IMAC material with polyacrylonitrile fiber as matrix and application of IMAC material

Abstract

The invention discloses an IMAC material with polyacrylonitrile fiber as a matrix and application of the IMAC material, and the IMAC material is prepared according to the following method: polyacrylonitrile fiber as a raw material is subjected to a polymerization reaction with a diamine compound to obtain an amino-functionalized polyacrylonitrile fiber matrix; introducing an aldehyde group on the surface of the substrate through a condensation reaction, and then modifying a phosphate group at an aldehyde group site; and finally modifying transition metal ions. The functional group content is high, the binding capacity of the material and phosphopeptides is greatly improved, particularly by means of the characteristics of high hydrophilicity and good flexibility of the polyacrylonitrile fiber, the IMAC material with the polyacrylonitrile fiber as the matrix can be easily made into an integrated tip device, the integrated tip device is used for achieving automation and in-situ enrichment, sample loss is reduced, and the cost is reduced. Therefore, high-efficiency and high-sensitivity enrichment of phosphopeptides is realized.

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. It participates in many important cellular functions and dynamically regulates various signaling pathways. Abnormal protein phosphorylation is closely related to the occurrence and development of many human diseases, such as cancer, nervous system diseases, and chronic inflammation. More than two-thirds of human proteins can be phosphorylated.

[0003] One of the difficulties in analyzing phosphorylated proteomics is the low abundance of phosphorylated proteins, the small proportion of phosphorylated peptides in the total peptide segment, the presence of a large number of non-specific peptide interferences, and the easy loss of samples in the complex phosphorylated proteomics workflow, resulting in a low mass spectrometry detection rate. Therefore, phosphorylation detection cannot be separated from efficient enrichment technology. Currently, the widely used phosphorylation enrichment materials such as Fe-NTA and TiO 2 The sample amount required is generally 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 phosphorylated proteomics analysis in complex biological sample systems, especially trace samples.

[0004] The methods reported in the literature for enriching phosphopeptides include metal oxide affinity chromatography (MOAC), immobilized metal ion affinity chromatography (IMAC), immunoaffinity chromatography (IAC) and chemical derivatization. Among them, IMAC uses the electrostatic chelation between transition metal cations fixed on the carrier and negatively charged phosphate groups to achieve the enrichment of phosphopeptides. It has been widely recognized and applied due to its advantages of: 1) tolerance to extreme acid and alkali enrichment conditions; 2) good chemical stability; 3) strong surface hydrophilicity (J.Ye, X.Zhang, C. Young, X.Zhao, Q Hao, L Cheng, J. ProteomeRes. 2010, 9, 3561-3573; D.Wang, J.Huang, H.Zhang, T.Gu, ACS Appl MaterInterfaces. 2023, 15, 47893-47901).

[0005] IMAC materials are mainly composed of transition metal ions, chelating ligands and fixed matrices. Traditional IMAC materials mostly use graphene, magnetic microspheres, silica microspheres and metal organic framework compounds as matrices. Due to their strong non-specificity, the enrichment selectivity and sensitivity of trace phosphorylated proteomes are still limited (D.Wang, J.Huang, H.Zhang, M Ma, M Xu, J. Proteome Res. 2023, 22, 2044-2054; J Huang, X Liu, DWang, Y Cui, X Shi, Anal. Chem., 2021, 93, 8568-8576). Polyacrylonitrile fiber materials have the advantages of low cost, strong hydrophilicity, high mechanical strength and good flexibility, but there are no reports on the use of IMAC materials based on polyacrylonitrile fiber 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 a matrix is ​​prepared according to the following method: polyacrylonitrile fiber (PAN) is used as a raw material, and a diamine compound is polymerized to obtain an amino-functionalized polyacrylonitrile fiber matrix; aldehyde groups are introduced on the surface of the matrix through a condensation reaction, and then the phosphate groups are modified at the aldehyde site; and finally, transition metal ions are modified.

[0008] Preferably, the preparation is carried out in the following steps: Step 1. Soak the polyacrylonitrile fiber in a 10-1000 mM diamine compound solution, react at 50-100°C for 4-24 h, and 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 pH 4.0-11.0 PBS buffer solution containing 5-50% glutaraldehyde by volume, react at room temperature for 4-24 hours, and wash away the unreacted reagent with pure water to obtain an amino-functionalized polyacrylonitrile fiber matrix containing aldehyde groups; Step 3. Immersing the amino-functionalized polyacrylonitrile fiber matrix containing aldehyde groups in a pH 4.0-11.0 PBS buffer solution containing sodium cyanoborohydride and a compound containing an amino group and a phosphate group, wherein the concentration of the sodium cyanoborohydride is 10-500 mM, reacting at room temperature for 4-24 hours, and washing away the unreacted reagent with pure water to obtain an amino-functionalized polyacrylonitrile fiber matrix modified with a phosphate group; Step 4. Soak the amino-functionalized polyacrylonitrile fiber matrix modified with the phosphate group in a 20-200 mM transition metal ion salt solution, react at room temperature for 4-24 h, and wash away the unreacted reagent with pure water to obtain an IMAC material with polyacrylonitrile fiber as the matrix.

[0009] 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.

[0010] A use of the IMAC material with polyacrylonitrile fiber as the matrix as claimed in claim 1, 2 or 3 in enriching phosphopeptides.

[0011] The preferred application of the IMAC material with polyacrylonitrile fiber as the matrix in enriching phosphopeptides is to make the IMAC material with polyacrylonitrile fiber as the matrix into an integrated tip device.

[0012] The preferred application of IMAC material with polyacrylonitrile fiber as the matrix in enriching phosphopeptides is as follows: First, a mixed solution of acetonitrile and ion additives is used as a loading buffer to balance 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 washing buffer I and washing buffer II, wherein the washing buffer I is a mixed solution of loading buffer and acetonitrile, and the washing 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.

[0013] The present invention adopts polyacrylonitrile fiber rich in nitrile groups as a matrix, and can prepare different types of transition metal IMAC materials with high functional group content, which greatly improves the binding ability of the material with phosphopeptides. In particular, by virtue of the strong hydrophilicity and good flexibility of polyacrylonitrile fiber, it is easy to make the IMAC material with polyacrylonitrile fiber as a matrix into an integrated tip device for realizing automation and in-situ enrichment, reducing sample loss, and thus realizing efficient and highly sensitive enrichment of phosphopeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The present invention is a flowchart of preparing an IMAC material with polyacrylonitrile fiber as a matrix.

[0015] Figure 2Schematic diagram of an integrated tip device prepared using the IMAC material with polyacrylonitrile fiber as the matrix in Example 1.

[0016] 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.

[0017] Figure 4 This is an EDS elemental analysis spectrum of the IMAC material with polyacrylonitrile fiber as the matrix according to Example 1 of the present invention.

[0018] Figure 5 This is a diagram showing the enrichment effect of the IMAC material with polyacrylonitrile fiber as the matrix on phosphopeptides and non-phosphopeptides according to Example 1 of the present invention.

[0019] Figure 6-8 This is a diagram showing the enrichment effect of the IMAC material with polyacrylonitrile fiber as the matrix on different amounts of Hela cell lysates in Example 1 of the present invention. DETAILED DESCRIPTION Example 1

[0020] 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: Step 1. Preparation of amine-functionalized polyacrylonitrile fiber matrix: soak polyacrylonitrile fiber (PAN) in 100 mM ethylenediamine solution, react at 60 °C for 8 h, wash away the unreacted solution with pure water and dry; Step 2. Preparation of aldehyde-modified polyacrylonitrile fiber matrix: Weigh 10 mg of amino-functionalized polyacrylonitrile fiber matrix, add 400 ul of 10% (v / v) glutaraldehyde, use 100 mM PBS (pH 8.0) as solvent, react at room temperature for 6 h, and wash the material with pure water to remove unreacted glutaraldehyde; Step 3. Preparation of phosphate-modified polyacrylonitrile fiber matrix: The amine-functionalized polyacrylonitrile fiber matrix containing aldehyde groups was immersed in a solution containing 10 mg / mL sodium cyanoborohydride (NaCNBH 3 ), 2 mg / mL aminomethylphosphonic acid (AMPA) in 100 mM PBS (pH 8.0) buffer solution, stirred at room temperature for 6 h, and then washed with pure water; 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(SO 4 ) 2 After reacting for 6 h, the material was washed with pure water.

[0021] 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.

[0022] 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.

[0023] 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

[0024] Immobilization of transition metal ions on the surface of polyacrylonitrile fiber matrix: Amine-functionalized polyacrylonitrile fiber matrix modified with phosphate groups was immersed in 100 mM ZrCl 4 After reacting for 6 h, the material was washed with pure water; the remaining steps were the same as in Example 1.

[0025] Embodiment 3: Immobilization of transition metal ions on the surface of polyacrylonitrile fiber matrix: Amine-functionalized polyacrylonitrile fiber matrix modified with phosphate groups was immersed in 100 mM FeCl 3 After reacting for 6 h, the material was washed with pure water; the remaining steps were the same as in Example 1.

[0026] experiment: 1. In order to investigate the selectivity and non-specific adsorption of the IMAC material based on polyacrylonitrile fiber for phosphopeptide enrichment, the following interference experiment was performed: 1. Preparation of peptide mixed solution The phosphopeptide (MAQPFS(phospho)LR) and the non-phosphopeptide (bovine serum albumin, BSA) were mixed at a mass ratio of 1:5000 and dissolved in a loading buffer, wherein the loading buffer is a mixture of 10-80% acetonitrile and ion additives; 2. Selective enrichment of phosphopeptides Take the IMAC material prepared in Example 1, make an integrated tip device of 10 μL, balance the integrated tip device with the loading buffer; add a mixed solution of phosphopeptide (MAQPFS (phospho) LR) and non-phosphopeptide (bovine serum albumin, BSA) for incubation; then, wash the integrated tip device with washing buffer I (loading buffer: acetonitrile = 1:1) and washing buffer II (0.5% volume fraction of ion additive aqueous solution); finally, elute with elution buffer (1-50% ammonia, 20-80% acetonitrile). After freeze-drying and re-dissolving the stock solution, loading effluent and eluate, MALDI-TOF MS identification was performed.

[0027] 3. MALDI-TOF MS analysis 2 μL of the reconstituted product and 2 μL of DHB matrix (20 mg / mL DHB dissolved in 60% acetonitrile solution containing 1% phosphoric acid and 0.1% trifluoroacetic acid) were spotted on the MALDI target plate in sequence, and mass spectrometry was performed after the sample spots were dried. MALDI-TOF MS experiments were performed on an Ultraflex Ⅲ TOF / TOF (Bruker Daltonics, Bremen, Germany), and linear positive ion mode was used for detection.

[0028] 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 stock 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 (eluent) by the IMAC material with polyacrylonitrile fiber as the matrix of the present invention, the signal intensity of the standard phosphopeptide (MAQPFS (phospho) LR) is significantly improved ( Figure 5 c) and the influence of nonspecific peptides was greatly eliminated. This indicates that the material of the present invention has extremely high selectivity for phosphopeptides, and only 10 ng of phosphopeptides can be enriched efficiently and sensitively.

[0029] 2. In order to investigate the enrichment ability of the IMAC material based on polyacrylonitrile fiber of the present invention on phosphopeptides, phosphorylated proteins and phosphorylation sites in different numbers of human cervical cancer cells (HeLa), the following experiments were conducted: 1. Hela cell lysis and protein extraction process Collect the cells, wash them three times with 0.9% NaCl solution, centrifuge at low speed (500 g) to remove the supernatant; resuspend the collected cells in 0.9% NaCl solution; 100 Hela cells: Take three copies of the corresponding number of Hela cells, add TEAB to make its concentration 100 mM, then add casein and Lys-C at a final concentration of 10 ng / μL, and enzymatically hydrolyze at 37℃ for 1.5 hours. The final protein enzymatic solution is stored at -80℃.

[0030] 1000 Hela cells: The treatment steps are the same as 100 Hela cells.

[0031] 10,000 Hela cells: Take three copies of the corresponding number of Hela cells, and disrupt them by ultrasonication in lysis buffer (100 mM TEAB, 1% phosphatase inhibitor) at 4°C for 20 min, with an energy of 400 W, ultrasonication for 30 s, and an interval of 30 s. Heat in a water bath at 95°C for 5 min to quench the lysis; add DTT to a final concentration of 2 mM, and reduce the protein at 37°C for 30 min; then add IAA to a final concentration of 5 mM, and react at room temperature in the dark for 30 min; add trypsin and Lys-C at a final concentration of 100 ng / μL respectively, and enzymatically hydrolyze them overnight at 37°C. The final protein enzymatic solution is stored at -80°C.

[0032] 10 5 Hela cells: treatment steps are the same as 10 4 HeLa cells.

[0033] 2. Selective Enrichment of Phosphopeptides The enrichment operation process was the same as that of Experiment 1. The eluate was freeze-dried and reconstituted with 0.1% FA water for LC-MS / MS analysis.

[0034] 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 capabilities of phosphopeptides, phosphorylated proteins, and phosphorylation sites were respectively as follows: Figure 6 , Figure 7 and Figure 8 As shown, 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 IMAC material with polyacrylonitrile fiber as the matrix 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 The preparation method is as follows: using polyacrylonitrile fiber as raw material, polymerizing with diamine compounds to obtain an amino-functionalized polyacrylonitrile fiber matrix; introducing aldehyde groups on the matrix surface through a condensation reaction, and then modifying the phosphate group at the aldehyde site; and finally modifying the transition metal ion.

2. The IMAC material based on polyacrylonitrile fiber according to claim 1, characterized in that Follow the steps to prepare: Step 1. Soak the polyacrylonitrile fiber in a 10-1000 mM diamine compound solution, react at 50-100°C for 4-24 h, and 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 pH 4.0-11.0 PBS buffer solution containing 5-50% glutaraldehyde by volume, react at room temperature for 4-24 hours, and wash away the unreacted reagent with pure water to obtain an amino-functionalized polyacrylonitrile fiber matrix containing aldehyde groups; Step 3. Immersing the amino-functionalized polyacrylonitrile fiber matrix containing aldehyde groups in a pH 4.0-11.0 PBS buffer solution containing sodium cyanoborohydride and a compound containing an amino group and a phosphate group, wherein the concentration of the sodium cyanoborohydride is 10-500 mM, reacting at room temperature for 4-24 hours, and washing away the unreacted reagent with pure water to obtain an amino-functionalized polyacrylonitrile fiber matrix modified with a phosphate group; Step 4. Soak the amino-functionalized polyacrylonitrile fiber matrix modified with the phosphate group in a 20-200 mM transition metal ion salt solution, react at room temperature for 4-24 h, and wash away the unreacted reagent with pure water to obtain an IMAC material with polyacrylonitrile fiber as the matrix.

3. The IMAC material based on polyacrylonitrile fiber according to claim 2, characterized in that: 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.

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

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

6. The use of the IMAC material based on polyacrylonitrile fiber in enriching phosphopeptides according to claim 5, 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 balance the IMAC material based on polyacrylonitrile fiber in the integrated tip device, and then the sample to be enriched is added for co-incubation; Then, the IMAC material with polyacrylonitrile fiber as the matrix is ​​washed in sequence with washing buffer I and washing buffer II, wherein the washing buffer I is a mixed solution of loading buffer and acetonitrile, and the washing 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 with polyacrylonitrile fiber as the matrix is ​​eluted with an alkaline elution buffer, and the eluate is freeze-dried.

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