Preparation method of directional pore channel integral material and method for constructing enrichment device and establishing phosphopeptide rapid enrichment by using directional pore channel integral material

Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a preparation method of a directional pore channel integral material and a method for constructing an enrichment device and establishing a rapid phosphopeptide enrichment method by using the directional pore channel integral material. The preparation method comprises the following steps: preparing a CS-PC integral material with directional pore channels from chitosan, zirconium amino phthalocyanine and polyethylene glycol diglycidyl ether through a directional freezing method and a chemical crosslinking method, and then modifying the CS-PC integral material with 5 '-phosphopyridoxal and zirconium oxychloride to obtain the CS-PC PLP integral material. The CS-PC-PLP integral material prepared by the invention has anisotropy and a directional pore channel structure, is convenient to fix in a pipette tip and is combined with a row pipettor for rapid enrichment; the CS-PC coated PLP integral material has the advantages of green and cheap raw materials and strong reusability.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a method for preparing a directional pore monolithic material and its use in constructing enrichment devices and establishing a rapid enrichment method for phosphopeptides. Background Technology

[0002] Among various post-translational modification (PTM) types, protein phosphorylation plays a regulatory role in cell proliferation, differentiation, motility, and apoptosis, and is closely related to various physiological processes and disease development in humans. Currently, phosphoproteomics, based on mass spectrometry and bottom-up analysis strategies, can reveal abnormal phosphorylation activity and modifications in biological samples, which is of great research significance for a comprehensive understanding of human diseases. However, due to the complexity of organisms, ionization efficiency, low phosphopeptide abundance, and various external interferences, direct analysis by mass spectrometry (MS) faces numerous challenges, including the absence of phosphorylation modifications at specific sites and long analysis times. To expand phosphoproteomics datasets and comprehensively analyze phosphorylation events in biological samples, it is necessary to perform batch sample pretreatment on biological samples.

[0003] Among the various phosphopeptide enrichment methods developed to date, immobilized metal affinity chromatography (IMAC) relies on the synergistic effect of electrostatic attraction and metal chelation between IMAC materials carrying positively charged metal ions and negatively charged target phosphopeptides to achieve enrichment. Its good selectivity and reproducibility have led to its widespread application. However, traditional IMAC materials, to enhance the modification density of metal ions, are mostly in powder form, exhibiting high mass transfer resistance. This necessitates additional equipment such as a constant-temperature metal bath and centrifuges for material dispersion and collection during the enrichment process. To shorten the mass transfer distance and reduce mass transfer resistance, the Ou group designed and fabricated hollow SiO2 nanospheres immobilized with Ti⁴⁺, reducing sample loading and elution time to within 1 minute. However, this still requires processing large numbers of samples individually in biological and clinical studies. IMAC materials using magnetic spheres as a matrix, combined with magnetic solid-phase microextraction technology and a magnetic rack, effectively avoid the use of centrifuges, meeting the needs for rapid and efficient sample preparation. However, this requires several hours for adsorption and desorption of the material. Furthermore, the preparation processes of these materials are complex and costly, involving large amounts of environmentally harmful raw materials and organic reagents. Monolithic materials, on the other hand, are easy to prepare, have high porosity, and low mass transfer resistance, giving them an advantage in separation science. Ye's group easily prepared a Ti4+-loaded monolithic material in a polypropylene pipette tip via photoinduced free radical polymerization. By combining this highly permeable monolithic material with a centrifuge, 1185 phosphorylation sites were identified from 5 μg of HeLa cell protein hydrolysate. On the other hand, most reported IMAC materials require ammonia to elute phosphopeptides, which inevitably results in the loss of some metal ions, thus limiting the reusability of the IMAC materials. The reproducible enrichment of phosphopeptides in some reported IMAC materials may be due to excessive initial material usage; after multiple cycles, sufficient eluent will cause the material's reusability to disappear. In view of the above challenges, it is a meaningful effort to design and prepare a monolithic material with green, inexpensive, and reusable raw materials for the enrichment of phosphopeptides in batches of biological samples. To this end, the present invention provides a method for preparing a directional pore monolithic material and its use in constructing an enrichment device and establishing a rapid phosphopeptide enrichment method. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a directional pore monolithic material and its use in constructing enrichment devices and establishing a rapid enrichment method for phosphopeptides. Addressing the shortcomings of the aforementioned background technology, this invention mainly solves the difficulties of long processing times for large-scale phosphorylation in clinical settings, high cost of enrichment materials, and poor reusability. The CS-PC@PLP monolithic material prepared by this method exhibits superior enrichment effects on phosphopeptides in biological samples; the raw materials for the CS-PC@PLP monolithic material are green, inexpensive, and have good reusability.

[0005] The specific technical solution adopted by this invention is as follows: A method for preparing a reusable monolithic directional channel material includes the following steps: Step 1: Preparation of CS-ZrPC monolithic material; 500-1000 mg of aminophthalide zirconium, 500-1000 mg of chitosan, and 50-100 mg of polyethylene glycol diglycidyl ether are stirred evenly in 30 mL of 0.5%-2% acetic acid-water solution. After ultrasonic degassing, the mixed solution is poured into a polytetrafluoroethylene mold with a thermally conductive copper plate at the bottom. Liquid nitrogen is added to make the aerogel grow along the direction perpendicular to the copper plate. The aerogel is freeze-dried for 24-48 h and then crosslinked in an oven for 6-12 h to obtain the CS-ZrPC monolithic material. The polytetrafluoroethylene mold has a thermally conductive copper plate at the bottom, which enables the monolithic material to grow in a directional manner. Step 2: The CS-ZrPC monolithic material prepared in Step 1, along with pyridoxal 5'-phosphate and sodium cyanoborohydride, are stirred in a phosphate buffer solution at pH 7.5 for 5 hours to obtain a phosphorylated CS-ZrPC monolithic material. Then, the phosphorylated CS-ZrPC monolithic material is added to zirconium salt containing an aqueous trifluoroacetic acid solution and stirred to fix Zr4+. After the process, it is washed three times with deionized water and vacuum dried at room temperature to obtain a reusable oriented channel monolithic material, namely the CS-ZrPC@PLP monolithic material.

[0006] Preferably, in step 2, the ratio of the amount of the CS-ZrPC monolithic material, pyridoxal 5'-phosphate, and sodium cyanoborohydride is 1:0.5-2g:0.5-2g; The concentration of the phosphate buffer solution is 100-500 mmol / L. ﹣1 ; The zirconium salt is ZrOCl2·8H2O, and the ratio of the phosphorylated modified CS-ZrPC monolithic material to the zirconium salt is 5-10 g: 100 mmol / L. ﹣1 ; The trifluoroacetic acid aqueous solution contains 0.1-1% by mass.

[0007] Preferably, the CS-ZrPC@PLP monolithic material is fixed on the pipette tip and combined with a series of pipettes to obtain an enrichment device; the CS-ZrPC@PLP monolithic material has low longitudinal mass transfer resistance and lateral compression resilience.

[0008] The CS-ZrPC@PLP monolithic material prepared by the above method is used for the large-scale and rapid enrichment of phosphopeptides. The CS-ZrPC@PLP monolithic material is used as a monolithic material for immobilized metal ion affinity chromatography directional pores.

[0009] A method for large-scale rapid enrichment of phosphopeptides includes: The standard protein β-casein was enzymatically digested, and the protein sample extracted from Hep G2 was denatured and enzymatically digested. Rapid enrichment of peptides in enzymatic hydrolysate was achieved using an immobilized metal ion affinity chromatography directional pore monolithic material. The acquired data were analyzed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry.

[0010] An enrichment device used in the enrichment method includes an integral material for directional channels fixed to a pipette tip and a series of pipettes that match the pipette tip. The device combining the pipette tip with the multi-pipette allows for free and independent switching between the three liquids: sample solution, eluent, and elution solution.

[0011] An application of the enrichment device described above in large-scale phosphopeptide enrichment includes: Immobilized bimetallic ion affinity chromatography directional channel monolithic material is fixed inside the pipette tip; The pipette tip with fixed directional channel monolithic material was combined with a series of pipettes. The sample solution was used to rinse the immobilized metal ion affinity chromatography monolithic material multiple times by blowing. Then, the protein hydrolysate was used to fully wet the immobilized metal ion affinity chromatography monolithic material in the same way to complete the sample loading. The non-phosphorylated peptides and salts were rinsed by repeated blowing and rinsing with the first and second rinsing solutions. Phosphorylated peptides retained on the immobilized metal ion affinity chromatography monolithic material are eluted by repeated pipetting using an eluent. The peptides in the eluent were analyzed using MALDI-TOF-MS or high-resolution mass spectrometry.

[0012] Preferably, the loading solution is a mixed solution consisting of 80%-85% acetonitrile, 0.1%-6% trifluoroacetic acid, and 9%-19.9% ​​water by volume. The first eluent consists of 30%-85% acetonitrile, 0.1%-6% trifluoroacetic acid, 9%-69.9% water, and 200 mmol / L. ﹣1 A mixed solution composed of NaCl; The second rinsing solution is a mixed solution consisting of 80%-85% acetonitrile, 0.1%-6% trifluoroacetic acid, and 9%-19.9% ​​water by volume. The eluent is a 10-15 wt% ammonia solution.

[0013] The technical effects achieved by this invention are as follows: The material prepared by this invention is simple to prepare, low in cost, and has good biocompatibility. The prepared CS-ZrPC@PLP monolithic material has directional channels, which can meet the requirements of rapid solution passage and achieve rapid enrichment of phosphopeptides. The material has certain compression resilience and can be loaded into pipette tips and combined with pipette arrays to achieve large-scale enrichment of phosphopeptides.

[0014] The enrichment method developed in this invention avoids the time-consuming steps such as shaking and centrifugation required by traditional enrichment methods, shortening the entire enrichment process to less than 5 minutes, thus achieving ultra-rapid enrichment of phosphopeptides. In particular, this invention can process 1-8 biological samples in batches at once, and its enrichment results complement those of the traditional IMAC method. Attached Figure Description

[0015] Figure 1 This is a flowchart of the overall material preparation process for CS-ZrPC@PLP.

[0016] Figure 2 This is a schematic diagram of the enrichment device.

[0017] Figure 3 The images show scanning electron microscope (SEM) images of the entire material, where a and c are cross-sections, and b and d are longitudinal sections; a and b represent the monolithic CS-ZrPC material, and c and d represent the monolithic CS-ZrPC@PLP material. Before phosphorylation, the surface of the monolithic material exhibits a smooth porous structure, while after treatment, the surface becomes rough, indicating successful phosphorylation and its ability to support more metal ions.

[0018] Figure 4 The images show the energy dispersive spectroscopy (EDS) spectra of the overall CS-ZrPC@PLP material, where a is a regional scanning electron microscope (SEM) image of CS-ZrPC@PLP, and b, c, and d are the EDS spectra of C, O, and Zr elements, respectively. This indicates a high density of zirconium ion modification in the material, which is beneficial for phosphopeptide enrichment as an IMAC material.

[0019] Figure 5 Thermogravimetric curves of ZrPC, CS-ZrPC monolithic materials, and CS-ZrPC@PLP monolithic materials are shown. The stability of the monolithic materials is enhanced after zirconium phthalocyanine and phosphorylation treatment.

[0020] Figure 6 The graph shows a comparison of column pressure between the CS-ZrPC@PLP monolithic material and a traditional C18 column at high flow rates. It indicates that the monolithic material still maintains low mass transfer resistance at high flow rates, which is beneficial for its combination with pipettes for large-scale phosphopeptide enrichment.

[0021] Figure 7The following are the MALDI-TOF mass spectra of the β-casein hydrolysate: (a) direct analysis, (b) enrichment with ZrPC, (c) enrichment with CS-ZrPC, (d) enrichment with CS@PLP, (e) enrichment with CS-ZrPC@PLP, and (f) secondary enrichment with TiO2.

[0022] Figure 8 MALDI-TOF mass spectra of β-casein hydrolysate enriched with Ti@Zr-PC at different loading amounts: (a) 100 fmol, (b) 10 fmol, and (c) 1 fmol.

[0023] Figure 9 MALDI-TOF mass spectra of the CS-ZrPC@PLP monolithic material after 10 repetitions enriching 100 fmol of β-casein hydrolysate.

[0024] Figure 10 MALDI-TOF mass spectra of CS-ZrPC@PLP monolithic material enriched with mixed samples of β-casein hydrolysate and bovine serum albumin hydrolysate at different mass ratios: (a) 1:10, (b) 1:50, and (c) 1:500.

[0025] Figure 11 The image shows a comparison of the number of unique phosphopeptides and phosphorylated proteins identified in two materials after enriching 500 μg of Hep G2 cell protein hydrolysate in commercially available silica microspheres and enriching 500 μg of Hep G2 cell protein hydrolysate in the CS-ZrPC@PLP monolithic material of this patent, using the same library search method. Detailed Implementation

[0026] To make the objectives and advantages of this invention clearer, the invention will be specifically described below with reference to embodiments. It should be understood that the following text is merely used to describe one or more specific embodiments of the invention and does not strictly limit the scope of protection specifically claimed by the invention.

[0027] The purpose of this invention is to develop materials and methods for rapid, large-scale enrichment of phosphopeptides. Using chitosan, zirconium aminophthalate, and polyethylene glycol diglycidyl ether as precursors, a monolithic CS-ZrPC material was obtained through directional freezing and chemical cross-linking. Subsequent modification involved further loading with zirconium ions to obtain a monolithic CS-ZrPC@PLP material with directional channels. The prepared CS-ZrPC@PLP material was then fixed within a pipette tip and combined with a multi-stage pipette, resulting in a method for phosphopeptide enrichment within 5 minutes. This overcomes the time-consuming challenge of large-volume phosphopeptide enrichment in clinical applications. Ultimately, the enrichment method developed in this invention enables the isolation and identification of phosphopeptides in complex biological samples such as Hep G2 cells, providing technical support for exploring their role in life activities.

[0028] The main contents of this invention include three parts: designing and preparing a reusable monolithic metal ion affinity chromatography material CS-ZrPC@PLP, designing and assembling an enrichment device, and developing a method for large-scale rapid enrichment of phosphopeptides. The monolithic metal ion affinity chromatography material CS-ZrPC@PLP is a reusable oriented channel monolithic material, abbreviated as CS-ZrPC@PLP monolithic material. The precursors selected in this invention include chitosan, which possesses good biocompatibility and modifiable functional groups, and zirconium phthalocyanine, a strong complexing agent for metal ions, facilitating the preparation of reproducible IMAC materials. First, chitosan, zirconium phthalocyanine, and polyethylene glycol diglycidyl ether are uniformly mixed in an acetic acid-water solution. The resulting CS-ZrPC monolithic material is prepared via directional freezing and chemical cross-linking. Then, phosphate groups are modified onto the material surface by condensing residual amino groups with pyridoxal phosphate and sodium cyanoborohydride. Finally, the phosphate groups modified on the surface of the CS-ZrPC monolithic material are chelated with zirconium ions to obtain the CS-ZrPC@PLP monolithic material. This CS-ZrPC@PLP monolithic material can be used to enrich phosphorylated peptides through immobilized metal ion affinity chromatography to interact with them.

[0029] The enrichment device designed and assembled in this invention can fix the CS-ZrPC@PLP material as a whole and complete the loading, rinsing and elution process of phosphopeptides in batches within 5 minutes, without the need to use traditional large equipment such as shakers, vortex mixers and centrifuges.

[0030] The CS-ZrPC@PLP monolithic material prepared in this invention can be used for rapid phosphopeptide enrichment, especially suitable for the batch enrichment of phosphopeptides in clinical samples. By coupling microcolumn liquid chromatography with high-resolution mass spectrometry, large-scale enrichment, analysis, and identification of phosphopeptides in simple samples (β-casein) and complex biological samples (Hep G2 cells, enzymatic digests of biological tissues, etc.) can be achieved. Specific implementation methods are as follows: Example

[0031] The preparation steps of the CS-ZrPC@PLP monolithic material are as follows: See Figure 1 As shown, the preparation of the monolithic CS-ZrPC@PLP material based on chitosan is as follows: Step 1: 900 mg zirconium phthalocyanine (ZrPC), 900 mg chitosan (CS), 90 mg polyethylene glycol diglycidyl ether (PEGDGE), and 200 μL acetic acid were added to 30 mL of deionized water and stirred until homogeneous. After ultrasonic removal of air bubbles, the mixture was poured into a polytetrafluoroethylene mold with a thermally conductive copper plate at the bottom. Liquid nitrogen was poured in to allow the aerogel to grow along the direction perpendicular to the copper plate. The aerogel was freeze-dried for 48 h and then dried overnight in a 60 °C oven to fully crosslink CS and ZrPC, obtaining CS-ZrPC. Step 2: The aerogel with oriented pore structure obtained in Step 1, 0.2 g of pyridoxal 5'-phosphate (PLP), and 0.2 g of NaBH3CN were added to a 100 mM phosphate buffer solution (pH 7.5) and stirred at room temperature for 5 h. Then, the phosphorylated material was added to 100 mM ZrOCl2·8H2O and stirred for 2 h to fix Zr4+. After completion, the mixture was washed three times with deionized water and vacuum dried at room temperature to obtain a reusable oriented pore monolithic material, namely CS-ZrPC@PLP monolithic material.

[0032] See Figure 2 As shown, the fabrication of a large-scale phosphopeptide enrichment device is as follows: First, the CS-ZrPC@PLP monolithic material is placed inside the pipette tip along the direction of ice crystal growth. The longitudinal directional channels and transverse compression and resilience of the CS-ZrPC@PLP monolithic material ensure that it is fixed inside the pipette tip while having low mass transfer resistance. The longitudinal mass transfer resistance of the CS-ZrPC@PLP monolithic material is less than 3.0 mL / min. ﹣1 The column pressure at the flow rate is 4.2 MPa.

[0033] Then, the CS-ZrPC@PLP monolithic material is combined with a series of pipettes to complete the preparation of a large-scale phosphopeptide enrichment device.

[0034] Preparation of β-casein and bovine serum albumin hydrolysate samples: First: Use a solution with a pH of 8.2 and containing 8 mol L... ﹣1 0.1 mol L of urea ﹣1 Dissolve 2 mg of β-casein in ammonium bicarbonate solution; Next: Add DL-1,4-dithiothreitol (80µmol) to the solution and incubate it at 37℃ for 2 hours; then add 2-iodoacetamide (40µmol) and react in the dark for 40 minutes. Finally: using 0.1 mol L ﹣1 The ammonium bicarbonate solution was used to dilute the urea contained in the previous step to 1 mol / L. ﹣1Add 80µg of trypsin, with the ratio of trypsin mass to β-casein mass being 1 / 25. Incubate at 37℃ for 16 hours to obtain β-casein or bovine serum albumin hydrolysate, and store at -20℃ for later use.

[0035] Enrichment of phosphorylated peptides: First: Use a pipette tip loaded with the CS-ZrPC@PLP monolithic material to blow the sample solution (ACN / H2O / TFA, v / v / v, 80 / 14 / 6) three times to allow the sample solution to quickly pass through the material and equilibrate. First: Redissolve the β-casein hydrolysate in the loading solution from the above steps, and use a pipette to draw 200 μL of the hydrolysate to fully wet the CS-ZrPC@PLP monolithic material and quickly complete the loading. Then: Prepare the first eluent: (ACN / H2O / TFA 200mM NaCl, v / v / v, 40 / 54 / 6), and pipette the eluent three times; then prepare the second eluent: (ACN / H2O / TFA, v / v / v, 40 / 54 / 6), and pipette the eluent three times to remove non-specifically adsorbed peptides and salts; Finally: Prepare a 10wt% ammonia solution as the eluent, and repeatedly pipette 200μL of the eluent three times to elute the phosphorylated peptides retained on the CS-ZrPC@PLP monolithic material.

[0036] MALDI-TOF-MS analysis: First: Add 0.5 μL of phosphorylated peptide sample to a mass spectrometry target plate and let it air dry. Then: pipette 0.5 μL of 2,5-dihydroxybenzoic acid (DHB) matrix solution (25 mg / mL). ﹣1 ACN / H2O / H3PO4, V / V / V, 70 / 29 / 1) is applied to the target sites of the mass spectrometry target plate prepared in the above steps as a matrix, and then allowed to air dry. Finally, the sample was analyzed using a MALDI-TOF mass spectrometer.

[0037] Material characterization: Scanning electron microscope images of CS-ZrPC and CS-ZrPC@PLP monolithic materials are shown below. Figure 3 The energy spectrum of the CS-ZrPC@PLP monolithic material is shown below. Figure 4 Thermogravimetric diagrams of ZrPC, CS-ZrPC, and CS-ZrPC@PLP integral materials are shown below. Figure 5 Enrichment diagrams of phosphorylated peptides in β-casein hydrolysates by ZrPC, CS-ZrPC, and CS-ZrPC@PLP are shown below. Figure 6MALDI-TOF mass spectra of β-casein hydrolysate at different loading amounts (100 fmol, 10 fmol, and 1 fmol) are shown below. Figure 7 .

[0038] Figure 4 The image shows the energy dispersive spectroscopy (EDS) spectrum of the Ti@Zr-PC material. This indicates that zirconium ions are uniformly loaded onto the material surface.

[0039] Figure 5 Thermogravimetric analysis (TGA) diagrams for ZrPC, CS-ZrPC, and CS-ZrPC@PLP bulk materials. From... Figure 4 It can be seen that the thermal stability of the CS-ZrPC@PLP material is enhanced after ZrPC and phosphorylation treatment.

[0040] Figure 6 The graph shows a comparison of column pressure between the CS-ZrPC@PLP monolithic material and a traditional C18 column at high flow rates. It indicates that the monolithic material still maintains low mass transfer resistance at high flow rates, which is beneficial for its combination with pipettes for large-scale phosphopeptide enrichment.

[0041] Figure 7 This diagram shows the enrichment capabilities of different IMAC materials for β-casein hydrolysates (phosphopeptides). After treatment with the monolithic material CS-ZrPC@PLP with high zirconium ion modification density, the enrichment of phosphopeptide targets in the sample can be completed even in a very short time, indicating that the developed large-scale rapid enrichment method is comparable to the enrichment capability of commercial TiO2.

[0042] Establishment and analysis of phosphopeptide enrichment methods: Figure 8 The sensitivity of the CS-ZrPC@PLP monolithic material prepared under enzyme hydrolysate volumes ranging from 1 to 100 fmol for rapid enrichment was investigated. Even with a β-casein hydrolysate volume as low as 1 fmol, three phosphorylated peptides could still be identified.

[0043] Figure 9 To investigate the reusability of the prepared CS-ZrPC@PLP monolithic material, the eluted material was eluted again. The second eluent showed virtually no phosphorylated peptide signals, indicating that the material's oriented pores provide low mass transfer resistance, and the rapid elution time meets the elution requirements. The same material was enriched 10 times using β-casein digest. Phosphorylated peptide signal peaks were still detected after 10 cycles, and the enrichment capacity remained consistent, indicating that the material has good reusability.

[0044] Figure 10Using mixed samples of different masses of β-casein hydrolysate and bovine serum albumin hydrolysate as simulants, this study demonstrates that the CS-ZrPC@PLP monolithic material and rapid enrichment method can enrich phosphopeptides from complex matrices. It was found that even at a dilution of 1:500, β-casein hydrolysate and bovine serum albumin hydrolysate could still identify three characteristic peaks of phosphopeptides, indicating the potential application of the developed method in complex clinical samples.

[0045] Figure 1 shows a comparison of the enrichment results of phosphopeptides in Hep G2 protein hydrolysate using 11 different materials (commercial TiO2 and CS-ZrPC@PLP monolithic material). Using the CS-ZrPC@PLP monolithic material, a total of 41,284 phosphopeptides were identified from 500 μg of Hep G2 protein hydrolysate in a single run. This included 3,123 unique phosphopeptides and 1,001 phosphorylated proteins. In comparison, this is an order of magnitude higher than the number identified by commercial TiO2, while being simpler to operate and significantly faster than traditional methods. In terms of enrichment time and the number of phosphopeptides identified, the CS-ZrPC@PLP monolithic material outperforms commercial TiO2. The Venn diagram of the identified phosphopeptides shows strong complementarity between the phosphopeptide enrichment results obtained by this method and those obtained by commercial TiO2.

[0046] As can be seen from the examples and figures, the preparation process of this material is simple, the raw materials are green and inexpensive, it has good thermal stability, a directional pore structure, low mass transfer resistance, and good reusability. Combined with an assembled enrichment device, it can be applied to the large-scale, rapid enrichment of phosphopeptides in clinical samples, providing a favorable method for analyzing and identifying phosphorylation sites from biological samples.

[0047] Example 2: Preparation of Hep G2 cell protease hydrolysate samples: Step 1: Culture of Hep G2 Cells: Human liver cancer cells (Hep G2) were obtained from the Cell Bank / Stem Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Hep G2 cells were cultured in a 37°C incubator with 5% CO2 aeration using Hep G2 complete culture medium. When the cell density reached over 90%, adherent cells were digested with trypsin for 5 min. After digestion was terminated with complete culture medium, the cells were collected by centrifugation, resuspended three times in pre-cooled PBS, and passaged. Cells were lysed using a cell lysis buffer in a cell disruptor under ice-water bath with sonication for 120 s (10 s on, 20 s off). The lysis buffer composition was as follows: 8M urea, 50mM Tris-HCl buffer, 65mM DTT, 1mM EDTA, 0.5mM EGTA, 2% protease inhibitor cocktail, 2% phosphatase inhibitor, and 1% Triton-X100 (v / v). After lysis, the cells were centrifuged at 16000g for 20 min at 4°C, and the supernatant was collected. Protein was precipitated from the supernatant using 5 volumes of acetone pre-chilled at -20°C overnight. After centrifugation, the precipitate was washed three times with pre-chilled acetone and ethanol, the supernatant was discarded, and the sample was air-dried to obtain the protein sample. The protein concentration was determined using a BCA kit.

[0048] Step 2: Enzymatic digestion of Hep G2 cells: Enzymatic digestion of the Hep G2 cell proteins obtained in Step 1: using a solution with pH 8.2 and containing 8 mol / L... ﹣1 0.1 mol L of urea ﹣1 Dissolve 2 mg of Hep G2 cell protein in ammonium bicarbonate solution, then add DL-1,4-dithiothreitol (80 µmol) and incubate the solution at 37 °C for 2 h; then add 2-iodoacetamide (40 µmol) and react in the dark for 40 min; finally, use 0.1 mol L... ﹣1 The ammonium bicarbonate solution was used to dilute the urea contained in step 2 to 1 mol / L. ﹣1 Then add 80µg of trypsin, with the ratio of trypsin mass to E. coli protein mass = 1 / 25, and incubate at 37℃ for 16h to obtain Hep G2 cell protease hydrolysate, freeze-dry and store at -20℃ for later use.

[0049] Enrichment of phosphorylated peptides: Step 1: Using a pipette tip loaded with the CS-ZrPC@PLP monolithic material, pipette the sample solution (ACN / H2O / TFA, v / v / v, 80 / 14 / 6) three times to allow the sample solution to quickly pass through the material and equilibrate. Step 2: Redissolve the Hep G2 cell protein hydrolysate in the loading solution from Step 1. Use a pipette to draw 200 μL of the protein hydrolysate to fully wet the CS-ZrPC@PLP monolithic material and quickly complete the loading. Step 3: Prepare the first eluent: (ACN / H2O / TFA 200mM NaCl, v / v / v, 40 / 54 / 6), and pipette the eluent three times; then prepare the second eluent: (ACN / H2O / TFA, v / v / v, 40 / 54 / 6), and pipette the eluent three times to remove non-specifically adsorbed peptides and salts; Step 4: Prepare a 10wt% ammonia solution as the eluent. Use a pipette to repeatedly pipette 200μL of the eluent three times to elute the phosphorylated peptides in the Hep G2 cell protein hydrolysate retained on the CS-ZrPC@PLP monolithic material.

[0050] LC-MS / MS Analysis and Data Retrieval: The mobile phase composition for reversed-phase chromatography separation in Micro-LC-MS / MS is as follows: Mobile phase A is H2O / FA ​​(99.9 / 0.1, v / v) solution, and mobile phase B is ACN / H2O / FA ​​(80 / 19.9 / 0.1, v / v) solution. Flow rate: 50 μL / min. The separation gradient is as follows: initially, the sample is loaded with 3% B phase, then the B phase is increased from 3% to 4% over 1 min, then from 4% to 32% B phase over 14 min, and then increased to 95% B phase over 2 min, and maintained for 2 min.

[0051] The raw data were analyzed using MaxQuant (version 2.6.3.0). Cysteine ​​alkylation (+71.0371 Da) was set as a fixed modification, while N-terminal acetylation (+42.0105 Da), methionine oxidation (+15.9949 Da), and phosphorylation of serine, threonine, tyrosine, histidine, arginine, and lysine (+79.7663 Da) were set as variable modifications. Trypsin was selected as the protease, allowing for two digestions. Search results were filtered, matched, and scored using modules embedded in the software.

[0052] In summary, this invention provides a method for preparing an immobilized bimetallic ion affinity monolithic material CS-ZrPC@PLP using chitosan as a precursor, and designs and assembles an enrichment device, providing a novel method and application for large-scale and rapid enrichment of phosphopeptides from clinical samples. This invention reveals that the prepared CS-ZrPC@PLP monolithic material exhibits superior enrichment effects for phosphopeptides in clinical samples. The CS-ZrPC@PLP monolithic material uses inexpensive and environmentally friendly raw materials, possesses directional pores and high permeability, high metal ion modification density, and good reusability. The designed enrichment device enables large-scale, rapid, and stable enrichment of phosphopeptides, requiring only 5 minutes to process 8 or more samples, significantly exceeding the pretreatment speed of conventional methods.

[0053] The key feature of this invention is its ability to simultaneously enrich large-scale clinical samples with phosphopeptides within a short timeframe, with enrichment results complementary to those reported by commercially available TiO2. Furthermore, the raw materials are environmentally friendly and inexpensive, the preparation process is simple, and it exhibits strong reusability. This invention provides a powerful method for analyzing and identifying phosphorylation sites in clinical samples.

[0054] It should be noted that, unless otherwise specified, the experimental methods used in this invention are all conventional methods; and the reagents and materials used, unless otherwise specified, are all commercially available.

[0055] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. Structures, devices, and operating methods not specifically described or explained in this invention are implemented according to conventional methods in the art unless otherwise specified or limited.

Claims

1. A method for preparing a reusable directional channel monolithic material, characterized in that, Includes the following steps: Step 1: Preparation of CS-ZrPC monolithic material; 500-1000 mg of aminophthalide zirconium, 500-1000 mg of chitosan, and 50-100 mg of polyethylene glycol diglycidyl ether are stirred evenly in 30 mL of 0.5%-2% acetic acid-water solution. After ultrasonic degassing, the mixed solution is poured into a polytetrafluoroethylene mold with a thermally conductive copper plate at the bottom. Liquid nitrogen is added to make the aerogel grow along the direction perpendicular to the copper plate. The aerogel is freeze-dried for 24-48 h and then crosslinked in an oven for 6-12 h to obtain the CS-ZrPC monolithic material. The polytetrafluoroethylene mold has a thermally conductive copper plate at the bottom, which enables the monolithic material to grow in a directional manner. Step 2: The CS-ZrPC monolithic material prepared in Step 1, along with pyridoxal 5'-phosphate and sodium cyanoborohydride, are stirred in a phosphate buffer solution at pH 7.5 for 5 hours to obtain a phosphorylated CS-ZrPC monolithic material. Then, the phosphorylated CS-ZrPC monolithic material is added to zirconium salt containing an aqueous trifluoroacetic acid solution and stirred to fix Zr4+. After the process, it is washed three times with deionized water and vacuum dried at room temperature to obtain a reusable oriented channel monolithic material, namely the CS-ZrPC@PLP monolithic material.

2. The method for preparing a reusable directional channel monolithic material according to claim 1, characterized in that: In step 2, the ratio of the amount of the CS-ZrPC monolithic material, pyridoxal 5'-phosphate, and sodium cyanoborohydride is 1:0.5-2g:0.5-2g; The concentration of the phosphate buffer solution is 100-500 mmol / L. ﹣1 ; The zirconium salt is ZrOCl2·8H2O, and the ratio of the phosphorylated modified CS-ZrPC monolithic material to the zirconium salt is 5-10 g: 100 mmol / L. ﹣1 ; The trifluoroacetic acid aqueous solution contains 0.1-1% by mass.

3. The method for preparing a reusable integral directional channel material according to claim 1, characterized in that: The monolithic CS-ZrPC@PLP material is fixed to the pipette tip and combined with a series of pipettes to form an enrichment device; the longitudinal mass transfer resistance of the monolithic CS-ZrPC@PLP material is less than 3.0 mL / min. ﹣1 At a flow rate of 4.2 MPa, the column pressure is 4.2 MPa, and it has compressive resilience in the transverse direction.

4. The application of the CS-ZrPC@PLP monolithic material prepared by the preparation method according to any one of claims 1-3 in the large-scale and rapid enrichment of phosphopeptides, characterized in that, CS-ZrPC@PLP monolithic material serves as a monolithic material for immobilized metal ion affinity chromatography directional channels.

5. A method for large-scale rapid enrichment of phosphopeptides, characterized in that, include: The standard protein β-casein was enzymatically digested, and the protein sample extracted from Hep G2 was denatured and enzymatically digested. Rapid enrichment of peptides in enzymatic hydrolysate was achieved using an immobilized metal ion affinity chromatography directional pore monolithic material. The acquired data were analyzed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry.

6. An enrichment apparatus used in the enrichment method of claim 5, characterized in that, This includes the integral material of the directional channel fixed to the pipette tip and the pipette assembly that matches the pipette tip; The device combining the pipette tip with the multi-pipette allows for free and independent switching between the three liquids: sample solution, eluent, and elution solution.

7. The application of the enrichment device according to claim 6 in large-scale phosphopeptide enrichment, characterized in that, include: Immobilized metal ion affinity chromatography directional channel monolithic material is fixed inside the pipette tip; The pipette tip with fixed directional channel monolithic material was combined with a series of pipettes. The sample solution was used to rinse the immobilized metal ion affinity chromatography monolithic material multiple times by blowing. Then, the protein hydrolysate was used to fully wet the immobilized metal ion affinity chromatography monolithic material in the same way to complete the sample loading. The non-phosphorylated peptides and salts were rinsed by repeated blowing and rinsing with the first and second rinsing solutions. Phosphorylated peptides retained on the immobilized metal ion affinity chromatography monolithic material are eluted by repeated pipetting using an eluent. The peptides in the eluent were analyzed using MALDI-TOF-MS or high-resolution mass spectrometry.

8. The application of the enrichment device according to claim 7 in large-scale phosphopeptide enrichment, characterized in that: The loading solution is a mixed solution consisting of 80%-85% acetonitrile, 0.1%-6% trifluoroacetic acid, and 9%-19.9% ​​water by volume. The first eluent consists of 30%-85% acetonitrile, 0.1%-6% trifluoroacetic acid, 9%-69.9% water, and 200 mmol / L. ﹣1 A mixed solution composed of NaCl; The second rinsing solution is a mixed solution consisting of 80%-85% acetonitrile, 0.1%-6% trifluoroacetic acid, and 9%-19.9% ​​water by volume. The eluent is a 10-15 wt% ammonia solution.

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