An IMAC material based on polyacrylonitrile fiber and its application

Abstract

The present invention discloses an IMAC material based on polyacrylonitrile fiber and its application. The material is prepared by the following method: using polyacrylonitrile fiber as a raw material, polymerizing it with a diamine compound to obtain an amino-functionalized polyacrylonitrile fiber matrix; introducing aldehyde groups on the matrix surface through a condensation reaction; then modifying the aldehyde sites with phosphate groups; and finally modifying them with transition metal ions. The high content of functional groups greatly improves the material's ability to bind phosphopeptides. In particular, by leveraging the strong hydrophilicity and good flexibility of polyacrylonitrile fiber, the IMAC material based on polyacrylonitrile fiber can be easily fabricated into an integrated tip device for automated and in-situ enrichment, reducing sample loss, and thus achieving efficient and highly sensitive enrichment of phosphopeptides.

Description

Technical Field

[0001] The invention belongs to the technical field of new materials, in particular to an IMAC material with polyacrylonitrile fiber as a matrix and application thereof. Background Art

[0002] Protein phosphorylation is one of the most common and important post-translational modifications, involved in many important cellular functions and dynamically regulating various signaling pathways. Abnormal protein phosphorylation is closely related to the occurrence and development of many human diseases, such as cancer, neurological diseases, and chronic inflammation. More than two-thirds of human proteins can be phosphorylated.

[0003] One of the difficulties in analyzing phosphoproteomics is the low abundance of phosphorylated proteins, the small proportion of phosphopeptides in the total peptides, the presence of a large amount of non-specific peptide interference, and the easy loss of samples in the complex phosphoproteomics workflow, resulting in a low mass spectrometry detection rate. Therefore, phosphorylation detection cannot be separated from efficient enrichment technology. Currently, widely used phosphoproteomic enrichment materials such as Fe-NTA and TiO2 generally require a sample amount of more than 200 μg, which cannot meet the analysis of some precious trace clinical samples that are difficult to obtain. Therefore, the development of efficient and low-residue enrichment materials is crucial for high-sensitivity phosphoproteomic analysis in complex biological sample systems, especially trace samples.

[0004] Reported methods for phosphopeptide enrichment include metal oxide affinity chromatography (MOAC), immobilized metal ion affinity chromatography (IMAC), immunoaffinity chromatography (IAC), and chemical derivatization. IMAC utilizes the electrostatic chelation between immobilized transition metal cations and negatively charged phosphate groups to enrich phosphopeptides. IMAC has gained widespread recognition and application due to its advantages: 1) tolerance to extreme acid and alkaline conditions; 2) excellent chemical stability; and 3) highly hydrophilic surface properties (J. Ye, X. Zhang, C. Young, X. Zhao, Q Hao, L Cheng, J. Proteome Res. 2010, 9, 3561-3573; D. Wang, J. Huang, H. Zhang, T. Gu, ACS Appl Mater Interfaces. 2023, 15, 47893-47901).

[0005] IMAC materials are primarily composed of transition metal ions, chelating ligands, and immobilization matrices. Traditional IMAC materials often use graphene, magnetic microspheres, silica microspheres, and metal-organic frameworks as matrices. However, due to their high nonspecificity, the selectivity and sensitivity of enrichment of trace phosphopeptides remain limited (D. Wang, J. Huang, H. Zhang, M Ma, M Xu, J. Proteome Res. 2023, 22, 2044-2054; J Huang, X Liu, D Wang, Y Cui, X Shi, Anal. Chem., 2021, 93, 8568-8576). Polyacrylonitrile fiber materials offer advantages such as low cost, strong hydrophilicity, high mechanical strength, and good flexibility. However, there are currently no reports on the use of polyacrylonitrile fiber-based IMAC materials for phosphopeptide enrichment. Summary of the Invention

[0006] The present invention aims to solve the above technical problems existing in the prior art and provides an IMAC material with polyacrylonitrile fiber as a matrix and its application.

[0007] The technical solution of the present invention is: an IMAC material with polyacrylonitrile fiber as the matrix is ​​prepared according to the following method: polyacrylonitrile fiber (PAN) is used as the raw material, and a diamine compound is polymerized to obtain an amino-functionalized polyacrylonitrile fiber matrix; aldehyde groups are introduced on the matrix surface through a condensation reaction, and then phosphate groups are modified at the aldehyde site; finally, transition metal ions are modified.

[0008] Preferably, the preparation is carried out in the following steps:

[0009] Step 1. Soak polyacrylonitrile fiber in a 10-1000 mM diamine compound solution at 50-100°C for 4-24 hours, and then wash away the unreacted reagent with pure water to obtain an amino-functionalized polyacrylonitrile fiber matrix.

[0010] Step 2. Immerse the amino-functionalized polyacrylonitrile fiber matrix in a 5-50% by volume glutaraldehyde-containing PBS buffer solution at pH 4.0-11.0, react at room temperature for 4-24 hours, and then wash away the unreacted reagent with pure water to obtain an amino-functionalized polyacrylonitrile fiber matrix containing aldehyde groups.

[0011] Step 3. Immersing the amine-functionalized polyacrylonitrile fiber matrix containing aldehyde groups in a PBS buffer solution (pH 4.0-11.0) containing sodium cyanoborohydride and a compound containing amino and phosphate groups, wherein the concentration of the sodium cyanoborohydride is 10-500 mM, the reaction is carried out at room temperature for 4-24 hours, and the unreacted reagent is washed away with pure water to obtain an amino-functionalized polyacrylonitrile fiber matrix modified with phosphate groups.

[0012] Step 4. Soak the amino-functionalized polyacrylonitrile fiber matrix modified with phosphate groups in a 20-200 mM transition metal ion salt solution and react at room temperature for 4-24 hours. Wash away the unreacted reagent with pure water to obtain an IMAC material based on polyacrylonitrile fiber.

[0013] Preferably, the diamine compound is ethylenediamine, 1,2-propylenediamine, hexamethylenediamine or p-phenylenediamine; the amino and phosphoric acid group compound is aminomethylphosphonic acid, aminoethylphosphonic acid or aminopropylphosphonic acid; and the transition metal ion salt solution is titanium sulfate, iron sulfate, titanium tetrachloride, zirconium sulfate or zirconium tetrachloride.

[0014] Use of the IMAC material with polyacrylonitrile fiber as a matrix as claimed in claim 1, 2 or 3 in enriching phosphopeptides.

[0015] The preferred application of the IMAC material with polyacrylonitrile fiber as the matrix in enriching phosphopeptides is to prepare the IMAC material with polyacrylonitrile fiber as the matrix into an integrated tip device.

[0016] The preferred application of IMAC material with polyacrylonitrile fiber as the matrix in enriching phosphopeptides is as follows:

[0017] First, a mixed solution of acetonitrile and an ion additive is used as a loading buffer to equilibrate the IMAC material based on polyacrylonitrile fiber in the integrated tip device, and then the sample to be enriched is added and incubated together; then, the IMAC material based on polyacrylonitrile fiber is washed in sequence with wash buffer I and wash buffer II, wherein the wash buffer I is a mixed solution of loading buffer and acetonitrile, and the wash buffer II is an ion additive with a volume fraction of 0.1-10%, and the ion additive is formic acid, trifluoroacetic acid, lactic acid, acetic acid or glyceric acid; finally, the IMAC material based on polyacrylonitrile fiber is eluted with an alkaline elution buffer, and the eluate is freeze-dried.

[0018] The present invention uses polyacrylonitrile fiber rich in nitrile groups as a matrix to prepare different types of transition metal IMAC materials. The high content of functional groups greatly improves the binding ability of the material with phosphopeptides. In particular, by leveraging the strong hydrophilicity and good flexibility of polyacrylonitrile fiber, the IMAC material based on polyacrylonitrile fiber can be easily made into an integrated tip device for realizing automation and in situ enrichment, reducing sample loss, and thus achieving efficient and highly sensitive enrichment of phosphopeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The flowchart of the preparation of IMAC material with polyacrylonitrile fiber as the matrix according to an embodiment of the present invention.

[0020] Figure 2 Schematic diagram of an integrated tip device prepared using the IMAC material with polyacrylonitrile fiber as the matrix in Example 1.

[0021] Figure 3 This is a scanning electron microscope image of the IMAC material based on polyacrylonitrile fiber according to Example 1 of the present invention.

[0022] Figure 4 This is an EDS elemental analysis spectrum of the IMAC material based on polyacrylonitrile fiber according to Example 1 of the present invention.

[0023] Figure 5 This is a diagram showing the enrichment effect of the IMAC material based on polyacrylonitrile fiber in Example 1 of the present invention on phosphopeptides and non-phosphopeptides.

[0024] Figure 6-8 This is a diagram showing the enrichment effect of the IMAC material with polyacrylonitrile fiber as the matrix in Example 1 of the present invention on different amounts of Hela cell lysates. DETAILED DESCRIPTION Example 1

[0025] The IMAC material based on polyacrylonitrile fiber of the present invention is as follows Figure 1 As shown, it is prepared according to the following process:

[0026] Step 1. Preparation of amino-functionalized polyacrylonitrile fiber matrix: Soak polyacrylonitrile (PAN) fiber in 100 mM ethylenediamine solution at 60°C for 8 h, then wash away the unreacted solution with pure water and dry.

[0027] Step 2. Preparation of aldehyde-modified polyacrylonitrile fiber matrix: Weigh 10 mg of amino-functionalized polyacrylonitrile fiber matrix, add 400 μL of 10% (v / v) glutaraldehyde in 100 mM PBS (pH 8.0) as solvent, and react at room temperature for 6 h. Wash the material with pure water to remove unreacted glutaraldehyde.

[0028] Step 3. Preparation of phosphate-modified polyacrylonitrile fiber matrix: Immerse the amine-functionalized polyacrylonitrile fiber matrix containing aldehyde groups in a 100 mM PBS (pH 8.0) buffer solution containing 10 mg / mL sodium cyanoborohydride (NaCNBH3) and 2 mg / mL aminomethylphosphonic acid (AMPA). Stir the reaction at room temperature for 6 h and then wash with pure water.

[0029] Step 4. Immobilization of transition metal ions on the surface of polyacrylonitrile fiber matrix: The amino-functionalized polyacrylonitrile fiber matrix modified with phosphate groups was immersed in 100 mM Ti(SO4)2 for 6 h, and then the material was washed with pure water.

[0030] The integrated tip device prepared by using the IMAC material with polyacrylonitrile fiber as the matrix obtained in Example 1 is as follows: Figure 2 shown.

[0031] The scanning electron microscope image of the IMAC material based on polyacrylonitrile fiber prepared in Example 1 is as follows: Figure 3 shown. Figure 3 Figure A is magnified 2000 times, and Figure B is magnified 250 times. Figure 3 It can be seen that a fibrous material is formed.

[0032] The EDS elemental analysis spectrum of the IMAC material based on polyacrylonitrile fiber in Example 1 is as follows: Figure 4 As shown in the figure, the distribution of Ti element was obtained by EDS element analysis. The results showed that the Ti element was successfully immobilized in the material. Example 2

[0033] Immobilization of transition metal ions on the surface of polyacrylonitrile fiber matrix: The amino-functionalized polyacrylonitrile fiber matrix modified with phosphate groups was immersed in 100 mM ZrCl4 and reacted for 6 h, and then the material was washed with pure water; the remaining steps were the same as in Example 1.

[0034] Example 3:

[0035] Immobilization of transition metal ions on the surface of polyacrylonitrile fiber matrix: The amino-functionalized polyacrylonitrile fiber matrix modified with phosphate groups was immersed in 100 mM FeCl3 for 6 h, and then the material was washed with pure water; the remaining steps were the same as in Example 1.

[0036] experiment:

[0037] 1. To investigate the selectivity and non-specific adsorption of the polyacrylonitrile fiber-based IMAC material for phosphopeptide enrichment, the following interference experiments were performed:

[0038] 1. Preparation of peptide mixed solution

[0039] Phosphopeptide (MAQPFS(phospho)LR) and non-phosphopeptide (bovine serum albumin, BSA) were mixed at a mass ratio of 1:5000 and dissolved in a loading buffer, which is a mixture of 10-80% acetonitrile and ion additives;

[0040] 2. Selective enrichment of phosphopeptides

[0041] The IMAC material prepared in Example 1 was fabricated into a 10 μL integrated tip device. The device was equilibrated with loading buffer and incubated with a mixed solution of phosphopeptide (MAQPFS(phospho)LR) and non-phosphopeptide (bovine serum albumin, BSA). The device was then washed sequentially with wash buffer I (loading buffer:acetonitrile = 1:1) and wash buffer II (0.5% aqueous solution of an ionic additive). Finally, the device was eluted with elution buffer (1-50% ammonia, 20-80% acetonitrile). The stock solution, loading effluent, and eluate were lyophilized and reconstituted for MALDI-TOF MS analysis.

[0042] 3. MALDI-TOF MS analysis

[0043] 2 μL of the reconstituted material and 2 μL of DHB matrix (20 mg / mL DHB dissolved in 60% acetonitrile containing 1% phosphoric acid and 0.1% trifluoroacetic acid) were spotted sequentially onto a MALDI target plate. After the sample spot dried, mass spectrometry analysis was performed. MALDI-TOF MS experiments were performed on an Ultraflex III TOF / TOF (Bruker Daltonics, Bremen, Germany) using linear positive ion mode.

[0044] The results of the original solution and the sample effluent were as follows Figure 5 As shown in a and b, the standard phosphopeptide (MAQPFS(phospho)LR) in the original solution and the sample effluent is extremely low in concentration, so that the mass spectrometry response signal is suppressed by BSA and cannot be detected; after enrichment (eluate) by the IMAC material based on polyacrylonitrile fiber of the present invention, the signal intensity of the standard phosphopeptide (MAQPFS(phospho)LR) is significantly improved ( Figure 5 c) and significantly eliminated the effects of nonspecific peptides. This indicates that the material of the present invention has extremely high selectivity for phosphopeptides and can achieve efficient and highly sensitive enrichment of phosphopeptides with only 10 ng.

[0045] 2. In order to investigate the enrichment ability of the polyacrylonitrile fiber-based IMAC material of the present invention for phosphopeptides, phosphoproteins, and phosphorylation sites in different numbers of human cervical cancer cells (HeLa), the following experiments were conducted:

[0046] 1. Hela cell lysis and protein extraction process

[0047] The cells were collected, washed three times with 0.9% NaCl solution, and centrifuged at low speed (500 g) to remove the supernatant; the collected cells were resuspended in 0.9% NaCl solution;

[0048] 100 HeLa cells: Take three aliquots of the corresponding number of HeLa cells and add TEAB to a concentration of 100 mM. Then add casein and Lys-C to a final concentration of 10 ng / μL. Incubate at 37°C for 1.5 hours to obtain a protein hydrolysate and store at -80°C.

[0049] 1000 HeLa cells: Perform the same treatment steps as 100 HeLa cells.

[0050] 10,000 HeLa cells: Take three aliquots of the corresponding number of HeLa cells and disrupt them in lysis buffer (100 mM TEAB, 1% phosphatase inhibitor) by ultrasonication at 4°C for 20 min, with an energy of 400 W, ultrasonication for 30 s, and an interval of 30 s. The lysis is quenched by heating in a water bath at 95°C for 5 min. DTT is added to a final concentration of 2 mM, and the protein is reduced at 37°C for 30 min. Then, IAA is added to a final concentration of 5 mM, and the reaction is carried out at room temperature in the dark for 30 min. Trypsin and Lys-C are added to a final concentration of 100 ng / μL respectively, and the enzymatic digestion is carried out at 37°C overnight. The resulting protein hydrolysate is stored at -80°C.

[0051] 10 5 HeLa cells: treatment steps are the same as 10 4 HeLa cells.

[0052] 2. Selective Enrichment of Phosphopeptides

[0053] The enrichment operation process was the same as that of Experiment 1. The eluate was lyophilized and then reconstituted with 0.1% FA water for LC-MS / MS analysis.

[0054] 100, 1000, 10 4 , 10 5 The enzymatic hydrolysates of HeLa cells were enriched using the integrated tip device made of the IMAC material of Example 1 of the present invention. Three technical replicates were performed for each sample, and the results were averaged. The enrichment abilities of phosphopeptides, phosphoproteins, and phosphorylation sites were respectively as follows: Figure 6 、 Figure 7 and Figure 8As shown in the figure, the results showed that 579 phosphorylated proteins, 1287 phosphorylated peptides, and 1394 phosphorylation sites were identified in the enzymatic hydrolysate of only 100 HeLa cells, indicating that the integrated tip device made of the polyacrylonitrile fiber-based IMAC material of the present invention can be used for efficient and highly sensitive enrichment of trace samples.

Claims

1. An IMAC material based on polyacrylonitrile fiber, characterized in that Prepare according to the following steps: Step 1. Soak polyacrylonitrile fibers containing methyl ester groups in a 10-1000 mM diamine compound solution at 50-100°C for 4-24 hours, and then wash away the unreacted reagent with pure water to obtain an amino-functionalized polyacrylonitrile fiber matrix. Step 2. Immerse the amino-functionalized polyacrylonitrile fiber matrix in a 5-50% by volume glutaraldehyde-containing PBS buffer solution at pH 4.0-11.0 at room temperature for 4-24 hours, and then wash away the unreacted reagent with pure water to obtain an amino-functionalized polyacrylonitrile fiber matrix containing aldehyde groups. Step 3. Immersing the amine-functionalized polyacrylonitrile fiber matrix containing aldehyde groups in a pH 4.0-11.0 PBS buffer solution containing sodium cyanoborohydride and a compound containing amino and phosphate groups, wherein the concentration of the sodium cyanoborohydride is 10-500 mM, the reaction is carried out at room temperature for 4-24 hours, and the unreacted reagent is washed away with pure water to obtain an amino-functionalized polyacrylonitrile fiber matrix modified with phosphate groups; Step 4. Soak the amino-functionalized polyacrylonitrile fiber matrix modified with phosphate groups in a 20-200 mM transition metal ion salt solution and react at room temperature for 4-24 hours. Wash away the unreacted reagent with pure water to obtain an IMAC material based on polyacrylonitrile fiber.

2. The IMAC material based on polyacrylonitrile fiber according to claim 1, characterized in that: The diamine compound is ethylenediamine, 1,2-propylenediamine, hexamethylenediamine or p-phenylenediamine; the compound containing amino and phosphoric acid groups is aminomethylphosphoric acid, aminoethylphosphoric acid or aminopropylphosphoric acid; and the transition metal ion salt solution is titanium sulfate, iron sulfate, titanium tetrachloride, zirconium sulfate or zirconium tetrachloride.

3. Use of the IMAC material with polyacrylonitrile fiber as a matrix as claimed in claim 1 or 2 in enriching phosphopeptides.

4. The use of the IMAC material based on polyacrylonitrile fiber in enriching phosphopeptides according to claim 3, characterized in that The IMAC material with polyacrylonitrile fiber as the matrix is ​​made into an integrated tip device.

5. The use of the IMAC material based on polyacrylonitrile fiber in enriching phosphopeptides according to claim 4, characterized in that The specific application method is as follows: first, a mixed solution of acetonitrile and ion additives is used as a loading buffer to equilibrate the IMAC material based on polyacrylonitrile fiber in the integrated tip device, and then the sample to be enriched is added and incubated together; The IMAC material based on polyacrylonitrile fiber is then washed sequentially with wash buffer I and wash buffer II, wherein wash buffer I is a mixed solution of loading buffer and acetonitrile, and wash buffer II is an aqueous solution of an ionic additive with a volume fraction of 0.1-10%, wherein the ionic additive is formic acid, trifluoroacetic acid, lactic acid, acetic acid, or glyceric acid. Finally, the IMAC material based on polyacrylonitrile fiber is eluted with an alkaline elution buffer, and the eluate is freeze-dried.

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