrane (I) obtained in (1) in 25 mL of an aqueous solution of titanium sulfate at 100 mg / mL, and incubate it at room temperature for 8 h; then wash the material with water 30 ml / time for a total of 3 times to remove excessive titanium ions; finally, freeze the washed material in liquid nitrogen for 10 min again and place it in a freeze dryer at -50 °C (vacuum degree 18 Pa) to dry for 12 h to obtain the modified tannic acid-chitosan composite membrane (I). 4+ 4+ modified tannic acid-chitosan composite membrane (I).

[0025] Ti 4+Application of the modified tannic acid-chitosan composite film (I):

[0026] Preparation of β-casein hydrolysate: Dissolve 2.0 mg of β-casein in 1.0 mL of a denaturing aqueous solution containing 8 M urea and 100 mM NH4HCO3; then add 7.0 mL of Tris-HCl buffer to dilute the urea concentration to 1 mol / L; add trypsin according to the enzyme-to-protein mass ratio of 1:25, react in a water bath at 37 °C for 18 h, desalt the obtained hydrolysate, and store it in a -20 °C refrigerator after lyophilization for later use.

[0027] Enrichment of phosphopeptides in β-casein hydrolysate: First, cut the prepared membrane material into rectangular sheets (length: 0.5 - 1.0 cm; width: 0.2 - 0.5 cm, 2 mg) and place them in a centrifuge tube; then, equilibrate the material twice with 200 μL of the loading solution (acetonitrile (ACN) / water (H2O) / trifluoroacetic acid (TFA), 80 / 14 / 6, v / v / v) at room temperature for 15 min each time, and centrifuge to discard the supernatant; after equilibrating the material, add 200 μL of the loading solution (ACN / H2O / TFA, 80 / 14 / 6, v / v / v) containing 10 μg of β-casein to the material, shake for 30 min and then centrifuge to remove the supernatant; then perform non-specific elution to remove non-phosphopeptides, use eluent A (ACN / 200 mM NaCl aqueous solution / TFA, 50 / 44 / 6, v / v / v) 200 μL each time for 15 min for a total of two times; eluent B (ACN / H2O / TFA, 30 / 69.9 / 0.1, v / v / v), 200 μL each time for 15 min for a total of two times; finally, add 100 μL of the elution solution (10% ammonia water by mass concentration) for specific elution, shake for 15 min, centrifuge, and take the supernatant (phosphopeptide enrichment solution) for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF / MS) analysis.

[0028] Repeat the above enrichment process for the used membrane material until the enrichment ability significantly decreases or the material is severely damaged and unable to continue the experiment.

[0029] Example 2: Ti 4+ Preparation and application of the modified tannic acid-chitosan composite film (II):

[0030] Ti 4+ Preparation of the modified tannic acid-chitosan composite film (II):

[0031] During the preparation of the chitosan composite film, increase the amount of tannic acid used in the reaction solution to 400 mg, and other preparation conditions and processes are the same as in Example 1Ti 4+ Preparation of the modified tannic acid-chitosan composite film (I).

[0032] Ti 4+ Application of the modified tannic acid-chitosan composite film (II) is the same as that of Example 1, Ti 4+ Application of the modified tannic acid-chitosan composite film (I).

[0033] Example 3: Ti 4+ Preparation and application of the modified tannic acid-chitosan composite film (III):

[0034] Ti 4+ Preparation of the modified tannic acid-chitosan composite film (III):

[0035] During the preparation of the chitosan composite film, the amount of chitosan used in the reaction solution was reduced to 250 mg, and the amount of tannic acid was reduced to 50 mg. Other preparation conditions and procedures were the same as those in Example 1, Ti 4+ Preparation of the modified tannic acid-chitosan composite film (I).

[0036] Ti 4+ Application of the modified tannic acid-chitosan composite film (III) is the same as that of Example 1, Ti 4+ Application of the modified tannic acid-chitosan composite film (I).

[0037] Comparative Example 1: Preparation and application of the chitosan composite film

[0038] During the preparation of the chitosan composite film, tannic acid was not added. Other preparation conditions and application procedures were the same as those in Example 1.

[0039] Comparative Example 2: Ti 4+ Preparation and application of the modified tannic acid-chitosan composite film (IV):

[0040] Ti 4+ Preparation of the modified tannic acid-chitosan composite film (IV):

[0041] During the preparation of the chitosan composite film, polyethylene glycol diglycidyl ether was not added. Other preparation conditions and procedures were the same as those in Example 1, Ti 4+ Preparation of the modified tannic acid-chitosan composite film (I).

[0042] Ti 4+ Application of the modified tannic acid-chitosan composite film (IV) is the same as that of Example 1.

[0043] Comparative Example 3: Commercial IMAC material for the separation and enrichment of phosphorylated peptides

[0044] For comparison, a commercial IMAC material (purchased from J&K Scientific Ltd., product name CAE-Ti-IMAC) was also used for the separation and enrichment of phosphopeptides in β-casein hydrolysate: 5 mg of the commercial IMAC material was weighed for phosphopeptide enrichment. The enrichment experiment process and method were the same as those in Example 1.

[0045] Product characterization

[0046] The cross-sections of the tannic acid-chitosan composite membranes (I-III) obtained in Examples 1-3 were scanned by helium ion microscopy. Figure 2 a-c show the structure of the tannic acid-chitosan composite membrane (I) in Example 1. From Figure 2 a and b, it can be clearly observed that there are a large number of dense hexagonal pores in the tannic acid-chitosan composite membrane (I), with pore diameters of 50-130 μm. These pores are closely connected to form a honeycomb structure, which is beneficial to improving the mass transfer efficiency of the material. The formation of this pore structure is due to the formation of an ice template during the freezing process of the prepolymer solution, and the sublimation of ice crystals during freeze-drying forms the pore structure, which is related to the viscosity of the prepolymer solution and the freezing conditions. Among them, the viscosity of the prepolymer solution affects the growth of ice crystals, and the freezing conditions determine the growth rate and growth direction of ice crystals during the freezing process. Under the combined influence of the two, different pore structures are formed. This microstructure is regarded as a "brick-bridge-mud" structure, and its microstructure includes: some flat structures arranged in parallel to form a layered structure can be regarded as "bricks"; there are some small protrusions between the upper and lower layers, which can be regarded as "mud", and when the protrusions connect the upper and lower layers, a "bridge" connection is formed. Figure 2 In b, it can be seen that there is a bridge structure between the two layers connecting the two layers to form a pore structure. At the same time Figure 2 In c, it can be more intuitively seen that the two layers are connected to each other. Figure 2 The structure of the tannic acid-chitosan composite membrane (II) obtained in Example 2 shown in d-f also has a pore structure similar to that in Example 1, with a pore diameter of 300-400 μm. Figure 2 In e, it can be observed that there are many protrusions between different layers that do not connect the two layers, and there are fewer bridge structures. This is because the increase in tannic acid content compared with Example 1 reduces the viscosity of the prepolymer solution, resulting in relatively fewer bridge structures formed, and thus the pore diameter becomes larger. Similarly, the viscosity of the prepolymer solution of the tannic acid-chitosan composite membrane (III) in Example 3 is lower than that in Example 2. Therefore, in Figure 2 h, only a layered structure is observed, and there are fewer protrusions on the layer surface, and the pore diameter (greater than 500 μm) of the formed pore structure is larger. In addition, by comparing Figure 2 c, f, and g, it can be observed that Figure 2 the layer surface in f is relatively rough. Figure 2The surface of g is relatively smooth because a large amount of tannic acid can form oligomers with chitosan. The amount of tannic acid added in Example 2 is the largest, and the amount of tannic acid in Example 3 is the least. Therefore, the surface of the layer structure in the tannic acid-chitosan composite film (II) is rough.

[0047] As Figure 3 shown, Fourier-transform infrared spectroscopy was used to characterize the chitosan composite film and three reaction monomers (chitosan, polyethylene glycol diglycidyl ether, tannic acid). From the spectra of chitosan and tannic acid-chitosan composite film (I), absorption peaks located at 3000-3600 cm -1 (-OH and -NH stretching vibrations), 2870 cm -1 (-CH stretching vibration), and 1020 cm -1 (C-O-C glycosidic bond bending vibration) can be observed simultaneously; in the infrared spectrum of tannic acid, there are -OH stretching vibrations (3000-3600 cm -1 ), -C=O on the benzene ring (1700 cm -1 ), skeletal vibrations of the benzene ring (1600, 1530 cm -1 ), and characteristic peaks of benzene ring substitution (753, 818 cm -1 ). In addition, the -C=O peak from tannic acid in the spectrum of tannic acid-chitosan composite film (I) shows a blue shift (1710 cm -1 ), and the -NH2 in chitosan (1590 cm -1 ) shows a red shift to 1540 cm -1 and the hydroxyl peak (3000-3600 cm -1 ) becomes significantly broader. The above results indicate that a large number of hydrogen bonds are formed between chitosan and tannic acid in the tannic acid-chitosan composite film. In addition, no epoxy characteristic peak from polyethylene glycol diglycidyl ether was found in the spectrum of tannic acid-chitosan composite film (I). Therefore, it is indicated that the chitosan monomer undergoes an epoxy-amine ring-opening reaction or an epoxy-phenol polycondensation reaction with the epoxy group. In summary, tannic acid in the tannic acid-chitosan composite film is mainly connected to chitosan by strong hydrogen bond forces, and the epoxy in polyethylene glycol diglycidyl ether undergoes a ring-opening reaction with the amino group in chitosan and the phenolic hydroxyl group in tannic acid to finally obtain the tannic acid-chitosan composite film.

[0048] Product application

[0049] Using β-casein hydrolysate as the sample and MALDI-TOF / MS as the analysis method, the phosphopeptide enrichment ability of the Ti 4+ -chitosan composite film was evaluated. As Figure 4As shown in a, the signal peaks with higher abundances in the mass spectrum before enrichment are all non-phosphopeptide signal peaks, and no phosphopeptide signal can be observed. Using the Ti 4+ modified tannic acid-chitosan composite membrane (I) prepared in Example 1 for enrichment, as Figure 4 shown in b, three phosphopeptide signals (2061, 2556, 3121, m / z) and their dephosphorylated fragment peak signals (1963, 2458, 3023, m / z) can be clearly observed in the mass spectrum, while the non-phosphopeptide signals basically disappear. This result indicates that the membrane material has a very high enrichment efficiency and selectivity for phosphopeptides. As Figure 4 shown in c and d, the Ti 4+ modified tannic acid-chitosan composite membranes (II) and (III) prepared in Example 2 and Example 3 also have similar enrichment effects, indicating that these two membrane materials also have good enrichment efficiency and selectivity for phosphopeptides in the enzymatic hydrolysate. By comparing the mass spectra after enrichment of the three membrane materials, it can be found that in the first enrichment, the phosphopeptide signal intensities after enrichment using the Ti 4+ modified tannic acid-chitosan composite membranes (I-III) are similar, indicating that the Ti 4+ modified tannic acid-chitosan composite membranes (I-III) prepared in Examples 1-3 all have excellent enrichment efficiency for phosphopeptides.

[0050] Due to the strong chelation force between Ti 4+ and the pyrogallol group, a stable coordination bond is formed, and Ti 4+ will not easily fall off from the material surface during the enrichment process. Therefore, the Ti 4+ modified tannic acid-chitosan composite membrane can be reused as an adsorbent. To investigate the reusability of the Ti 4+ modified tannic acid-chitosan composite membranes (I-III), a 10 μg β-casein enzymatic hydrolysate was used as a sample for a cyclic enrichment experiment. As Figure 5 shown in a, in 16 cycles of enrichment, the signal intensities of the three characteristic phosphopeptides (2061, 2556, 3121, m / z) enriched using the Ti 4+ modified tannic acid-chitosan composite membrane (I) prepared in Example 1 hardly decreased significantly, showing its stable enrichment efficiency and proving that the material has excellent reusability. As Figure 5 shown in b, the Ti 4+ modified tannic acid-chitosan composite membrane (II) prepared in Example 2 can show stable enrichment efficiency in 14 consecutive enrichments, but due to material swelling, it cannot complete the fifteenth enrichment, and its material stability is slightly worse than that in Example 1. In addition, Figure 5 c shows the Ti prepared in Example 34+ The modified tannic acid-chitosan composite membrane (III) also has a certain enrichment effect and reusability for phosphopeptides. However, compared with the enrichment effects of Examples 1 and 2, its phosphopeptide signal is significantly lower, and the number of times of reuse is only 5 times, which is much less than that of Examples 1 and 2. Therefore, the above results indicate that the Ti prepared in Example 1 4+ The number of times of reuse of the modified tannic acid-chitosan composite membrane (I) is much greater than that of Example 3, so it has the most excellent reusability.

[0051] In Comparative Example 1, the chitosan membrane without using tannic acid was applied to the enrichment of phosphopeptides in β-casein hydrolysate, and the results are as Figure 6 shown in a. No phosphopeptide signal was detected in the mass spectrometry analysis of its eluate, indicating that the chitosan membrane prepared in Comparative Example 1 does not have the ability to enrich phosphopeptides. This is because the trihydroxybenzene group in tannic acid in Examples 1-3 is the key group, while tannic acid was not used in Comparative Example 1, resulting in the inability of the prepared chitosan membrane to provide groups for chelating metal ions. Therefore, it does not have the ability to enrich phosphopeptides. This highlights the decisive role of tannic acid monomers in the application of materials in the present invention.

[0052] Figure 6 The recycling results of the Ti 4+ modified tannic acid-chitosan composite membrane (IV) in Comparative Example 2 are shown in b and c. It can be seen that the Ti 4+ modified tannic acid-chitosan composite membrane (IV) can complete 2 full processes of enriching phosphopeptides. Although Figure 6 b shows that it has good enrichment ability during its first use, but Figure 6 the signal intensity of the phosphopeptides enriched in the second time shown in c is significantly reduced, indicating that its enrichment ability has significantly decreased. This is because polyethylene glycol diglycidyl ether, the cross-linking agent, was not added in Comparative Example 2. Therefore, the material was partially dissolved and damaged in the solution, resulting in a decrease in its enrichment ability and no stable reusability. This shows that polyethylene glycol diglycidyl ether in the present invention enhances the stability of the material structure and properties, and not using it will affect the reusability of the material.

[0053] For further comparison, in Comparative Example 3, the reusability of the commercially available Ti 4+ -IMAC material (CAE-Ti-IMAC) for phosphopeptide enrichment was investigated. As Figure 6As shown in Fig. d, three phosphopeptide signals (2061, 2556, 3121, m / z) and their dephosphorylated fragment signals can also be observed in the enriched mass spectrum, and there is no obvious interference from non-phosphopeptide signals, indicating that CAE-Ti-IMAC also has good enrichment efficiency and selectivity for phosphopeptides. To verify the reusability of the CAE-Ti-IMAC material, after the first enrichment was completed, the material was washed and then subjected to the second enrichment. No phosphopeptide signal peaks were detected in the enriched mass spectrum ( Figure 6 e), indicating that the CAE-Ti-IMAC lost its ability to enrich phosphopeptides after the first use, suggesting that the commercial IMAC material does not have reusability. After the chelating groups on the surface of the IMAC material are chelated with titanium ions, the titanium ions interact with the phosphate groups in the phosphopeptides to fix the phosphate groups on the material surface. However, under alkaline conditions, the titanium ions dissociate from the phosphate ions to achieve the enrichment purpose. But since the chelating group of this commercial IMAC is phosphate, when releasing the phosphopeptides, the titanium ions will also dissociate from the material surface and cannot be reused. In the present invention, tannic acid is used as the chelating agent, and pyrogallol in tannic acid is used as the chelating group. Its chelating force with titanium ions is stronger than the force between titanium ions and phosphate ions. Therefore, when the titanium ions dissociate from the phosphate groups, the force between the titanium ions and pyrogallol can be maintained without being destroyed, that is, the Ti 4+ -modified tannic acid-chitosan composite membrane material in the application of phosphopeptide enrichment, when releasing the phosphopeptides, Ti 4+ will not dissociate from the material surface, so it still has the enrichment ability and is reusable.

[0054] In the present invention, chitosan and tannic acid are used as raw materials, and a novel low-cost and reusable Ti 4+ -modified tannic acid-chitosan composite membrane material is successfully fabricated by the green and pollution-free freeze-drying method. This composite membrane can be used as an immobilized titanium ion affinity chromatography adsorbent for the separation and enrichment of phosphopeptides in biological samples. The polyethylene glycol diglycidyl ether used increases the acid and alkali resistance of the material in solution; the three-dimensional supermacroporous structure in the Ti 4+ -modified tannic acid-chitosan composite membrane structure provides more adsorption sites for phosphopeptide enrichment, showing excellent enrichment effects; and it uses a large number of pyrogallol groups provided by tannic acid to form stable chelation bonds with Ti 4+ , making the material reusable when enriching phosphopeptides.

Claims

1. A Ti 4+ method for preparing a modified tannic acid-chitosan composite film, characterized in that: First, chitosan, tannic acid, and polyethylene glycol diglycidyl ether were dissolved in water as precursors. After freeze-drying, the obtained solid film was crosslinked at 50 - 80 °C to obtain a tannic acid-chitosan composite film. Finally, it was chelated with titanium ions to obtain a Ti 4+ -modified tannic acid-chitosan composite film.

2. The preparation method according to claim 1, characterized in that: Ti 4+ The preparation of the modified tannic acid-chitosan composite film can be carried out according to the following steps: (1) Preparation of tannic acid-chitosan composite film: Disperse 50-500 mg of chitosan in 10-100 mL of water, and carry out mechanical stirring at a rotation speed of 50-150 r / min for 1-10 min; then add 100-500 μL of acetic acid and continue stirring to form a clear and homogeneous solution; then add 50-200 mg of polyethylene glycol diglycidyl ether and 50-500 mg of tannic acid, and continue stirring for 3-15 min until completely dissolved; Then transfer the solution to a flat-bottomed metal mold or a metal plate, freeze it in liquid nitrogen for 5-25 min to completely freeze the solution into a solid film, place it in a freeze dryer and dry it for 12-24 h, and finally place it at 50-80 °C and react for 2-6 h to obtain the tannic acid-chitosan composite film; (2) Ti 4+ Preparation of Ti-modified tannic acid-chitosan composite film: Immerse the tannic acid-chitosan composite film obtained in (1) in 15 - 100 mL of an aqueous titanium sulfate solution with a concentration of 40 - 200 mg / mL, incubate at room temperature for 4 - 12 h, and then wash the material with water 15 - 100 mL / time for 3 - 5 times to remove excess titanium ions; finally, freeze the washed material in liquid nitrogen again for 5 - 25 min and then dry it in a freeze dryer for 12 - 24 h to obtain the Ti 4+ -modified tannic acid-chitosan composite film.

3. The preparation method according to claim 2, characterized in that: Carry out freeze drying in a freeze dryer, and the freeze drying temperature condition is -40~-55 °C, and the vacuum degree is lower than 20 Pa.

4. Ti prepared by the method according to any one of claims 1 to 3 4+ modified tannic acid-chitosan composite film.

5. Use of the tannic acid-chitosan composite film modified with Ti as claimed in claim 4 4+ which is used as an adsorbent or stationary phase for immobilized metal ion affinity chromatography for enriching phosphopeptides in biological samples.

6. The application according to claim 5, characterized in that: The biological sample is one or more of human and / or animal body fluids, tissue enzymolysis solutions, and cell enzymolysis solutions.

7. The application according to claim 5, wherein: Ti 4+ The modified tannic acid-chitosan composite membrane can be reused. The usage process is that after the material undergoes a single enrichment process including equilibration, sample loading, non-specific elution, and specific elution, after re-equilibration, the material still has an enrichment effect on phosphopeptides when repeating the above enrichment process; among them, the solution used for equilibration is ACN / H2O / TFA, 80 / 14 / 6, v / v / v, the sample loading solution is ACN / H2O / TFA containing 10 μg β-casein, 80 / 14 / 6, v / v / v, the non-specific elution solution includes: eluent A and eluent B, eluent A is: ACN / 200 mM NaCl aqueous solution / TFA, 50 / 44 / 6, v / v / v; eluent B is: ACN / H2O / TFA, 30 / 69.9 / 0.1, v / v / v, the specific elution is also 10% ammonia water, and the re-equilibration solution is ACN / H2O / TFA, 80 / 14 / 6, v / v / v.

Images