A fractured solid-phase alkylation material and its preparation and application
The use of acid-breakable solid-phase alkylation materials has solved the problems of low recovery rate and poor anti-interference ability of clinical proteomics samples, enabling high-throughput and flexible protein detection and expanding detection coverage.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- DALIAN INSTITUTE OF CHEMICAL PHYSICS CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2023-09-28
- Publication Date
- 2026-06-30
AI Technical Summary
In existing technologies, clinical proteomics sample preparation has low recovery rates and poor resistance to matrix interference, resulting in low analytical coverage, and thiol-containing peptides cannot be effectively collected and detected.
By employing acid-breakable solid-phase alkylation materials, proteins are covalently bound to thiol groups on the material surface. The linker arms are then broken using an acidic solution, enabling high-recovery capture of proteins and non-destructive release of thiol-containing peptides, supporting TOP-DOWN and BOTTOM-UP studies.
It improves the throughput and anti-interference ability of proteomics sample preparation, increases the coverage of protein detection, expands the detection range, and supports flexible research methods.
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Figure CN119708512B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a fractured solid-phase alkylation material that can be applied to the selective reaction and pretreatment of thiol proteins in cells, tissues, body fluids, hair, or other protein-rich samples. Background Technology
[0002] Clinical sample proteomics studies are of great significance for the discovery of potential biomarkers, drug target screening, early diagnosis and treatment of diseases, and prognosis. Samples such as plasma, urine, tissue sections, and circulating tumor cells are widely used in clinical testing. Most clinical samples are characterized by small sample volume, complex matrices, and numerous interfering substances. Therefore, developing efficient sample preparation methods is a prerequisite for achieving high-throughput, high-precision, and in-depth coverage of clinical proteomics analysis.
[0003] To address the issues of low recovery rates, poor resistance to matrix interference, and low throughput in clinical proteomics sample preparation, resulting in low analytical coverage, the inventors developed a solid-phase alkylation (SPA) material using atom transfer radical polymerization. This material utilizes the covalent interaction between iodine on the material surface and the thiol groups on cysteine residues exposed after protein denaturation and reduction to achieve protein capture on the material surface. This invention significantly improves the throughput of proteomics sample preparation and exhibits strong resistance to interference. This invention has been granted a patent: Preparation of Protein Solid-Phase Alkylation Reagents and Their Applications (ZL201510531298.3).
[0004] During the preparation of proteomic samples using SPA materials, we found that while most of the cleaved peptides were collected after enzymatic digestion, thiol-containing peptides were not collected or detected due to binding to the SPA material, resulting in a loss of this information. Based on this, we invented an acid-cleavable solid-phase alkylation material. Protein thiol groups can covalently bind to the material. After sample processing, adding an acidic solution to the reaction system breaks the linker arms, releasing the protein or thiol peptides without damage. Compared to previous solid-phase alkylation materials, the new material increases the enrichment of thiol-containing peptides, further improving the coverage of protein detection; it can also be applied to top-down studies targeting intact thiol proteins. Summary of the Invention
[0005] The purpose of this invention is to provide a solid-phase alkylation material that can be used for protein pretreatment. This material not only achieves high-recovery protein capture, rapid separation from other small molecules, and reduces the complexity of protein samples; but also, because the amino linkers of compounds with acid-cleavable amino groups on both sides (ACA) can be cleaved under acidic conditions, subsequent processing methods can employ either a BOTTOM-UP study approach (collecting peptides after enzymatic digestion for mass spectrometry analysis without loss of thiol-containing peptides) or a TOP-DOWN study using intact thiol-containing proteins. To achieve this objective, the technical solution of this invention is as follows:
[0006] 1) Epoxy microspheres (silica gel or magnetic sphere matrix) are sequentially modified with polyethyleneimine (PEI), ACA and iodoacetic acid-N-succinamide ester (NIS) via covalent bonding;
[0007] A) Hydrophilic active groups are bonded to the surface of epoxy-based microspheres. The process is as follows: 0.1-100g of epoxy-based microspheres are dispersed in phosphate buffer solution, 0.1-100L of PEI aqueous solution containing 1-1000g of PEI is added, and the reaction is stirred for 6-24 hours. The total reaction system is 0.1-100L, and amino-modified microspheres are obtained.
[0008] B) Disperse 0.1-100 g of the amino-modified microspheres obtained in the previous step in a phosphate buffer solution, add 0.1-100 mol of the intermediate compound, which is dissolved in methanol and / or dimethyl sulfoxide (DMSO) solution, and stir the reaction for 0.5-6 hours. The reaction system consists of 45-90% methanol (v / v), 5-10% 50 mM phosphate buffer (pH 8.0, v / v), and 0-50% DMSO (v / v), with a volume of 0.1-100 L, to obtain active ester-modified microspheres.
[0009] C): Disperse 0.1-100 g of the active ester-modified microspheres obtained in the previous step in a phosphate buffer solution, add 0.2-200 mol of ACA (dissolved in methanol, phosphate buffer, or DMSO), and stir the reaction for 0.5-6 hours. The reaction solution system consists of 45-90% methanol (v / v), 5-10% 50 mM phosphate buffer (pH 8.0, v / v), and 0-50% DMSO (v / v), with a volume of 0.1-100 L, to obtain acid-cleavable amino-modified microspheres.
[0010] D) Disperse 0.1-100 g of the acid-cleavable amino-modified microspheres obtained in the previous step in a phosphate buffer solution, add 0.1-100 g of iodoacetic acid-N-succinamide ester, dissolve NIS in a mixture of methanol and phosphate buffer solution, stir and react in the dark for 6-24 hours. The reaction solution system is 50-90% methanol (v / v) and 10-50% 50mM phosphate buffer (pH 8.0, v / v), with a volume of 0.1-100 L, to obtain a cleavable solid-phase alkylated material under acidic conditions.
[0011] E) The epoxy microspheres mentioned in A) above can be either silica-based epoxy microspheres or magnetic iron oxide-based epoxy microspheres. The intermediate compound mentioned in B) above can be succinyl succinimide ester, glutaraldehyde, or any other compound that can provide an active ester group. The ACA mentioned in C) above can be any compound with amino groups on both sides, providing bifunctional linkers, and capable of cleavage under acidic conditions, with the following structural formula:
[0012] Where n is an integer from 1 to 20.
[0013] 2) Applying fractured solid-phase alkylation materials to selective reactions or pretreatment of thiol-containing proteins in cells, tissues, body fluids, hair or other protein-rich samples.
[0014] The present invention has the following advantages:
[0015] 1. The prepared protein solid-phase alkylation material can realize in-situ pretreatment of proteins in micro-tissues or cells (including alkylation, removal of small molecule interfering substances, and enzymatic hydrolysis of particles);
[0016] 2. Building upon the original advantages of SPA materials, such as high throughput and anti-interference, the material exhibits the characteristic of breaking down under acidic pH conditions in the reaction environment. This enhances the enrichment of thiol-containing peptides and further expands the coverage of protein detection.
[0017] 3. In our previous development, the SPA material reacted with proteins, requiring enzymatic hydrolysis to release the proteins into peptides, which were then collected for mass spectrometry analysis—a BOTTOM-UP study method. The cleavable SPA material involved in this invention reacts with proteins, releasing proteins through the cleavage of the connective arms under acidic conditions. Subsequent analysis can either involve enzymatic hydrolysis to collect peptides for BOTTOM-UP studies, or direct analysis of the released proteins—a TOP-DOWN study. Users can flexibly design experiments according to their research needs, greatly expanding the application scenarios of solid-phase alkylation materials. Attached Figure Description
[0018] Figure 1 The structural formula of a compound with acid-breakable amino linkage arms on both sides (ACA).
[0019] Figure 2 , 2 2'-(propane-2,2-dimethylbis(oxy))bis(ethane-1-amine) 1H NMR and 1C NMR spectra.
[0020] Figure 3 A schematic diagram of solid-phase alkylation reagent materials and their application process.
[0021] Figure 4 The application of fractured solid-phase alkylation materials was investigated using BSA as a model protein. Detailed Implementation
[0022] The following examples are illustrative of the above-mentioned patent and not limiting.
[0023] Example 1
[0024] The synthetic route for 2,2'-(propane-2,2-diylbis(oxy))bis(ethane-1-amine), i.e. (ACA), is as follows:
[0025]
[0026] Its synthesis process is as follows:
[0027] 1. Compound 1 (2-(2-hydroxyethyl)isoindoline-1,3-dione, 20.0 g, 105 mmol, 2.0 eq) was dissolved in anhydrous toluene (200 mL), followed by the addition of compound 2 (2,2-dimethoxypropane, 5.4 g, 52.5 mmol, 1.0 eq) and p-TSA (0.9 g, 5.25 mmol, 0.1 eq). The mixture was then refluxed for 4 h. After the reaction was complete, the mixture was cooled to room temperature, concentrated under reduced pressure, and column-secured (PE:EA = 4:1) to give 9.9 g of compound 3 (Y = 45%).
[0028] 2. Compound 3 obtained in step 1 was dissolved in 100 mL of 6 N NaOH and refluxed for 8 h. After cooling to room temperature, it was extracted with DCM / MeOH (10:1), and the organic phase was evaporated to dryness under reduced pressure. The crude product was column-sected (DCM:MeOH = 20:1) to give 2.2 g of compound 4, namely 2,2'-(propane-2,2-dimethylbis(oxy))bis(ethane-1-amine) (Y = 59%).
[0029] 3. The 1H and 1C NMR spectra of the finished product ACA [2,2'-(propane-2,2-dimethylbis(oxy))bis(ethane-1-amine)] are attached. Figure 2 .
[0030] Example 2
[0031] Synthesis of fracture-resistant solid-phase alkylation materials via epoxy-based magnetic sphere matrix and succinyl succinimide ester pathway: 1. Purchase 0.1 g of commercially available epoxy-based magnetic spheres (Suzhou Beaver, catalog number P07990027, which are superparamagnetic particles with epoxy groups on the surface and a particle size range of 300-500 nm) and disperse them in 90 mL of 50 mM phosphate buffer solution (pH 8.0); dissolve 1 g of PEI in water to prepare a 100 mg / mL solution; add the PEI solution dropwise to the solution containing epoxy magnetic spheres while stirring at room temperature, and stop the reaction after stirring for 15 hours. Wash the magnetic spheres with water 3 times to obtain amino-modified magnetic spheres for the next reaction.
[0032] 2. 0.15 mol of succinyl succinimide ester was dissolved in 90 mL of a mixture containing 5% 50 mM phosphate buffer (pH 8.0, v / v), 45% methanol (v / v), and 50% DMSO (v / v). 0.1 g of amino-modified magnetic beads were fully dispersed in 10 mL of 50 mM phosphate buffer (pH 8.0). 90 mL of the succinyl succinimide ester solution was added while stirring at room temperature. The reaction was stopped after stirring for 0.5 hours. The magnetic beads were washed three times with the mixture containing the dissolved succinyl succinimide ester and twice with 50 mM phosphate buffer (pH 8.0) to obtain the active ester-modified magnetic beads for the next reaction.
[0033] 3. 0.3 mol of ACA prepared in Example 1 was dissolved in 90 mL of a mixture containing 10% 50 mM phosphate buffer (pH 8.0, v / v) and 90% methanol (v / v). 0.1 g of the magnetic beads from the previous reaction were fully dispersed in 10 mL of 50 mM phosphate buffer (pH 8.0). 90 mL of ACA solution was added while stirring at room temperature. The reaction was stopped after stirring for 0.5 hours. The magnetic beads were washed three times with the mixture containing dissolved ACA and twice with 50 mM phosphate buffer (pH 8.0) to obtain amino-modified magnetic beads for further reaction.
[0034] 4. Dissolve 0.1 g of iodoacetic acid-N-succinamide ester (NIS) in 10 mL of a mixture containing 10% 50 mM phosphate buffer (pH 8.0, v / v) and 90% methanol (v / v); after the previous reaction, dissolve 0.1 g of magnetic beads in 90 mL of 50 mM phosphate buffer (pH 8.0), add 10 mL of NIS solution while stirring at room temperature, and stir and react for 15 hours in the dark to stop the reaction. Wash the magnetic beads 3 times with the NIS-dissolved mixture and 2 times with 50 mM phosphate buffer (pH 8.0) to obtain fracture-resistant solid-phase alkylation material 1.
[0035] Example 3
[0036] Synthesis of fracture-resistant solid-phase alkylation materials via epoxy silica sphere matrix and glutaraldehyde pathway
[0037] 1. 0.1 g of epoxy-modified silica spheres were synthesized in-house (preparation method referred to Example 1 of our research team's authorized patent: ZL201510531298.3) and dispersed in 90 mL of 50 mM phosphate buffer solution (pH 8.0); 1 g of PEI was prepared into a 100 mg / mL solution with water; the PEI solution was added dropwise to the solution containing the epoxy-modified silica spheres while stirring at room temperature. After stirring for 15 hours, the reaction was stopped. The silica spheres were washed with water 3 times to obtain amino-modified silica spheres for the next reaction.
[0038] 2. A commercially available 50% glutaraldehyde solution (v / v) was diluted to a 10% solution (v / v) with 50 mM phosphate buffer (pH 8.0). After the previous reaction, 0.1 g of silica spheres were fully dispersed in 90 mL of 50 mM phosphate buffer (pH 8.0). 10 mL of 10% glutaraldehyde solution was added dropwise while stirring at room temperature. The reaction was stopped after stirring for 3 hours. The silica spheres were washed 3 times each with 50 mM phosphate buffer (pH 8.0) and methanol alternately to obtain active ester-modified silica spheres for the next reaction.
[0039] 3. 0.3 mol of ACA prepared in Example 1 was dissolved in 10 mL of 50 mM phosphate buffer solution (pH 8.0); 0.1 g of the silicon spheres after the previous reaction were fully dispersed in 90 mL of 50 mM phosphate buffer solution (pH 8.0), and 10 mL of ACA solution was added under stirring at room temperature. The reaction was stopped after stirring for 3 hours. The silicon spheres were washed 3 times with 50 mM phosphate buffer solution (pH 8.0) to obtain amino-modified silicon spheres that can be broken down, which were prepared for the next reaction.
[0040] 4. Dissolve 0.1 g of iodoacetic acid-N-succinamide ester (NIS) in 10 mL of a mixture containing 10% 50 mM phosphate buffer (pH 8.0, v / v) and 90% methanol (v / v); after the reaction in the previous step, disperse 0.1 g of silicon spheres in 90 mL of 50 mM phosphate buffer (pH 8.0), add 10 mL of NIS solution while stirring at room temperature, and stir and react for 15 hours in the dark to stop the reaction. Wash the silicon spheres 3 times with the NIS-dissolved mixture and 2 times with 50 mM phosphate buffer (pH 8.0) to obtain fracture-resistant solid-phase alkylation material 2.
[0041] Example 4
[0042] Synthesis of fracture-resistant solid-phase alkylation materials using an optimized pathway with epoxy-based magnetic sphere matrix and glutaraldehyde: 1. Purchase 1g of commercially available epoxy-based magnetic spheres (Suzhou Beaver, catalog number P07990027, which are superparamagnetic particles with epoxy groups on their surface and a particle size range of 300-500nm) and disperse them in 450mL of 20mM phosphate buffer solution (pH 8.0); dissolve 5g of PEI in water to prepare a 100mg / mL solution; add the PEI solution dropwise to the solution containing the epoxy magnetic spheres while stirring at room temperature, and stop the reaction after stirring for 24 hours. Wash the magnetic spheres with water 3 times to obtain amino-modified magnetic spheres for the next reaction.
[0043] 2. A commercially available 50% glutaraldehyde solution (v / v) was diluted to a 10% solution (v / v) with 20 mM phosphate buffer (pH 8.0); sodium cyanoborohydride was prepared to a 10 mg / mL solution with 20 mM phosphate buffer (pH 8.0); 1 g of silica spheres from the previous reaction were thoroughly dispersed in 300 mL of 20 mM phosphate buffer (pH 8.0), and 100 mL of 10% glutaraldehyde solution and 100 mL of sodium cyanoborohydride solution were added dropwise with stirring at room temperature. The reaction was stopped after stirring for 1 hour. The silica spheres were washed three times each with alternating 20 mM phosphate buffer (pH 8.0) and methanol to obtain active ester-modified silica spheres for the next reaction.
[0044] 3. The 3 mol of ACA prepared in Example 1 was dissolved in 100 mL of 25 mM phosphate buffer solution (pH 8.0); sodium cyanoborohydride was prepared into a 10 mg / mL solution using 20 mM phosphate buffer solution (pH 8.0); 1 g of silicon spheres after the previous reaction were fully dispersed in 300 mL of 20 mM phosphate buffer solution (pH 8.0), and 100 mL of ACA solution and 100 mL of sodium cyanoborohydride solution were added while stirring at room temperature. The reaction was stopped after stirring for 1 hour. The silicon spheres were washed 3 times with 20 mM phosphate buffer solution (pH 8.0) to obtain amino-modified silicon spheres that can be broken down, which were prepared for the next reaction.
[0045] 4. Dissolve 0.5 g of iodoacetic acid-N-succinamide ester (NIS) in 50 mL of a mixture containing 10% 20 mM phosphate buffer (pH 8.0, v / v) and 90% methanol (v / v); after the reaction in the previous step, disperse 1 g of silicon spheres in 450 mL of 20 mM phosphate buffer (pH 8.0), add 50 mL of NIS solution while stirring at room temperature, and stop the reaction after stirring in the dark for 24 hours. Wash the silicon spheres 3 times with the mixture containing NIS and 2 times with 20 mM phosphate buffer (pH 8.0) to obtain fracture-resistant solid-phase alkylation material 3.
[0046] Example 5
[0047] The application of cleavable alkylation materials was investigated using bovine serum albumin (BSA) as the target.
[0048] 1. Prepare a 1 mg / mL BSA solution using a 50 mM ammonium bicarbonate solution;
[0049] 2. Add 100 μL of the above BSA solution to a 1.5 mL EP tube, the BSA content is 100 μg; 3. Add 1 μL of TCEP (prepared with 1 mol, 50 mM ammonium bicarbonate solution);
[0050] 4. 95℃, shake for 10 minutes, shaking speed 1500 rpm;
[0051] 5. Add 100 μL of the material 3 solution prepared in Example 4 (containing 1 mg of material 3 and 50 mM ammonium bicarbonate solution);
[0052] 6. Shake at 40℃, away from light, for 4 hours at a shaking speed of 1500 rpm;
[0053] 7. Magnetic removal of supernatant;
[0054] 8. Wash the material 3 prepared in Example 4 once with 20mM ammonium bicarbonate solution, 200ul / time, and remove the supernatant by magnetic adsorption after shaking or blowing.
[0055] 9. Wash the material prepared in Example 4 once with 50% methanol (prepared with 50mM phosphate buffer solution (pH 8.0, v / v), 200ul / time, and remove the supernatant by shaking or blowing.
[0056] Wash the material prepared in Example 4 once with 20mM ammonium bicarbonate solution, 200ul / time, and remove the supernatant by magnetic adsorption after shaking or blowing.
[0057] 11. Add 100 μL of 0.1% formic acid solution (pH 3) (v / v), shake at room temperature for 30 min at a shaking speed of 1500 rpm;
[0058] The supernatant was collected by magnetic absorption and subjected to 12.5% gel electrophoresis. The electrophoresis results are attached. Figure 3 The arrow indicates the BSA released under acidic conditions after the alkylated material is captured.
Claims
1. A process for the preparation of a rupturable solid phase alkylated material, characterized by: Using epoxy-based microspheres as a carrier, hydrophilic compound polyethyleneimine (PEI), acid-breakable linker arms, and iodoacetic acid-N-succinamide ester (NIS) were sequentially modified via covalent bonding to prepare a breakable solid-phase alkylated material. Specifically, the following steps are included: (1) The epoxy-based microsphere carrier is mixed with PEI and reacted to introduce hydrophilic active reactive groups on the surface of the microspheres to prepare amino-modified microspheres; (2) The amino-modified microspheres obtained in step (1) are reacted with succinimide succinimide or glutaraldehyde to obtain modified microspheres; (3) The modified microspheres prepared in step (2) are reacted with the compound of the acid-severable linker arm to obtain acid-severable amino-modified microspheres; (4) Acid-crackable amino-modified microspheres were reacted with iodoacetic acid-N-succinamide ester (NIS) under light-protected stirring to prepare an acid-crackable solid-phase alkylated material. The reaction system in step (3) uses the following amounts: 0.1-100 g of the modified microspheres prepared in step (2), 0.2-200 mol of the acid-cleavable linker compound ACA, and a reaction solution consisting of a mixture of 45-90% methanol, 5-10% pH 8.0 50 mM phosphate buffer, and 0-50% DMSO, with a volume of 0.1-100 L. The reaction time is 0.5-6 hours. The structural formula of the acid-severable linker compound ACA is: ， Where n is an integer from 1 to 20.
2. The method for preparing the fractured solid-phase alkylation material according to claim 1, characterized in that: In step (1), 0.1-100 g of epoxy microspheres are dispersed in phosphate buffer solution, and 1-1000 g of PEI is added to 0.1-100 L of water to prepare an aqueous solution. The two solutions are mixed and stirred for 6-24 hours. The epoxy microspheres are either epoxy magnetic spheres or epoxy silica microspheres.
3. The method for preparing the fractured solid-phase alkylation material according to claim 1, characterized in that, The reaction system in step (2) is as follows: 0.1-100 g of amino-modified microspheres, 0.1-100 mol of succinyl succinimide ester or glutaraldehyde, and a mixed solution of 45-90% methanol, 5-10% 50 mM phosphate buffer at pH 8.0, and 0-50% dimethyl sulfoxide, with a volume of 0.1-100 L. The reaction time is 0.5-6 hours.
4. The method for preparing the fractured solid-phase alkylation material according to claim 1, characterized in that: The reaction system in step (4) is as follows: 0.1-100 g of amino-modified microspheres and 0.1-100 g of NIS. The reaction solution system is a mixed solution of 45-90% methanol, 5-10% pH 8.0 50 mM phosphate buffer, and 0-50% DMSO, with a volume ratio of 0.1-100 L. The reaction is carried out by stirring in the dark for 6-24 hours.
5. A fractured solid-phase alkylated material prepared by the preparation method according to any one of claims 1-4, characterized in that: The acid-breakable connecting arm of this material can break under acidic conditions with a pH < 7.
6. An application of the fractured solid-phase alkylation material according to claim 5, characterized in that: After the fractured solid-phase alkylating material reacts with thiol proteins in biological samples, it can release thiol proteins or thiol peptides under acidic conditions, i.e., pH < 7. The biological samples are selected from one or more of the following: cells, tissues, body fluids, hair, or other protein-rich samples.