Fracturable solid-phase alkylation material and preparation and application thereof

Abstract

The invention relates to a breakable solid-phase alkylation material as well as preparation and application thereof, epoxy microspheres are taken as a carrier, polyethyleneimine, an acid breakable connecting arm and iodoacetic acid-N-succinamide ester are sequentially modified in a covalent bonding manner, and the breakable solid-phase alkylation material is prepared. The material selectively reacts with sulfydryl on protein, efficient enrichment and treatment of protein in complex biological samples such as cells, tissues and body fluid can be achieved, lossless release of protein and peptide fragments can be achieved by breaking connecting arms under the acidic condition, and important technical support is expected to be provided for proteome deep coverage analysis and protein variant analysis.

Description

Technical Field

[0001] The invention relates to a cleavable solid phase alkylation material, which can be applied to the selective reaction and pretreatment of thiol proteins in cells, tissues, body fluids, hair or other samples rich in proteins. Background Art

[0002] Clinical sample proteomic research is of great significance for the discovery of potential biomarkers, screening of drug targets, early diagnosis and treatment of diseases, and prognosis judgment. Samples such as plasma, urine, tissue sections, and circulating tumor cells have been widely used in clinical testing. Most clinical samples have the characteristics of small sample volume, complex matrix, and many interferences. Therefore, the development of efficient sample preparation methods is a prerequisite for achieving high-throughput, high-precision, and deep-coverage clinical proteomic analysis.

[0003] In order to solve the problems of low recovery rate, poor anti-matrix interference ability and low throughput in the preparation of clinical proteome samples, which lead to low analytical coverage, the inventors used the principle of atom transfer radical polymerization to prepare solid phase alkylation (SPA) materials, and used the covalent interaction between iodine on the surface of the material and the thiol group on the cysteine ​​exposed after protein denaturation and reduction to achieve protein capture on the surface of the material. This invention significantly improves the preparation throughput of proteome samples and has strong anti-interference ability. This invention has been granted patents: Preparation of protein solid phase alkylation reagents and solid phase alkylation reagents and applications (ZL201510531298.3).

[0004] In the process of preparing proteome samples using SPA materials, we found that by collecting the supernatant after enzymatic hydrolysis, most of the peptides after enzymatic digestion were collected, while the peptides containing thiol groups were not collected and detected because they were bound to the SPA material, resulting in the loss of this part of information. Based on this, we invented an acid-cleavable solid-phase alkylation material. The protein thiol group can be covalently bound to the material. After sample treatment, the linker arm is broken by adding an acidic solution to the reaction system, and the protein or thiol peptide segment is released without loss. Compared with the previous solid-phase alkylation material, the new material increases the enrichment of thiol-containing peptide segments and further improves the coverage of detected proteins; at the same time, it can be applied to TOP-DOWN studies on intact thiol proteins. Summary of the invention

[0005] The purpose of the present invention is to provide a solid phase alkylation material that can be used for protein pretreatment. This material can not only achieve high recovery rate capture of proteins, achieve rapid separation from other small molecules, and reduce the complexity of protein samples; at the same time, because the amino linker of the compound with acid-cleavable amino linkers on both sides (ACA) can be broken under acidic conditions, the subsequent treatment method can adopt a BOTTOM-UP research method of collecting peptides after enzymatic hydrolysis for mass spectrometry analysis (without losing thiol-containing peptides), or a TOP-DOWN study can be performed on intact thiol-containing proteins. In order to achieve this purpose, the technical solution of the present invention is:

[0006] 1) Epoxy microspheres (silica gel or magnetic sphere matrix) are sequentially modified with polyethyleneimine (PEI), ACA and iodoacetic acid-N-succinamide ester (NIS) by covalent bonding;

[0007] A) bonding hydrophilic active groups to the surface of epoxy microspheres, the process is as follows: 0.1-100 g of epoxy microspheres are dispersed in a phosphate buffer solution, 0.1-100 L of a PEI aqueous solution containing 1-1000 g of PEI is added, and the mixture is stirred for reaction for 6-24 hours, with a total reaction system of 0.1-100 L, to obtain amino-modified microspheres;

[0008] B) 0.1-100 g of the amino-modified microspheres obtained in the previous step are dispersed in a phosphate buffer solution, and 0.1-100 mol of the intermediate compound is added, and the intermediate compound is dissolved in methanol and / or dimethyl sulfoxide (DMSO) solution. The reaction is stirred for 0.5-6 hours. The reaction system is 45-90% methanol (v / v), 5-10% 50 mM phosphate buffer (pH 8.0, v / v), 0-50% DMSO (v / v), and the volume is 0.1-100 L to obtain active ester-modified microspheres.

[0009] C): 0.1-100 g of the active ester modified microspheres obtained in the previous step are dispersed in a phosphate buffer solution, 0.2-200 mol of ACA is added, ACA is dissolved in methanol or phosphate buffer or DMSO, and the reaction is stirred for 0.5-6 hours. The reaction solution system is 45-90% methanol (v / v), 5-10% 50 mM phosphate buffer (pH 8.0, v / v), 0-50% DMSO (v / v), and the volume is 0.1-100 L to obtain acid-cleavable amino-modified microspheres.

[0010] D) 0.1-100 g of the acid-cleavable amino-modified microspheres obtained in the above reaction are dispersed in a phosphate buffer solution, 0.1-100 g of iodoacetic acid-N-succinamide ester is added, and NIS is dissolved in a mixed system of methanol and phosphate buffer solution. The mixture is stirred and reacted for 6-24 hours in the dark. The reaction solution system is 50-90% methanol (v / v), 10-50% 50 mM phosphate buffer (pH 8.0, v / v), and the volume is 0.1-100 L to obtain a solid-phase alkylation material that can be cleaved under acidic conditions.

[0011] E) The epoxy microspheres described in A) above can be epoxy microspheres based on silica gel or epoxy microspheres based on magnetic ferroferric oxide. The intermediate compound described in B) above can be succinyl succinimide ester, glutaraldehyde or any other compound that can provide an active ester group. The ACA described in C) above can be any compound with amino groups on both sides, can provide a bifunctional linker, and can break under acidic conditions, and its structural formula is as follows:

[0012] Wherein n is an integer from 1 to 20.

[0013] 2) Applying the cleavable solid phase alkylation material to the selective reaction 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 the in situ pretreatment of proteins in trace tissues or cells (including alkylation, removal of small molecule interfering substances, and particle enzymatic hydrolysis);

[0016] 2. On the basis of the original high-throughput and anti-interference advantages of SPA materials, because of its characteristic of breaking under acidic pH conditions in the reaction environment, it increases the enrichment of thiol-containing peptides and further expands the coverage of detected proteins;

[0017] 3. After the SPA material we developed earlier reacts with protein, the protein can only be hydrolyzed into peptides by enzymatic hydrolysis to be released from the SPA material, and then the peptides are collected for mass spectrometry analysis, which is a BOTTOM-UP research method. After the cleavable SPA material involved in the present invention reacts with protein, the connecting arm breaks under acidic conditions to release the protein. Subsequently, it can be hydrolyzed and the peptides can be collected for BOTTOM-UP research, or the released protein can be directly analyzed, which is a TOP-DOWN study. Users can flexibly design experiments according to research needs, which greatly expands the application scenarios of solid-phase alkylation materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 , the structural formula of the compound with acid-cleavable amino linking arms on both sides (ACA).

[0019] Figure 2 , 2 ,2'-(propane-2,2-diylbis(oxy))bis(ethane-1-amine) H NMR spectrum and C NMR spectrum.

[0020] Figure 3 , solid phase alkylation reagent materials and application process diagram.

[0021] Figure 4 , using BSA as a model protein to investigate the application of cleavable solid-phase alkylation materials. DETAILED DESCRIPTION

[0022] The following examples are provided to illustrate but not to limit the above patent.

[0023] Example 1

[0024] The synthetic route of 2,2'-(propane-2,2-diylbis(oxy))bis(ethane-1-amine) (ACA) is:

[0025]

[0026] The synthesis process is as follows:

[0027] 1. Dissolve compound 1 (2-(2-hydroxyethyl)isoindoline-1,3-dione, 20.0 g, 105 mmol, 2.0 eq) in anhydrous toluene (200 mL), then add 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), and reflux for 4 h after addition. After the reaction, cool to room temperature, concentrate under reduced pressure and pass through a column (PE:EA=4:1), to obtain 9.9 g of compound 3 (Y=45%).

[0028] 2. Compound 3 obtained in step 1 was dissolved in 6N NaOH (100 mL) and refluxed for 8 h. After cooling to room temperature, it was extracted with DCM / MeOH (10:1), the organic phase was dried under reduced pressure, and the crude product was passed through a column (DCM:MeOH = 20:1) to obtain 2.2 g of compound 4, namely 2,2'-(propane-2,2-diylbis(oxy))bis(ethane-1-amine) (Y = 59%).

[0029] 3. The H NMR and C NMR spectra of the finished product ACA [2,2'-(propane-2,2-diylbis(oxy))bis(ethane-1-amine)] are attached. Figure 2 .

[0030] Example 2

[0031] Synthesis of cleavable solid-phase alkylation materials via epoxy magnetic sphere matrix and succinyl succinimide ester pathway 1. Purchase 0.1 g of commercial epoxy magnetic spheres (Suzhou Beaver BEAVER, item number P07990027, 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); prepare 1 g of PEI with water to make a 100 mg / mL solution; add the PEI solution dropwise to the solution containing epoxy magnetic spheres at room temperature while stirring. After stirring for 15 hours, terminate the reaction, 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 mixed system containing 5% 50 mM phosphate buffer solution (pH 8.0, v / v), 45% methanol (v / v), and 50% DMSO (v / v); 0.1 g of amino-modified magnetic spheres was fully dispersed in 10 mL 50 mM phosphate buffer solution (pH 8.0), and 90 mL of succinyl succinimide ester solution was added under stirring at room temperature. The reaction was terminated after stirring for 0.5 hours, and the magnetic spheres were washed 3 times with the mixed system dissolving succinyl succinimide ester and washed 2 times with 50 mM phosphate buffer solution (pH 8.0) to obtain active ester-modified magnetic spheres for the next reaction.

[0033] 3. 0.3 mol of ACA prepared in Example 1 was dissolved in 90 mL of a mixed system containing 10% 50 mM phosphate buffer solution (pH 8.0, v / v) and 90% methanol (v / v); 0.1 g of the magnetic spheres after the previous reaction were fully dispersed in 10 mL of 50 mM phosphate buffer solution (pH 8.0), and 90 mL of ACA solution was added under stirring at room temperature. The reaction was terminated after stirring for 0.5 hour, and the magnetic spheres were washed 3 times with the mixed system in which ACA was dissolved and washed 2 times with 50 mM phosphate buffer solution (pH 8.0) to obtain cleavable amino-modified magnetic spheres for the next reaction.

[0034] 4. 0.1 g of iodoacetic acid-N-succinamide ester (NIS) was dissolved in 10 mL of a mixed system containing 10% 50 mM phosphate buffer solution (pH 8.0, v / v) and 90% methanol (v / v). 0.1 g of the magnetic sphere after the previous reaction was fully dispersed in 90 mL of 50 mM phosphate buffer solution (pH 8.0), and 10 mL of NIS solution was added under stirring at room temperature. The reaction was stirred in the dark for 15 hours and then terminated. The magnetic sphere was washed 3 times with the mixed system containing dissolved NIS and washed 2 times with 50 mM phosphate buffer solution (pH 8.0) to obtain the cleavable solid phase alkylation material 1.

[0035] Example 3

[0036] Synthesis of cleavable solid-phase alkylation materials using epoxy-silica sphere matrix and glutaraldehyde route

[0037] 1. 0.1 g of epoxy silica spheres were synthesized by ourselves (for the preparation method, refer to the patent authorized by our research team: Example 1 of 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 epoxy silica spheres at room temperature while stirring, and the reaction was terminated after stirring for 15 hours. The silica spheres were washed with water for 3 times to obtain amino-modified silica spheres for the next reaction.

[0038] 2. Commercial 50% glutaraldehyde solution (v / v) was diluted with 50mM phosphate buffer solution (pH 8.0) to a 10% solution (v / v); 0.1g of silica spheres after the previous reaction were fully dispersed in 90mL 50mM phosphate buffer solution (pH 8.0), and 10mL 10% glutaraldehyde solution was added dropwise under stirring at room temperature. The reaction was terminated after stirring for 3 hours, and the silica spheres were washed alternately with 50mM phosphate buffer solution (pH8.0) and methanol for 3 times each 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 silica 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 terminated after stirring for 3 hours, and the silica spheres were washed 3 times with 50 mM phosphate buffer solution (pH 8.0) to obtain cleavable amino-modified silica spheres for the next reaction.

[0040] 4. 0.1 g of iodoacetic acid-N-succinamide ester (NIS) was dissolved in 10 mL of a mixed system containing 10% 50 mM phosphate buffer solution (pH 8.0, v / v) and 90% methanol (v / v). 0.1 g of the silica spheres after the previous reaction were fully dispersed in 90 mL of 50 mM phosphate buffer solution (pH 8.0), and 10 mL of NIS solution was added under stirring at room temperature. The reaction was stirred for 15 hours in a dark place to terminate the reaction. The silica spheres were washed 3 times with the mixed system for dissolving NIS and 2 times with 50 mM phosphate buffer solution (pH 8.0) to obtain the cleavable solid phase alkylation material 2.

[0041] Example 4

[0042] Synthesis of breakable solid-phase alkylation materials using epoxy magnetic sphere matrix and glutaraldehyde optimized pathway 1. Purchase 1 g of commercial epoxy magnetic spheres (Suzhou Beaver BEAVER, item number P07990027, superparamagnetic particles with epoxy groups on the surface and a particle size range of 300-500 nm) and disperse them in 450 mL of 20 mM phosphate buffer solution (pH 8.0); prepare 5 g of PEI with water to make a 100 mg / mL solution; add the PEI solution dropwise to the solution containing epoxy magnetic spheres at room temperature while stirring. After stirring for 24 hours, terminate the reaction, wash the magnetic spheres with water 3 times to obtain amino-modified magnetic spheres for the next reaction.

[0043] 2. Commercial 50% glutaraldehyde solution (v / v) was diluted with 20 mM phosphate buffer solution (pH 8.0) to a 10% solution (v / v); sodium cyanoborohydride was prepared into a 10 mg / mL solution with 20 mM phosphate buffer solution (pH 8.0); 1 g of silica spheres after the previous reaction were fully dispersed in 300 mL 20 mM phosphate buffer solution (pH 8.0), and 100 mL 10% glutaraldehyde solution and 100 mL sodium cyanoborohydride solution were added dropwise under stirring at room temperature. The reaction was terminated after stirring for 1 hour, and the silica spheres were washed alternately with 20 mM phosphate buffer solution (pH 8.0) and methanol for 3 times each to obtain active ester-modified silica spheres for the next reaction.

[0044] 3. 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 with 20 mM phosphate buffer solution (pH 8.0); 1 g of silica 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 under stirring at room temperature. The reaction was terminated after stirring for 1 hour, and the silica spheres were washed 3 times with 20 mM phosphate buffer solution (pH 8.0) to obtain cleavable amino-modified silica spheres for the next reaction.

[0045] 4. 0.5 g of iodoacetic acid-N-succinamide ester (NIS) was dissolved in 50 mL of a mixed system containing 10% 20 mM phosphate buffer solution (pH 8.0, v / v) and 90% methanol (v / v). 1 g of silica spheres after the previous reaction was fully dispersed in 450 mL of 20 mM phosphate buffer solution (pH 8.0), and 50 mL of NIS solution was added under stirring at room temperature. The reaction was stirred in the dark for 24 hours and then terminated. The silica spheres were washed 3 times with the mixed system for dissolving NIS and 2 times with 20 mM phosphate buffer solution (pH 8.0) to obtain the cleavable solid phase alkylation material 3.

[0046] Example 5

[0047] Study on the application of cleavable alkylation materials using bovine serum albumin (BSA) as the object

[0048] 1. Prepare 1 mg / mL BSA solution with 50 mM ammonium bicarbonate solution;

[0049] 2. Take 100 μL of the above BSA solution and add it to a 1.5 mL EP tube. The BSA content is 100 μg. 3. Add 1 μL TCEP (1 mol, prepared with 50 mM ammonium bicarbonate solution).

[0050] 4. 95°C, shake for 10 min, shaking speed 1500 rpm;

[0051] 5. Add 100 μL of the material 3 solution prepared in Example 4 (containing 1 mg of material 3, prepared in 50 mM ammonium bicarbonate solution);

[0052] 6. 40℃, dark-proof, shake for 4h, shaking speed 1500rpm;

[0053] 7. Remove the supernatant by magnetic suction;

[0054] 8. Wash the material 3 prepared in Example 4 once with 20 mM ammonium bicarbonate solution, 200 ul / time, shake or pipette, and remove the supernatant by magnetic aspiration;

[0055] 9. Wash the material 3 prepared in Example 4 once with 50% methanol (prepared with 50 mM phosphate buffer solution (pH 8.0), v / v), 200 ul / time, shake or pipette, and remove the supernatant by magnetic aspiration;

[0056] 10. Wash the material 3 prepared in Example 4 once with 20 mM ammonium bicarbonate solution, 200 ul / time, shake or pipette, and then remove the supernatant by magnetic aspiration;

[0057] 11. Add 100 μL of 0.1% formic acid (pH 3) solution (v / v), shake at room temperature for 30 min at a shaking speed of 1500 rpm;

[0058] Collect the supernatant by magnetic absorption, and perform 12.5% ​​gel electrophoresis. The electrophoresis results are attached. Figure 3 The arrow shows BSA released under acidic conditions after being captured by the alkylated material.

Claims

1. A method for preparing a cleavable solid phase alkylation material, characterized in that: Using epoxy microspheres as carriers, hydrophilic compound polyethyleneimine (PEI), acid-cleavable linker and iodoacetic acid-N-succinamide (NIS) were sequentially modified by covalent bonding to prepare a cleavable solid-phase alkylation material.

2. The method for preparing a cleavable solid phase alkylation material according to claim 1, characterized in that: The specific steps include: (1) The epoxy microsphere carrier is mixed with PEI to introduce hydrophilic reactive groups on the surface of the microspheres to obtain amino-modified microspheres; (2) subjecting the amino-modified microspheres obtained in step (1) to an esterification reaction with succinyl succinimide ester or glutaraldehyde to obtain active ester-modified microspheres; (3) reacting the active ester modified microspheres with a compound having acid-cleavable amino linker arms on both sides to prepare acid-cleavable amino modified microspheres; (4) The acid-cleavable amino-modified microspheres were reacted with iodoacetic acid-N-succinamide ester (NIS) in the dark with stirring to obtain a solid-phase alkylated material that can be cleaved under acidic conditions.

3. The method for preparing a cleavable solid phase alkylation material according to claim 2, characterized in that: In step (1), 0.1-100 g of epoxy microspheres are dispersed in a phosphate buffer solution, 1-1000 g of PEI is added to 0.1-100 L of water to form an aqueous solution, and the two solutions are mixed and stirred for reaction for 6-24 hours. The epoxy microspheres are one of epoxy magnetic microspheres or epoxy silica microspheres.

4. The method for preparing a cleavable solid phase alkylation material according to claim 2, characterized in that: The reaction system in step (2) is: 0.1-100 g of amino-modified microsphere particles, 0.1-100 mol of succinyl succinimide ester or glutaraldehyde, and the reaction solvent is a mixed solution of 45-90% methanol (v / v), 5-10% 50 mM phosphate buffer (pH 8.0, v / v), and 0-50% dimethyl sulfoxide (DMSO, v / v), with a volume of 0.1-100 L and a stirring reaction time of 0.5-6 hours.

5. The method for preparing a cleavable solid phase alkylation material according to claim 2, characterized in that: The amount of the reaction system in step (3) is: 0.1-100 g of active ester modified microspheres, 0.2-200 mol of a compound with acid-cleavable amino linking arms on both sides (ACA for short), a reaction solution system of 45-90% methanol (v / v), 5-10% 50 mM phosphate buffer (pH 8.0, v / v), and 0-50% DMSO (v / v) mixed solution, the volume is 0.1-100 L, and the reaction time is 0.5-6 hours.

6. The method for preparing a cleavable solid phase alkylation material according to claim 2, characterized in that: The reaction system in step (4) is: 0.1-100 g of cleavable amino-modified microspheres, 0.1-100 g of NIS, and a reaction solution system of 45-90% methanol (v / v), 5-10% 50 mM phosphate buffer (pH 8.0, v / v), and 0-50% DMSO (v / v) mixed solution, with a volume of 0.1-100 L, and the reaction is stirred in the dark for 6-24 hours.

7. The method for preparing a cleavable solid phase alkylation material according to claim 5, characterized in that: The structural formula of the compound with acid-cleavable amino linker arms on both sides (ACA) is: Wherein n is an integer from 1 to 20.

8. A cleavable solid-phase alkylation material prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The amino connecting arm of the material can be broken under acidic conditions of pH <7.

9. An application of the cleavable solid phase alkylation material according to claim 8, characterized in that: After the cleavable solid phase alkylation material reacts with the thiol protein in the biological sample, the thiol protein or thiol peptide can be released under acidic conditions (pH value <7). The biological sample is selected from one or more of cells, tissues, body fluids, hair or other protein-rich samples.

10. The use according to claim 8, characterized in that: The cleavable solid phase alkylation material can be applied to the selective reaction or pretreatment of thiol-containing proteins in cells, tissues, body fluids, hair or other protein-rich samples.

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