Thienyl microspheres containing phosphonate functional groups and their preparation and application

Abstract

The present invention relates to the preparation of a thienyl microsphere containing a phosphonate functional group and its application in the enrichment of phosphopeptides. Specifically, the thienyl microsphere containing a phosphonate is prepared by using a Friedel-Crafts reaction, using diethyl 2-(thienylmethyl)phosphonate (Diethyl (thiophen-2-ylmethyl) phosphonate, DTYP) and thiophene (Thiophene, Th) as comonomers, and the phosphonate on the surface of the microsphere can be degraded to a phosphate after acidification with hydrochloric acid, and further chelated with titanium ions to obtain an adsorbent that can be used as an immobilized ion affinity chromatography (Immobilized metal affinity chromatography, IMAC) for the highly selective enrichment of phosphopeptides. The method is characterized in that the reaction conditions are mild, the requirements for the reaction equipment are low, the reaction system does not require an initiator, a dispersant, a surfactant, etc., the material is cleaner, the biocompatibility is good, the functionalization step is simple, and the phosphate functional group is directly introduced into the microsphere without additional modification.

Description

Technical Field

[0001] The present invention relates to a thienyl microsphere containing a phosphonate functional group prepared based on Friedel-Crafts reaction and its application in phosphopeptide enrichment. Specifically, the thienyl microsphere containing a phosphonate is first prepared by precipitation polymerization, and the phosphonate on the surface of the microsphere is degraded into phosphate after acidification with hydrochloric acid, and then chelated with titanium ions to finally obtain Ti ions that can be used for phosphopeptide enrichment in complex biological samples. 4+ -IMAC microspheres. Background Art

[0002] Phosphorylation modification is an extremely important type of post-transcriptional modification in proteomics. Studies have confirmed that the phosphorylation process of proteins participates in and regulates a variety of important life activities (Reference 1, Zhang Y. et.al, "Fishing the PTMproteome with chemical approaches using functional solid phases", Chemical Society Reviews, 2015, 44, 8260-8287). The bottom-up proteomics method based on mass spectrometry is one of the most accurate and efficient tools for studying phosphorylated proteomics. However, due to the extremely low abundance of phosphopeptides in proteolytic products and their very complex composition, high requirements are placed on the enrichment of phosphopeptides to match the high sensitivity and high accuracy of mass spectrometry.

[0003] IMAC is a commonly used method for enriching phosphopeptides and is widely used in phosphopeptide proteomics research (Reference 2, Zhai Guijin et al., "Separation and enrichment of serum phosphopeptides and mass spectrometry quantification and evaluation as potential tumor markers", Chromatography, 2016, 34 (12), 1192-1198). In recent years, functional IMAC materials have been greatly developed, especially IMAC materials based on polymer microspheres, which have the advantages of good selectivity, simple preparation, good dispersibility, wide pH tolerance range, stable material chemical properties, and high practical value. However, the preparation of polymer microspheres often depends on the free radical polymerization of styrene and methacrylates, and the commonly used preparation methods such as suspension polymerization and seed swelling polymerization have complex preparation processes, harsh reaction conditions, and introduce initiators, dispersants and surfactants that are difficult to remove, which is not conducive to large-scale production and practical applications. Therefore, it is still of great positive significance to develop a polymer microsphere IMAC material with a simple preparation method, cleanliness, and high specificity. Friedel-Crafts reaction is a typical alkylation and acylation reaction of aromatic organic compounds, which is widely used in organic synthesis. Wood et al. (Document 3, Wood C. et al., "Microporous organic polymers formethane storage, Advanced Materials, 2008, 20, 1916-1921) prepared a super cross-linked polystyrene material with vinylbenzyl chloride as a monomer for the first time through Friedel-Crafts reaction, and Li et al. (Document 4, Li, B. et al., "A new strategy to microporous polymers: knitting rigid aromatic building blocks by external cross-linker", Macromolecules, 2011, 44, 2410-2414) proposed a strategy of "knitting" Friedel-Crafts super cross-linked polymers with dimethoxymethane as a cross-linking agent. The advantage of this synthetic strategy is that it does not require the initiator, dispersant and surfactant required for free radical reactions. Although these materials have great application prospects in the fields of gas adsorption, no one has reported Friedel-Crafts polymer materials that can be used in the field of proteomics. Summary of the invention

[0004] The main contents of the present invention include a preparation of thienyl microspheres containing phosphonate based on Friedel-Crafts reaction and its application in phosphopeptide enrichment. First, thienyl microspheres containing phosphonate are prepared by precipitation polymerization. The phosphonate on the surface of the microspheres is degraded into phosphate after acidification with hydrochloric acid, and then chelated with titanium ions to finally obtain Ti 4+ -IMAC microspheres. Specifically include the following steps:

[0005] (1) Preparation of thienyl microspheres containing phosphonate functional groups: 40-80 μL of diethyl 2-(thienylmethyl)phosphonate, 20-170 μL of thiophene, and 100-400 μL of dimethoxymethane are added to 2-4 mL of tetrahydrofuran, and then 2-4 mL of a tetrahydrofuran solution containing 0.05-0.2 g / mL of ferric chloride catalyst is added, mixed and transferred to a 10-20 mL ampoule; the ampoule is placed in liquid nitrogen for 2-5 minutes to freeze the system into a solid state, and then the tube mouth is sealed with a flame spray gun. After the sample returns to room temperature, it is reacted at 70-90° C. for 12-24 hours, and the product is washed with tetrahydrofuran and ethanol for 2-4 times, respectively, and the residual solvent is removed by filtration to obtain thienyl microspheres containing phosphonate functional groups;

[0006] (2) Preparation of phosphate-functionalized thienyl microspheres: First, prepare an acid hydrolysis solution by mixing 100-200 mL of water and 150-250 mL of methanol to make a water / methanol volume ratio of 0.4-0.6, and slowly add 20-70 mL of concentrated hydrochloric acid with a concentration of 10-12 mol / L to make the hydrochloric acid concentration in the solution 1-2 mol / L; after the prepared acid hydrolysis solution is cooled, take 5-30 mL and add it to 50-100 mg of thienyl microspheres, ultrasonicate for 5-10 minutes to make it evenly dispersed, and then heat it in a water bath at a reaction temperature of 50-70°C and a reaction time of 24-72 hours; after the reaction is completed, wash the material with water until it is neutral to obtain phosphate-functionalized thienyl microspheres;

[0007] (3) Ti 4+ Preparation of IMAC microspheres: Add the phosphate-functionalized thienyl microspheres obtained in step (2) to 10-20 mL of a titanium sulfate solution having a concentration of 50-200 mg / mL, with the mass ratio of the microspheres to the titanium sulfate being 1:10-1:100; shake the reaction at room temperature for 4-12 h, wash with deionized water, and dry to finally obtain Ti 4+ -IMAC microspheres.

[0008] The advantages of the preparation method of thienyl microspheres containing phosphonate functional groups are:

[0009] (1) The reaction conditions are mild and the requirements for reaction equipment are low;

[0010] (2) The reaction system does not require initiators, dispersants, surfactants, etc., and the materials are cleaner and have better biocompatibility;

[0011] (3) The functionalization procedure is simple, and the desired functional groups are directly introduced into the microspheres without the need for additional modification.

[0012] The Ti 4+ -IMAC microsphere material has the following advantages:

[0013] (1) Uniform particle size and strong acid and alkali resistance;

[0014] (2) It has good enrichment specificity and selectivity for phosphopeptides.

[0015] The present invention firstly uses diethyl 2-(thienylmethyl)phosphonate and thiophene as comonomers and dimethoxymethane as a crosslinking agent, and prepares thienyl microspheres containing phosphonate by precipitation polymerization based on the Friedel-Crafts reaction mechanism. The phosphonate on the surface of the microspheres can be degraded into phosphate after acidification with hydrochloric acid, and then directly chelates titanium ions to obtain Ti ions that can be used to enrich phosphopeptides. 4+ -IMAC microspheres. This method is based on the highly efficient Friedel-Crafts reaction. In addition to the catalyst, no additional initiator, dispersant and surfactant are required, which avoids the problem of difficulty in removing small molecule harmful substances in free radical reactions. The phosphate-functionalized thiophene microspheres are used as the matrix, and no functional group modification is required, which eliminates the complicated modification steps of the traditional method. The obtained Ti 4+ -IMAC materials are highly selective for phosphopeptides and can be used for highly sensitive and selective enrichment of phosphopeptides in biological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the preparation process of thienyl microspheres containing phosphonate functional groups and the principle of phosphopeptide enrichment.

[0017] Figure 2 Helium ion scanning electron microscopy images of Poly(DTYP-co-Th)-1, Poly(DTYP-co-Th)-2, Poly(DTYP-co-Th)-3, Poly(DTYP) and Poly(Th) microspheres.

[0018] Figure 3 Comparison of Fourier transform infrared spectra of thiophene, diethyl 2-(thienylmethyl)phosphonate and acid-hydrolyzed Poly(DTYP-co-Th)-1 microspheres.

[0019] Figure 4 Ti 4+ -X-ray photoelectron spectroscopy analysis of Poly(DTYP-co-Th)-1 microspheres, (a) full spectrum scan; (b) P2p spectrum; (c) Ti 2p spectrum.

[0020] Figure 5 MALDI-TOF-MS comparison of 10 μg β-casein hydrolyzed sample before and after enrichment, (a) before enrichment; (b) after enrichment 4+ -Poly(DTYP-co-Th)-1 microspheres, (c)Ti 4+ -Poly(DTYP-co-Th)-2 microspheres, (d)Ti4+ -Poly(DTYP-co-Th)-3 microspheres, (e)Ti 4+ -Poly(DTYP) microspheres, (f)Ti 4+ -Poly(Th) microspheres and (g) commercial Ti 4 + -IMAC microspheres. DETAILED DESCRIPTION

[0021] Example 1

[0022] Preparation of Poly(DTYP-co-Th)-1 Microspheres and Their Application in Enrichment of Phosphopeptides from β-Tyrosine Hydrolyzed Samples

[0023] (1) 50 μL of diethyl 2-(thienylmethyl)phosphonate, 65 μL of thiophene and 250 μL of dimethoxymethane were added to 2 mL of tetrahydrofuran, so that the molar ratio of diethyl 2-(thienylmethyl)phosphonate, thiophene and dimethoxymethane was 1:3:12, and then 2 mL of a tetrahydrofuran solution containing 0.1 g / mL ferric chloride catalyst was added, mixed and transferred to a 10 mL ampoule;

[0024] (2) The ampoule was placed in liquid nitrogen for 3 minutes to freeze the system into a solid state, and then the mouth of the ampoule was sealed with a flame spray gun. After the sample in the ampoule returned to room temperature, the reaction was carried out at 80° C. for 24 hours. The product was washed with tetrahydrofuran and ethanol three times in sequence, and the residual solvent was removed by filtration to obtain thienyl microspheres containing phosphonate;

[0025] (3) Preparation of acid hydrolysis solution: Mix 150 mL of water and 150 mL of methanol, and slowly add 43 mL of 12 mol / L concentrated hydrochloric acid to make the hydrochloric acid concentration in the solution 1.25 mol / L;

[0026] (4) After the prepared acid hydrolysis solution is cooled to room temperature, 15 mL of the acid hydrolysis solution is added to 80 mg of thiophene microspheres, and ultrasonicated for 10 minutes to make them evenly dispersed, and then heated in a water bath at a reaction temperature of 65° C. for a reaction time of 36 hours. After the reaction is completed, the material is washed with water until it is neutral to obtain phosphate-functionalized thiophene microspheres, which are recorded as Poly(DTYP-co-Th)-1 microspheres;

[0027] (5) Poly(DTYP-co-Th)-1 microspheres were added to 15 mL of a 100 mg / mL titanium sulfate solution, with the mass ratio of microspheres to titanium sulfate being 1:10; the reaction was oscillated at room temperature for 7 h, washed with deionized water, and dried in a vacuum oven at 60 °C for 8 h to obtain Ti 4+ -Poly(DTYP-co-Th)-1 microspheres.

[0028] Preparation of β-casein enzymatic digestion samples:

[0029] Dissolve 2 mg of β-casein in 1 mL of 0.1 M ammonium bicarbonate solution containing 8 M urea (pH = 8.2), add 80 μmol DL-1,4-dithiothreitol, keep constant temperature at 37°C for 2 h, then add 40 μmol 2-iodoacetamide, react for 40 min in the dark, dilute the urea concentration to 1 M with 0.1 M ammonium bicarbonate solution, add 80 μg of trypsin according to the mass ratio of trypsin to protein of 1:25, keep constant temperature at 37°C for 16 h, obtain the proteolysis sample, and store it in a refrigerator at -20°C for use.

[0030] Enrichment of phosphopeptides:

[0031] Weigh 3 mg Ti 4+ -Poly(DTYP-co-Th)-1 microspheres were placed in a centrifuge tube, and the material was balanced with 200 μL of loading solution (80% acetonitrile, 6% trifluoroacetic acid, and the rest was water); then 200 μL of loading solution (containing 10 μg of β-casein hydrolyzate) was added, and the mixture was shaken at room temperature for 30 min; then centrifuged for 5 min (15,000 r / min), and the supernatant solution was removed; the mixture was washed three times with the loading solution to remove nonspecific adsorbed peptides; then 100 μL of 10% (mass percentage) ammonia water was used to elute the phosphopeptides adsorbed on the microsphere material (room temperature, 1500 r / min, and shaken for 15 min). After elution, the mixture was centrifuged for 5 min (15,000 r / min) to separate the eluate and the material, and the eluate was analyzed by matrix-assisted laser desorption / ionization-time of flight mass spectrometry (MALDI-TOF MS).

[0032] MALDI-TOF-MS mass spectrometry analysis:

[0033] 0.5 μL of sample solution was added to the MALDI target, and after it was naturally dried, 0.5 μL of 2,5-dihydroxybenzoic acid solution (25 mg / mL) was covered on the sample point as a matrix. After it was completely naturally dried, it was sent to the MALDI-TOF mass spectrometer for analysis. The analysis spectrum is shown in Figure 5 shown.

[0034] Example 2

[0035] Preparation of Poly(DTYP-co-Th)-2 and its application in enrichment of phosphopeptides from β-tyrosine hydrolyzed samples

[0036] (1) 50 μL of diethyl 2-(thienylmethyl)phosphonate, 20 μL of thiophene and 125 μL of dimethoxymethane were added to 2 mL of tetrahydrofuran, so that the molar ratio of diethyl 2-(thienylmethyl)phosphonate, thiophene and dimethoxymethane was 1:1:6, and then 2 mL of a tetrahydrofuran solution containing 0.1 g / mL ferric chloride catalyst was added, mixed and transferred to a 10 mL ampoule;

[0037] Steps (2) to (5) are the same as those in Example 1. The obtained phosphate-functionalized thienyl microspheres are denoted as Poly(DTYP-co-Th)-2, and the microspheres after chelating titanium ions are denoted as Ti 4+ -Poly(DTYP-co-Th)-2. The preparation of β-tyrosine hydrolyzed samples, enrichment of phosphopeptides and MALDI-TOF-MS mass spectrometry analysis were the same as in Example 1.

[0038] Example 3

[0039] Preparation of Poly(DTYP-co-Th)-3 and its application in enrichment of phosphopeptides from β-tyrosine hydrolyzed samples

[0040] (1) 50 μL of diethyl 2-(thienylmethyl)phosphonate, 108 μL of thiophene and 300 μL of dimethoxymethane were added to 2 mL of tetrahydrofuran, so that the molar ratio of diethyl 2-(thienylmethyl)phosphonate, thiophene and dimethoxymethane was 1:5:18, and then 2 mL of a tetrahydrofuran solution containing 0.1 g / mL ferric chloride catalyst was added, mixed and transferred to a 10 mL ampoule;

[0041] Steps (2) to (5) are the same as those in Example 1. The obtained phosphate-functionalized thienyl microspheres are denoted as Poly(DTYP-co-Th)-3, and the microspheres after chelating titanium ions are denoted as Ti 4+ -Poly(DTYP-co-Th)-3. The preparation of β-tyrosine hydrolysis samples, enrichment of phosphopeptides and MALDI-TOF-MS mass spectrometry analysis were the same as in Example 1.

[0042] Example 4

[0043] Preparation of Poly(DTYP) and its application in enrichment of phosphopeptides from β-tyrosine hydrolyzed samples

[0044] (1) During the preparation of thienyl microspheres, 50 μL of diethyl 2-(thienylmethyl)phosphonate and 63 μL of dimethoxymethane were added to 2 mL of tetrahydrofuran, so that the molar ratio of diethyl 2-(thienylmethyl)phosphonate to dimethoxymethane was 1:3, and then 2 mL of a tetrahydrofuran solution containing 0.1 g / mL ferric chloride catalyst was added, mixed and transferred to a 10 mL ampoule;

[0045] Steps (2) to (5) are the same as those in Example 1. The obtained phosphate functionalized thienyl microspheres are denoted as Poly(DTYP), and the microspheres after chelating titanium ions are denoted as Ti 4+ The preparation of β-tyrosine hydrolyzed samples, the enrichment of phosphopeptides and the MALDI-TOF-MS mass spectrometry analysis were the same as those in Example 1.

[0046] Comparative Example 1

[0047] Preparation of Poly(Th) and its application in enrichment of phosphopeptides from β-tyrosine hydrolyzed samples

[0048] (1) During the preparation of thiophene-based microspheres, 87 μL of thiophene and 170 μL of dimethoxymethane were added to 2 mL of tetrahydrofuran, so that the molar ratio of thiophene to dimethoxymethane was 1:3, and then 2 mL of tetrahydrofuran solution containing 0.1 g / mL ferric chloride catalyst was added, mixed and transferred to a 10 mL ampoule;

[0049] Steps (2) to (5) are the same as those in Example 1. The obtained thienyl microspheres are denoted as Poly(Th), and the microspheres after chelating titanium ions are denoted as Ti 4+ The preparation of β-tyrosine hydrolyzed samples, the enrichment of phosphopeptides and the MALDI-TOF-MS mass spectrometry analysis were the same as those in Example 1.

[0050] Comparative Example 2

[0051] Commercial SPE-Ti-IMAC material enriches phosphopeptides from β-casein enzymatic digestion samples:

[0052] For comparison, the commercial Ti 4+ -IMAC microspheres (SPE-Ti-IMAC, purchased from Beijing Bailingwei Technology Co., Ltd., product number 2749381) were used as adsorbents, and the phosphopeptides in the β-casein enzymatic hydrolyzed samples were enriched under the same operating conditions as in Example 1.

[0053] Product characterization:

[0054] The morphology of the thiophene-based microspheres was characterized by helium ion scanning electron microscopy. Figure 2 As shown, it can be seen that the Poly(DTYP-co-Th)-1 microspheres prepared in Example 1 ( Figure 2a) presents relatively uniform and well-shaped micron-sized microspheres, with a particle size distribution of 2.5 to 5.0 μm; the Poly(DTYP-co-Th)-2 material prepared in Example 2 presents a cluster of microspheres with a particle size of less than 1 μm, and the particle size distribution of the clusters is 2.0 to 4.0 μm; the Poly(DTYP-co-Th)-3 material prepared in Example 3 is a larger microsphere with a wider particle size distribution of 3.0 to 8.0 μm; the particle size distribution of the microspheres prepared by Poly(DTYP) in Example 4 is very uneven, and is not a complete sphere, ranging from 0.1 to 10 μm, and there are many amorphous particles; the Poly(Th) ( Figure 2 c) is also spherical, but some microspheres are deformed, and the particle size distribution is 0.5-3.0 μm. This shows that using 2-(thienylmethyl)phosphonic acid diethyl ester as a monomer alone cannot obtain a very ideal microsphere morphology, while adding a certain amount of thiophene can effectively improve and adjust the size and morphology of the microspheres. When the ratio of 2-(thienylmethyl)phosphonic acid diethyl ester to thiophene is 1:1 (Example 2), the material is clustered, and when the ratio is 1:3 (Example 1) and 1:5 (Example 3), microspheres of different particle sizes are presented.

[0055] Fourier transform infrared spectroscopy was used to characterize diethyl 2-(thienylmethyl)phosphonate, thiophene and acid-hydrolyzed Poly(DTYP-co-Th)-1 microspheres. Figure 3 As shown in the infrared spectrum of thienyl microspheres, the -1 A strong and broad peak appears in the range, which should be from the OH stretching vibration of the phosphate group and dimethoxymethane residue. This characteristic peak does not appear in the spectra of the two monomers, indicating that the phosphonate functional group is acid-lyzed into a phosphate group rich in -OH. The wave numbers are 2979 and 2927 cm -1 The two peaks at 1392 cm-1 can be assigned to the CH symmetric and antisymmetric stretching vibrations of the CH3 group in the ethylphosphonate group, and the peak at 1392 cm-1 corresponds to the antisymmetric deformation vibration of P-CH2. These two CH vibration peaks appear simultaneously in the spectra of diethyl 2-(thienylmethyl)phosphonate and thienyl microspheres, indicating the presence of P-CH2 bonds from diethyl 2-(thienylmethyl)phosphonate in the thienyl microspheres. In addition, it can be observed that absorption peaks attributed to the out-of-plane swinging vibration of the CH on the thiophene ring appear in both the thiophene and diethyl 2-(thienylmethyl)phosphonate spectra, but their wavenumbers and intensities are different. Thiophene appears at 707 cm-1. -1 , while diethyl 2-(thienylmethyl)phosphonate appears at 697 cm -1 , but these two characteristic peaks disappeared in the spectrum of thiophene-based microspheres, indicating that the Friedel-Crafts electrophilic substitution reaction between the two monomers led to alkylation of the CH site on the thiophene ring.

[0056] Analysis of Ti by X-ray Photoelectron Spectroscopy 4+ -Elemental composition of Poly(DTYP-co-Th)-1 microspheres. Figure 4 As shown in a, in Ti 4+ In the full scan spectrum of -Poly(DTYP-co-Th)-1 microspheres, not only the S1s peak from the thiophene matrix was found, but also the peaks of P 2s, P 2p and Ti 2p were found, among which the S element exists in both monomers, the P element comes from diethyl 2-(thienylmethyl)phosphonate, and the Ti element comes from the chelated titanium ion. This proves that the thiophene-based microspheres are copolymerized from diethyl 2-(thienylmethyl)phosphonate and thiophene. Figure 4 b, Ti 4+ -IMAC material has a P 2p peak at 133.5 eV, indicating the presence of phosphonate functional groups. Figure 4 c shows two spin-orbit peaks of Ti 2p, Ti 2p 3 / 2 and 2p 1 / 2 The ions appeared at 459.0 and 464.8 eV, respectively, which indicated that the titanium ions were fixed on the thienyl microspheres.

[0057] Ti 4+ -Poly(DTYP-co-Th) microspheres were used as adsorbents for immobilized metal ions to separate and enrich phosphopeptides in β-casein hydrolyzed samples. The MALDI-TOF-MS comparison before and after enrichment is shown in the figure below. Figure 5 As shown. A large number of non-phosphopeptide signal peaks appeared in the spectrum of the pre-enrichment digest, and the phosphopeptide signal was strongly suppressed, and only one phosphopeptide signal (2061, m / z) was detected ( Figure 5 a). Using the Ti prepared in Example 1 4+ After enrichment with -Poly(DTYP-co-Th)-1 microspheres, the enriched non-phosphopeptide signal was significantly reduced, and the phosphopeptide signal was significantly enhanced. Three phosphopeptide signals (2061, 2556 and 3012, m / z) and their dephosphorylated fragment peaks were clearly detected, and there was almost no interference from non-phosphopeptides in the spectrum ( Figure 5 b), the signal intensity of phosphopeptide is up to 4800, indicating that the material has high selectivity and enrichment efficiency for phosphopeptide. 4+ After enrichment with -Poly(DTYP-co-Th)-2 microspheres, three complete phosphopeptide signals also appeared in the mass spectrum, but the signal intensity was 2200, which was lower than that of Ti 4+ -Poly(DTYP-co-Th)-1 microspheres are low, indicating that the microspheres also have good selectivity, but the adsorption capacity is low ( Figure 5c); Ti prepared in Example 3 4+ After enrichment with -Poly(DTYP-co-Th)-3 microspheres, although the signal intensity was relatively high, reaching 3600, only two phosphopeptide signals (2061 and 2556, m / z) appeared in the mass spectrum, accompanied by a small amount of non-phosphopeptide signal interference ( Figure 5 d), indicating that the microspheres have a large adsorption capacity but poor selectivity. 4+ After -Poly(DTYP) enrichment, although three complete phosphopeptides were detected, a large number of interfering peaks from material degradation appeared ( Figure 5 e) This is because Ti 4+ The oligomers in Ti-Poly(DTYP) were degraded by 10% ammonia, indicating that the material is not suitable for use as an IMAC adsorbent. 4+ After enrichment of -Poly(Th), no obvious phosphopeptide signal was detected ( Figure 5 f), indicating that the material has no obvious enrichment effect on phosphopeptides. The thienyl microspheres in Examples 1 to 4 were all prepared under the condition that the molar ratio of the total amount of monomers (thiophene and diethyl 2-(thienylmethyl)phosphonate) to the crosslinking agent (dimethoxymethane) was 1:3. The main difference was that the molar ratio of diethyl 2-(thienylmethyl)phosphonate to thiophene in the preparation system was different: in Example 1, the molar ratio of diethyl 2-(thienylmethyl)phosphonate to thiophene was 1:3, in Example 2, the ratio was 1:1, and in Example 3, the ratio was 1:5. Example 4 did not contain diethyl 2-(thienylmethyl)phosphonate, and Comparative Example 1 did not contain diethyl 2-(thienylmethyl)phosphonate. From the results of mass spectrometry analysis, it can be seen that since the phosphonate functional group is provided by diethyl 2-(thienylmethyl)phosphonate, the lower the content of diethyl 2-(thienylmethyl)phosphonate in the preparation system, the fewer phosphate functional groups on the surface of the microspheres after acid degradation, the fewer titanium ion chelating sites that can be provided, and the lower the enrichment efficiency of phosphopeptides. 4+ -Poly(DTYP-co-Th)-2 has a relatively low enrichment efficiency due to its poor morphology. 4+ -Poly(DTYP) has interference peaks, Ti 4+ -Poly(DTYP-co-Th)-3 has a low phosphate content, while Ti 4+ -Poly(DTYP-co-Th)-1 has both suitable morphology and high phosphate content, thus showing the best enrichment performance.

[0058] For comparison, commercial Ti 4+ -IMAC material was used to enrich the phosphopeptides in β-casein hydrolysate under the same conditions. Figure 5g. Commercial Ti 4+ -IMAC microspheres can enrich three characteristic phosphopeptides, but their abundance is generally lower than that of Ti 4+ -Poly(DTYP-co-Th)-1 microspheres are low, indicating that Ti 4+ -Poly(DTYP-co-Th)-1 has a higher enrichment efficiency for phosphopeptides.

[0059] The thienyl microspheres containing phosphonate prepared by the Friedel-Crafts reaction can be subjected to acid degradation and titanium ion chelation to obtain Ti for phosphopeptide enrichment. 4+ -IMAC microspheres. The microspheres show high selectivity and specificity for phosphopeptides, comparable to commercial IMAC microspheres. The entire preparation process does not require additional grafting functional groups and is suitable for large-scale preparation.

Claims

1. A method for preparing thienyl microspheres containing phosphonate functional groups, characterized in that: Thiophene-based microspheres containing phosphonate were prepared by Friedel-Crafts reaction using diethyl 2-(thienylmethyl)phosphonate and thiophene as comonomers and dimethoxymethane as a crosslinker. The phosphonate on the surface of the microspheres can be degraded into phosphate, which then chelates with titanium ions to obtain Ti 4+ -IMAC microspheres; You can follow the steps below, (1) Preparation of thienyl microspheres containing phosphonate functional groups: 40-80 μL of diethyl 2-(thienylmethyl)phosphonate, 20-170 μL of thiophene, and 100-400 μL of dimethoxymethane were added to 2-4 mL of tetrahydrofuran, and then 2-4 mL of a tetrahydrofuran solution containing 0.05-0.2 g / mL of ferric chloride catalyst was added. After mixing, the mixture was transferred to a 10-20 mL ampoule. The ampoule was placed in liquid nitrogen for 2-5 minutes to freeze the system into a solid state. The mouth of the ampoule was then sealed with a flame spray gun. After the sample in the ampoule returned to room temperature, the reaction was carried out at 70-90 °C for 12-24 h. The product was washed with tetrahydrofuran and ethanol for 2-4 times, respectively, and the residual solvent was removed by filtration to obtain thienyl microspheres containing phosphonate functional groups. (2) Preparation of phosphate-functionalized thienyl microspheres: First, prepare an acid hydrolysis solution by mixing 100-200 mL of water and 150-250 mL of methanol, and add 20-70 mL of 10-12 mol / L concentrated hydrochloric acid to make the hydrochloric acid concentration in the solution 1-2 mol / L; after the prepared acid hydrolysis solution is cooled to room temperature, take 5-30 mL and add it to 50-100 mg of thienyl microspheres, ultrasonicate for 5-10 minutes to make them evenly dispersed, and then heat in a water bath at a reaction temperature of 50-70 °C and a reaction time of 24-72 h; after the reaction is completed, wash the material with water until it is neutral to obtain phosphate-functionalized thienyl microspheres; (3) Ti 4+ Preparation of IMAC microspheres: Add the phosphate-functionalized thienyl microspheres obtained in step (2) to 10-20 mL of a 50-200 mg / mL titanium sulfate solution, with the mass ratio of microspheres to titanium sulfate being 1:10-1:100; shake the reaction at room temperature for 4-12 h, wash with deionized water, and dry to obtain Ti 4+ -IMAC microspheres.

2. The Ti prepared by the preparation method according to claim 1 4+ -IMAC microspheres.

3. Ti according to claim 2 4+ -Application of IMAC microspheres, characterized by: The Ti 4+ -IMAC microsphere material is used as IMAC adsorbent to separate and enrich phosphopeptides in biological samples.

4. The use according to claim 3, characterized in that: The biological sample is one or more of human and / or animal body fluids, tissue enzymatic hydrolysate or cell enzymatic hydrolysate samples.

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