WO2022213656A1 - 功能化磁珠及采用其的生物正交化学的大分子一步捕获方法 - Google Patents

功能化磁珠及采用其的生物正交化学的大分子一步捕获方法 Download PDF

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WO2022213656A1
WO2022213656A1 PCT/CN2021/138134 CN2021138134W WO2022213656A1 WO 2022213656 A1 WO2022213656 A1 WO 2022213656A1 CN 2021138134 W CN2021138134 W CN 2021138134W WO 2022213656 A1 WO2022213656 A1 WO 2022213656A1
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magnetic beads
magnetic
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magnetic bead
aqueous solution
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李楠
王蕾
王维杰
赵文娟
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Shenzhen Institute of Advanced Technology of CAS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads

Definitions

  • the invention relates to the technical field of biological macromolecule capture, in particular to a functionalized magnetic bead and a bioorthogonal chemistry macromolecule one-step capture method using the same.
  • the intracellular signaling and intercellular communication of each cell mainly depend on the function of proteins and their specific interactions. Therefore, the study of protein interactions is the key to deciphering the life activities of cells.
  • the interacting proteins are usually low in abundance, and the content is less than 1%. It will be a huge challenge to deeply identify the entire proteome of the cell.
  • Even today's state-of-the-art mass spectrometers cannot achieve complete identification of low-abundance proteins due to limited detection speed and dynamic detection range. If temporally and spatially characterized subproteomes are to be identified, this will be a more difficult task, and the key point is subproteome isolation and enrichment.
  • BONCAT bioorthogonal non-natural amino acid tagging technology
  • Orthogonal unnatural amino acids can be used to label, identify and enrich newly synthesized protein subsets, and can also combine with subcellular separation technology to obtain the information of newly synthesized proteins and their interacting proteins in the spatial distribution of proteins.
  • RNA and protein In the process of regulating life activities, in addition to the interaction between proteins, RNA and protein also interact to regulate each other's functions. The interaction between RNA and protein is very important for maintaining cell homeostasis, and Interfering with the interaction between the two can lead to cell dysfunction and related diseases. Therefore, studying and characterizing RNA interaction networks can help scientists solve specific research questions.
  • the latest research method is also applied to bioorthogonal non-natural nucleotide labeling technology, which uses non-natural nucleotides to be embedded in newly transcribed RNA during DNA transcription, which can be used to label newly transcribed RNA or newly transcribed RNA- Protein complexes are labeled, identified, and enriched, followed by RNA sequencing and mass spectrometry analysis to identify and obtain information about RNA and its interacting proteins.
  • Click chemistry reaction is a chemical synthesis concept introduced by chemist K.B. Sharpless in 2001, and its representative reaction is azide alkyne cycloaddition reaction.
  • azide (azide) group The desired functional group is linked by the reaction of the group with an alkyne to form a triazole ring.
  • Affinity purification technology By adding tags to proteins, such as: His, GST, HA and FLAG, etc., using the specific affinity between tags and their specific ligands, the tagged proteins are subjected to specific ligand-modified solid-phase support. capture, so as to achieve the purpose of separation and purification.
  • Biotin and streptavidin can be specifically combined, and the labeled newly synthesized protein can be analyzed by Western blot or affinity purification using a solid-phase carrier containing streptavidin, and the protein information can be identified by mass spectrometry.
  • Document 2 “Capturing the interactome of newly transcribed RNA” (DOI: 10.1038/nmeth.4595) discloses a method for capturing bioorthogonal non-natural nucleotide-labeled newly transcribed RNA interactomes, as shown in Figure 2, using The alkynyl uracil analog EU (5-ethynyluridine) labels newly transcribed RNAs in cell culture, biotinylates EU-labeled RNAs by click reaction, and immobilizes them by streptavidin. Affinity purification of biotinylated RNA by Streptavidin-coated beads, RNA sequencing and proteome mass spectrometry identification, the RNA and its interactome information can be obtained.
  • EU alkynyl uracil analog EU
  • an unnatural amino acid is used to replace the corresponding natural amino acid.
  • Amino acids with alkynyl or azide groups are analogs of natural amino acids. They are used as natural amino acids in the process of protein synthesis and are embedded into newly generated proteins, so that the new proteins have alkynyl or azide groups.
  • Molecular probes with azide groups or alkynyl groups can be linked to these proteins through click chemistry reactions.
  • the reporter group at the end of the molecular probes contains biotin, which can specifically interact with the corresponding streptavidin.
  • Document 3 "Selective enrichment of newly synthesized proteins for quantitative secretome analysis” (DOI: 10.1038/nbt.2356) discloses a resin containing an alkynyl group, by adding an azide unnatural amino acid to a biological macromolecule, an azide group The reaction between the alkynyl group and the alkynyl group of the resin does not involve antigen-antibody reaction, which avoids the production of non-specific binding proteins.
  • the capture of biological macromolecules can be realized by one-step method, but the reaction time of this method is as long as more than a dozen. Hour.
  • the purpose of the present invention is to provide a method for synthesizing functionalized magnetic beads and a one-step capture method for macromolecules of protein bioorthogonal chemistry, which are used for proteomics research, and the functionalized magnetic beads used are modified by unconjugated antibodies and small molecules.
  • the method does not involve antigen-antibody reaction, adopts the principle of azide alkyne cycloaddition reaction in click chemistry, avoids the production of non-specific binding proteins, and adopts a one-step method to capture protein biomacromolecules, which is more direct, efficient and background. Low, and suitable for protein capture under denaturing conditions, with wider applicability.
  • the present invention provides an alkynyl magnetic bead, which is synthesized by coupling NH 3 -PEG4-Alkyne and carboxyl magnetic beads.
  • the preparation method of the alkynylated magnetic beads is as follows:
  • the suspension of carboxyl magnetic beads is magnetically separated, and the supernatant is removed;
  • the second step add an organic solvent to the separated product, use a pipette to slowly blow for several times to resuspend the magnetic beads, magnetically separate again, remove the supernatant, repeat the washing for several times to separate the product, and resuspend the magnetic beads in the organic solvent , to obtain a magnetic bead suspension;
  • the third step configure (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) first solution for activating the carboxyl group;
  • the fourth step configure (NH 3 -PEG4-Alkyne) second solution
  • the first solution obtained in the third step is added to the magnetic bead suspension obtained in the second step;
  • the sixth step adding the second solution to the magnetic bead suspension obtained in the fifth step;
  • the mixed solution obtained in the sixth step is placed in a shaker at 37°C and shaken for incubation;
  • the eighth step magnetic separation, remove the supernatant
  • the ninth step adding ethanol for resuspending by pipetting, magnetic separation, removing the supernatant, repeating the washing of the separated product, and finally resuspending the magnetic beads in an aqueous ethanol solution;
  • the tenth step is to store at 4°C.
  • the invention provides an azide magnetic bead, which is synthesized by coupling azidopropylamine and carboxyl magnetic beads.
  • the preparation method of the azide magnetic beads is as follows:
  • the suspension of carboxyl magnetic beads is magnetically separated, and the supernatant is removed;
  • the second step add an organic solvent to the separated product, use a pipette to slowly blow for several times to resuspend the magnetic beads, magnetically separate again, remove the supernatant, repeat the washing for several times to separate the product, and resuspend the magnetic beads in the organic solvent , to obtain a magnetic bead suspension;
  • the third step configure (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) first solution for activating the carboxyl group;
  • the 4th step configure the second solution of azidopropylamine
  • the first solution obtained in the third step is added to the magnetic bead suspension obtained in the second step;
  • the sixth step adding the second solution to the magnetic bead suspension obtained in the fifth step;
  • the mixed solution obtained in the sixth step is placed in a shaker at 37°C and shaken for incubation;
  • the eighth step magnetic separation, remove the supernatant
  • the ninth step adding ethanol for resuspending by pipetting, magnetic separation, removing the supernatant, repeating the washing of the separated product, and finally resuspending the magnetic beads in an aqueous ethanol solution;
  • the tenth step is to store at 4°C.
  • the present invention also provides a bioorthogonal chemical macromolecule one-step capture method, which is obtained by using functionalized magnetic beads and biological macromolecules through click chemical reaction.
  • the functionalized magnetic beads can be azide magnetic beads or acetylenic magnetic beads prepared above.
  • the biomacromolecules can be proteins with alkynyl or azide unnatural amino acids embedded, and nucleic acids with alkynyl or azide unnatural nucleotides embedded.
  • the click chemistry reaction is an azide alkyne cycloaddition reaction.
  • the method includes:
  • the first step is to configure copper sulfate aqueous solution, sodium ascorbate aqueous solution, (tris (3-hydroxypropyl triazomethyl) amine) aqueous solution, sodium lauryl sulfate aqueous solution, 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution , urea aqueous solution, acetonitrile aqueous solution;
  • the second step is to take the magnetic bead suspension prepared above, replace it with protein or nucleic acid buffer, and resuspend it in protein buffer;
  • the third step copper sulfate aqueous solution, sodium ascorbate aqueous solution, (tris(3-hydroxypropyltriazomethyl)amine) aqueous solution, the protein sample to be captured, the magnetic bead suspension prepared in the second step, and the Ultra-pure water;
  • the fourth step after configuring the reaction mixture, put it on a constant temperature mixer and incubate;
  • the fifth step magnetic separation
  • the sixth step reductive alkylation
  • the seventh step is to wash the magnetic beads
  • the proteins on the magnetic beads were trypsinized for LC-MS analysis.
  • the existing two-step technology is to enrich the protein through antigen-antibody reaction.
  • the magnetic beads need to be used under mild conditions.
  • the magnetic beads synthesized by the present invention are small-molecule-modified magnetic beads.
  • the process of protein capture and impurity removal after capture can be carried out under strong denaturing conditions (such as SDS, urea, guanidine hydrochloride, etc.), which has a wider range of applications and can better remove non-specificity Combined, the background signal is lower;
  • the antibody-modified magnetic beads and molecular probes used in the existing two-step technology are relatively expensive, and the magnetic beads and reactants required by the present invention are inexpensive, and a large amount of synthesis can be stored and used for a long time, which reduces the experimental cost;
  • the existing reference 3 adopts a one-step method, but the alkynylated resin provided by it has poor operability, which is reflected in the need for centrifugation during solid-liquid separation, and it is easy to adhere to the wall of the centrifuge tube, and the resin is lost during the pipetting operation.
  • the solid-liquid separation is not complete, and the liquid cannot be completely removed. Therefore, more washing steps are required when washing the resin.
  • the alkynylated magnetic beads of the present invention can be used for solid-liquid separation. High efficiency, saving time and reagents;
  • the existing one-step method requires a long click chemistry reaction time (18 hours), and the present invention only needs to react for 1-2 hours, which greatly saves the experimental time.
  • the present invention provides an alkynylated magnetic bead, which is synthesized by coupling NH 3 -PEG4-Alkyne and carboxyl magnetic beads.
  • the invention also provides an azide magnetic bead, which is synthesized by coupling azidopropylamine and carboxyl magnetic beads.
  • the core part of the carboxyl magnetic beads is ferric tetroxide, and the exterior is coated with a resin containing carboxyl groups, preferably commercially available carboxyl magnetic beads.
  • the preparation method of the alkynylated magnetic beads is as follows:
  • the suspension of carboxyl magnetic beads is magnetically separated, and the supernatant is removed;
  • the second step add an organic solvent to the separated product, use a pipette to slowly blow for several times to resuspend the magnetic beads, magnetically separate again, remove the supernatant, repeat the washing for several times to separate the product, and resuspend the magnetic beads in the organic solvent , to obtain a magnetic bead suspension;
  • the third step configure (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) first solution for activating the carboxyl group;
  • the fourth step configure (NH 3 -PEG4-Alkyne) second solution
  • the first solution obtained in the third step is added to the magnetic bead suspension obtained in the second step;
  • the sixth step adding the second solution to the magnetic bead suspension obtained in the fifth step;
  • the mixed solution obtained in the sixth step is placed in a shaker at 37°C and shaken for incubation;
  • the eighth step magnetic separation, remove the supernatant
  • the ninth step adding ethanol for resuspending by pipetting, magnetic separation, removing the supernatant, repeating the washing of the separated product, and finally resuspending the magnetic beads in an aqueous ethanol solution;
  • the tenth step is to store at 4°C.
  • the preparation method is further specifically:
  • the carboxyl magnetic bead suspension is placed in a centrifuge tube, placed on a magnetic stand for 1-3 minutes, preferably 2 minutes, magnetic separation is performed, and the supernatant is sucked away with a pipette and discarded;
  • an organic solvent is added to the separated product, preferably DMF, and 1 ml of the organic solvent is used for each 1 ml of the carboxyl magnetic bead suspension used in the first step.
  • the third step configure (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) first solution, the concentration is 5-15mg/mL, more preferably 10mg/mL ;
  • the fourth step configure (NH3-PEG4-Alkyne) second solution, the concentration is 150mg/mL-250mg/mL, more preferably 200mg/mL;
  • the first solution obtained in the third step is added to the magnetic bead suspension obtained in the second step, and the addition amount is 1 ml of the magnetic bead suspension corresponding to 5 ml of the first solution;
  • the second solution is added to the magnetic bead suspension obtained in the fifth step, and the addition amount is 1 ml of the magnetic bead suspension corresponding to 10 ⁇ l of the second solution;
  • the mixed solution obtained in the sixth step is placed in a shaker at 37°C, shaken and incubated for 1-3 hours, preferably 2 hours;
  • the eighth step magnetic separation 1-3min, preferably 2min, remove the supernatant
  • the ninth step adding an ethanol solution with an ethanol concentration of 10%-30%, preferably 20%, resuspending by pipetting, magnetic separation for 1-3min, more preferably 2min, removing the supernatant, and repeating the washing of the separated product 4-6 times, Finally, resuspend the magnetic beads in an alcohol concentration of 10%-30%, preferably 20% ethanol aqueous solution;
  • the tenth step, the magnetic bead suspension is stored at 4°C to avoid freezing.
  • the preparation method of the alkynylated magnetic beads is as follows:
  • the suspension of carboxyl magnetic beads is magnetically separated, and the supernatant is removed;
  • the second step add an organic solvent to the separated product, use a pipette to slowly blow for several times to resuspend the magnetic beads, magnetically separate again, remove the supernatant, repeat the washing for several times to separate the product, and resuspend the magnetic beads in the organic solvent , to obtain a magnetic bead suspension;
  • the third step configure (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) first solution for activating the carboxyl group;
  • the 4th step configure the second solution of azidopropylamine
  • the first solution obtained in the third step is added to the magnetic bead suspension obtained in the second step;
  • the sixth step adding the second solution to the magnetic bead suspension obtained in the fifth step;
  • the mixed solution obtained in the sixth step is placed in a shaker at 37°C and shaken for incubation;
  • the eighth step magnetic separation, remove the supernatant
  • the ninth step adding ethanol for resuspending by pipetting, magnetic separation, removing the supernatant, repeating the washing of the separated product, and finally resuspending the magnetic beads in an aqueous ethanol solution;
  • the tenth step is to store at 4°C.
  • the preparation method is further specifically:
  • the carboxyl magnetic bead suspension is placed in a centrifuge tube, placed on a magnetic stand for 1-3 minutes, preferably 2 minutes, magnetic separation is performed, and the supernatant is sucked away with a pipette and discarded;
  • an organic solvent is added to the separated product, preferably DMF, and 1 ml of the organic solvent is used for each 1 ml of the carboxyl magnetic bead suspension used in the first step.
  • the third step configure (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) first solution, the concentration is 5-15mg/mL, more preferably 10mg/mL ;
  • the 4th step configure the second solution of azidopropylamine, the concentration is 150mg/mL-250mg/mL, more preferably 200mg/mL;
  • the first solution obtained in the third step is added to the magnetic bead suspension obtained in the second step, and the addition amount is 1 ml of the magnetic bead suspension corresponding to 5 ml of the first solution;
  • the second solution is added to the magnetic bead suspension obtained in the fifth step, and the addition amount is 1 ml of the magnetic bead suspension corresponding to 10 ⁇ l of the second solution;
  • the mixed solution obtained in the sixth step is placed in a shaker at 37°C, shaken and incubated for 1-3 hours, preferably 2 hours;
  • the eighth step magnetic separation 1-3min, preferably 2min, remove the supernatant
  • the ninth step adding an ethanol solution with an ethanol concentration of 10%-30%, preferably 20%, resuspending by pipetting, magnetic separation for 1-3min, more preferably 2min, removing the supernatant, and repeating the washing of the separated product 4-6 times, Finally, resuspend the magnetic beads in an alcohol concentration of 10%-30%, preferably 20% ethanol aqueous solution;
  • the tenth step, the magnetic bead suspension is stored at 4°C to avoid freezing.
  • the present invention also provides a bioorthogonal chemical macromolecule one-step capture method, which is obtained by using functionalized magnetic beads and biological macromolecules through click chemical reaction.
  • the functionalized magnetic beads can be azide magnetic beads or acetylenic magnetic beads prepared above.
  • the biomacromolecules can be proteins with alkynyl or azide unnatural amino acids embedded, and nucleic acids with alkynyl or azide unnatural nucleotides embedded.
  • the click chemistry reaction is an azide alkyne cycloaddition reaction.
  • the one-step capture method specifically includes:
  • the first step is to configure copper sulfate aqueous solution, sodium ascorbate aqueous solution, (tris (3-hydroxypropyl triazomethyl) amine) aqueous solution, sodium lauryl sulfate aqueous solution, 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution , urea aqueous solution, acetonitrile aqueous solution;
  • the second step take the alkynyl magnetic bead suspension or azide magnetic bead suspension prepared above, replace it with protein or nucleic acid buffer, and resuspend in protein buffer;
  • the third step copper sulfate aqueous solution, sodium ascorbate aqueous solution, (tris(3-hydroxypropyltriazomethyl)amine) aqueous solution, the protein sample to be captured, the magnetic bead suspension prepared in the second step, and the Ultra-pure water;
  • the fourth step after configuring the reaction mixture, put it on a constant temperature mixer and incubate;
  • the fifth step magnetic separation
  • the sixth step reductive alkylation
  • the seventh step is to wash the magnetic beads
  • the proteins on the magnetic beads were trypsinized for LC-MS analysis.
  • the above-mentioned capturing method provided by the present invention further specifically includes:
  • the first step configure 300-600mmol/L, further preferred 500mmol/L copper sulfate aqueous solution, 0.5mol/L-1.5mol/L, further preferred 1mol/L sodium ascorbate aqueous solution, 50mmol/L-150mmol/L preferred 100mmol/L (Tris(3-hydroxypropyltriazomethyl)amine) aqueous solution, 0.5%-1.5%, more preferably 1% aqueous sodium dodecyl sulfate solution, 30mmol/L-60mmol/L, more preferably 50mmol/L 4-Hydroxyethylpiperazine ethanesulfonic acid aqueous solution (pH value is 8), 6mol/L-10mol/L, further preferably 8mol/L urea solution, 20%-40%, further preferably 30% acetonitrile aqueous solution;
  • the volume ratio is preferably 0.1-0.3:1:0.3-0.5:1-1.5:15-25:20-25, more preferably 0.2:1:0.5:1.25:20:23.8;
  • the fourth step after configuring the reaction mixture, put it on a constant temperature mixer, the temperature is set to 25°C, the rotation speed is 1200rpm-2000rpm, preferably 1600rpm, and incubated for 0.5-1.5 hours, preferably 1 hour;
  • the fifth step the reaction mixture is placed on a magnetic stand for magnetic separation, 1-3min, preferably 2min, remove the supernatant;
  • the sixth step adding a reductive alkylation reagent to the sample, including: 500 mM tris(2-carboxyethyl) phosphine, 1 M chloroacetamide, 10% SDS, 50 mM 4-hydroxyethylpiperazineethanesulfonic acid, at 37 Incubate at °C for 30 min, and then remove the supernatant by magnetic separation;
  • a reductive alkylation reagent including: 500 mM tris(2-carboxyethyl) phosphine, 1 M chloroacetamide, 10% SDS, 50 mM 4-hydroxyethylpiperazineethanesulfonic acid, at 37 Incubate at °C for 30 min, and then remove the supernatant by magnetic separation;
  • the seventh step successively use sodium dodecyl sulfate aqueous solution to wash 3-5 times, urea solution to wash 3-5 times, acetonitrile to wash 3-5 times, and 4-hydroxyethylpiperazine ethanesulfonic acid aqueous solution to wash 3-5 times magnetic beads;
  • the proteins on the magnetic beads were trypsinized for LC-MS analysis.
  • the urea solution is obtained by dissolving urea by using a 50mmol/L 4-hydroxyethylpiperazine ethanesulfonic acid aqueous solution (pH value is 8) as a buffer solution.
  • Embodiments of the present invention are described in detail below by using examples, so as to fully understand and implement the implementation process of how the present invention applies technical means to solve technical problems and achieve technical effects.
  • the first step take 1 mL of the carboxyl magnetic beads produced by Beaverbeads TM , model 70102-5, in a 2.0 mL centrifuge tube, put it on a magnetic stand for 2 min, and remove the supernatant after magnetic separation; in the second step, add 1 mL of DMF (N,N-dimethylformamide), use a pipette to slowly and repeatedly blow and suck 10 times to suspend and evenly disperse the magnetic beads in the solvent (resuspend), remove the supernatant after magnetic separation for 2 min, and repeat washing for 3 Second, finally resuspend the magnetic beads in 1 mL DMF; third step, prepare the first solution: 10 mg/mL EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide salt In the fourth step, prepare the second solution: 200mg/mL NPA (NH3-PEG4-Alkyne) in DMF solution; in the fifth step, add 5m
  • Example 2 The same preparation method as in Example 1 was adopted, except that the DMF solution of 200 mg/mL azidopropylamine was used to replace the DMF solution of 200 mg/mL NPA (NH3-PEG4-Alkyne).
  • C10102 was used to label the protein with azide; shake it evenly, put it into a cell incubator at 37°C, and incubate for 6 hours ; Wash the cells in the culture dish once with 5 mL of PBS buffer; gently scrape the cells with a cell scraper, collect them in a 1.5 ml centrifuge tube, and remove the supernatant by centrifuging at 200 rcf for 5 min; add 200 ⁇ L of RIPA cells to the cell pellet Lysis buffer (from Thermo Scientific, Cat. No.
  • the alkynylated resin suspension was prepared using the method in document 3.
  • the one-step capture of macromolecules by bioorthogonal chemistry was carried out using the acetylenated magnetic beads prepared in Example 1 of the present invention and the acetylated resin provided in Comparative Example 1, as shown in Table 1 below.
  • FIG. 1 The experimental process is shown in Figure 1.
  • FIG. 2A and 2B show the response of the alkynyl magnetic beads (red) and the alkynyl magnetic beads (green) with a 488nm fluorescent group in the fluorescence channel (FITC), respectively.
  • Figure 2C shows that More than 99% of magnetic beads incorporate fluorescence. Indicates that the capture is complete.

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Abstract

一种功能化磁珠及采用其的生物正交化学的大分子一步捕获方法，其中，功能化磁珠为非偶联抗体小分子修饰的磁珠，利用该磁珠捕获大分子的方法不涉及抗原抗体反应，采用点击化学中的叠氮炔环加成反应原理，避免了非特异性结合蛋白的产生，一步法捕获蛋白质生物大分子，更加直接、高效、背景更低，且适用于变性条件下的蛋白捕获，适用性更广泛。

Description

功能化磁珠及采用其的生物正交化学的大分子一步捕获方法 技术领域
本发明涉及生物大分子捕获技术领域，具体涉及一种功能化磁珠及采用其的生物正交化学的大分子一步捕获方法。
背景技术
每个细胞的细胞内信号传递和细胞间通信主要依赖于蛋白质的功能及其特定的相互作用，因此，研究蛋白质相互作用是破译细胞生命活动的关键。然而一个哺乳动物细胞约有一万种不同的蛋白质，发生相互作用的蛋白质通常是低丰度的，含量只有不到1％，对于深入鉴定细胞的整个蛋白质组来说将会是巨大的挑战，即使是当今最先进的质谱仪，由于有限的检测速度和动态检测范围，也无法做到低丰度蛋白质的完全鉴定。如果要鉴定时间和空间方面特征的亚蛋白质组，那么这将会是一项更加困难的任务，关键之处在于亚蛋白质组的分离和富集。为了对新合成的蛋白质进行分离，已有研究者开发了一种生物正交非天然氨基酸标记技术(BONCAT,bioorthogonal noncanonical amino acid tagging)，这种技术是利用细胞自身翻译机制，使人们能够用生物正交非天然氨基酸来标记、识别和富集新合成的蛋白质亚群,也能结合亚细胞分离技术获得蛋白质在空间分布上的新合成蛋白质及其互作蛋白信息。
在调控生命活动的过程中，除了蛋白质之间的相互作用，RNA和蛋白质之间也会发生互作，从而调节彼此的功能，RNA与蛋白质的相互作用对于维持细胞稳态是非常重要的，而干扰两者的相互作用会造成细胞功能失调和相关疾病的发生。因此研究和鉴定RNA的相互作用网络，有助于科学家解决特定研究问题。而最新的研究方法也应用到生物正交非天然核苷酸标记技术，是利用非天然核苷酸在DNA转录时嵌入到新转录的RNA中，可以对新转录的RNA或新转录的RNA-蛋白质复合物进行标记、识别和富集，后续进行RNA测序以及质谱分析，可以鉴定并得到RNA及其互作蛋白的信息。
上述两种生物大分子标记技术涉及了点击化学(click chemistry)反应和亲和纯化技术。
点击化学(click chemistry)反应是由化学家K.B.Sharpless在2001年引入的一种化学合成概念，其代表反应是叠氮炔环加成反应，在亚铜离子的催化下，叠氮(azide)基团和炔基(alkyne)反应生成三唑环(triazole)，从而把想要的官能团链接起来。亲和纯化技术：通过给蛋白质添加标签，例如：His、GST、HA及FLAG等，利用标签与其特异性配体之间的特异亲和力，使用特异性配体修饰的固相载体对标签化蛋白进行捕获，从而达到分离纯化的目的。
文献1“BONCAT:Metabolic Labeling,Click Chemistry,and Affinity Purification of Newly Synthesized Proteomes”(DOI:org/10.1007/978-1-4939-2272-7_14)公开了生物正交非天然氨基酸标记的蛋白质相互作用的捕获方法，如图1所示，细胞在培养时添加叠氮化非天然氨基酸，新合成的蛋白质(橙色)会含有叠氮基团，可通过点击化学反应将带有炔基的生物素亲和标签进行标记。生物素和链霉亲和素可以发生特异性结合，标记的新合成蛋白可以通过Western blot分析或使用含有链霉亲和素的固相载体进行亲和纯化、质谱鉴定蛋白质信息。
文献2“Capturing the interactome of newly transcribed RNA”(DOI：10.1038/nmeth.4595)公开了生物正交非天然核苷酸标记的新转录RNA相互作用组的捕获方法，如图2所示，使用含有炔基的尿嘧啶类似物EU(5-ethynyluridine)在细胞培养时对新转录的RNA进行标记，利用点击化学反应(click reaction)对EU标记的RNA进行生物素化，通过链霉亲和素固相载体(Streptavidin-coated beads)对生物素化的RNA进行亲和纯化，对RNA测序和蛋白质组质谱鉴定，即可得到RNA及其相互作用组信息。
如上述两篇文献所介绍，在现阶段生物大分子，如蛋白质组、RNA组学研究中，在细胞培养阶段的某一时间节点下，用一种非天然氨基酸替代对应的天然氨基酸，非天然氨基酸带有炔基或叠氮基团，是天然氨基酸的类似物，在蛋白质合成的过程中会被当作天然氨基酸，嵌入到新生成的蛋白质中，从而使新生蛋白带有炔基或叠氮基团，通过点击化学反应，可以将带有叠氮基团或炔基的分子探针链接在这些蛋白质上，分子探针末端的报告基团含有生物素，能够特异性地与对应链霉亲和素磁珠结合，形成蛋白质-探针-磁珠的复合体，对复合体进行磁性分离、洗去杂质和蛋白洗脱，即可实现新生蛋白 的富集与纯化，最后进行LC-MS分析获得新生蛋白质组信息。
但是，现有的这种生物大分子捕获研究中，蛋白质需要先与探针结合，再利用探针与磁珠的特异性反应，通过两步法将蛋白连接到磁珠上。其主要有以下缺点：1、所使用的磁珠一般是偶联抗体的磁珠，大部分抗体无法耐受变性条件，从而限制了磁珠的使用范围，且抗原抗体的反应偶有非特异性结合的发生；2、所采用的两步法中涉及的分子探针、磁珠，价格均较为昂贵，同时实验需要投入的时间也更久。
文献3“Selective enrichment of newly synthesized proteins for quantitative secretome analysis”(DOI：10.1038/nbt.2356)公开了一种含有炔基的树脂，通过在生物大分子添加上叠氮化非天然氨基酸，叠氮基团与树脂的炔基基团发生反应，不涉及抗原抗体反应，避免了非特异性结合蛋白的产生，采用一步法即可实现生物大分子的捕获，但是采用这种方法反应时间长达十几个小时。
发明内容
本发明的目的提供一种功能化磁珠的合成方法及蛋白质生物正交化学的大分子一步捕获方法，用于蛋白质组学研究，其所使用的功能化磁珠为非偶联抗体小分子修饰的磁珠，方法不涉及抗原抗体反应，采用点击化学中的叠氮炔环加成反应原理，避免了非特异性结合蛋白的产生，采用一步法捕获蛋白质生物大分子，更加直接、高效、背景更低，且适用于变性条件下的蛋白捕获，适用性更广泛。
为解决上述技术问题，本发明提供一种炔基化磁珠，其采用NH 3-PEG4-Alkyne与羧基磁珠偶联合成。
该炔基化磁珠的制备方法如下：
第一步，羧基磁珠悬液进行磁性分离，除去上清液；
第二步，在分离产物中加入有机溶剂，使用移液器缓慢吹打多次，以重悬磁珠，再次磁性分离，去除上清液，重复洗涤多次分离产物，重悬磁珠在有机溶剂中，获得磁珠悬液；
第三步，配置(N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐)第一溶液，用于活化羧基；
第四步，配置(NH 3-PEG4-Alkyne)第二溶液；
第五步，将第三步获得的第一溶液加入第二步获得的磁珠悬液中；
第六步，将第二溶液加入第五步获得的磁珠悬液中；
第七步，将第六步获得的混合液置于37℃摇床中，摇晃孵育；
第八步，磁性分离，去除上清液；
第九步，加入乙醇进行吹打重悬，磁性分离，去除上清液，对分离产物重复洗涤，最后重悬磁珠在乙醇水溶液中；
第十步，在4℃的条件下保存。
本发明提供一种叠氮化磁珠，其采用叠氮基丙胺与羧基磁珠偶联合成。
该叠氮化磁珠的制备方法如下：
第一步，羧基磁珠悬液进行磁性分离，除去上清液；
第二步，在分离产物中加入有机溶剂，使用移液器缓慢吹打多次，以重悬磁珠，再次磁性分离，去除上清液，重复洗涤多次分离产物，重悬磁珠在有机溶剂中，获得磁珠悬液；
第三步，配置(N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐)第一溶液，用于活化羧基；
第四步，配置叠氮基丙胺第二溶液；
第五步，将第三步获得的第一溶液加入第二步获得的磁珠悬液中；
第六步，将第二溶液加入第五步获得的磁珠悬液中；
第七步，将第六步获得的混合液置于37℃摇床中，摇晃孵育；
第八步，磁性分离，去除上清液；
第九步，加入乙醇进行吹打重悬，磁性分离，去除上清液，对分离产物重复洗涤，最后重悬磁珠在乙醇水溶液中；
第十步，在4℃的条件下保存。
本发明还提供一种生物正交化学的大分子一步捕获方法，其采用功能化磁珠与生物大分子通过点击化学反应获得。
所述功能化磁珠可以为上述制备的叠氮化磁珠或炔基化磁珠。
所述生物大分子可以为嵌入了炔基或叠氮非天然氨基酸的蛋白质，以及嵌入了炔基或叠氮非天然核苷酸的核酸。
所述点击化学反应为叠氮炔环加成反应。
该方法具体包括：
第一步，配置硫酸铜水溶液、抗坏血酸钠水溶液、(三(3-羟丙基三氮基甲基)胺)水溶液、十二烷基硫酸钠水溶液、4-羟乙基哌嗪乙磺酸水溶液、尿素水溶液、乙腈水溶液；
第二步，取上面制备的磁珠悬液，使用蛋白或核酸缓冲液置换，重悬于蛋白缓冲液中；
第三步，按顺序依次加入硫酸铜水溶液、抗坏血酸钠水溶液、(三(3-羟丙基三氮基甲基)胺)水溶液、待捕获的蛋白样品、第二步制备的磁珠悬液和超纯水；
第四步，配置好反应混合液后，放在恒温混匀仪上，孵育；
第五步，磁性分离；
第六步，还原烷基化；
第七步，洗涤磁珠；
第八步，磁珠上的蛋白经胰蛋白酶消化，用于LC-MS分析。
本发明的有益效果
1、现有两步法技术是通过抗原抗体反应来富集蛋白质，为了保证抗体的反应性，磁珠需要在温和的条件下使用，本发明合成的磁珠是小分子修饰的磁珠，不涉及抗原抗体反应，在蛋白质捕获过程中以及捕获后的除杂过程都能够在强变性条件(如含SDS、尿素、盐酸胍等)下进行，适用范围更广，同时能够更好地去除非特异性结合，背景信号更低；
2、现有技术通过两步法富集蛋白质，本发明只需要一步法，操作简便高效，节省实验时间；
3、现有两步法技术使用的抗体修饰磁珠、分子探针价格较为昂贵，本发明所需的磁珠与反应物价格低廉，一次大量合成可以长期保存和使用，降低了实验成本；
4、现有参考文献3采用了一步法，但是其提供的炔基化树脂，操作性差，体现在固液分离时需要进行离心，并且容易粘附在离心管壁，进行移液操作时树脂丢失率高，固液分离不完全，无法完全移除液体，因此在洗涤树脂时需要进行更多的洗涤步骤，本发明的炔基化磁珠可利用磁性进行固液分离，无需繁琐的离心，分离效率高，节省时间及试剂；
5、现有一步法要求点击化学反应时间较长(18小时)，而本发明只需 反应1-2小时，大大节省实验时间。
附图说明
图1炔基化磁珠与Azide-488反应流程；
图2荧光结合效率的检测结果。
具体实施方式
本发明提供一种炔基化磁珠，其采用NH 3-PEG4-Alkyne与羧基磁珠偶联合成。
本发明还提供一种叠氮化磁珠，其采用叠氮基丙胺与羧基磁珠偶联合成。
所述羧基磁珠的核心部分为四氧化三铁，外部包覆含有羧基的树脂，优选市售的羧基磁珠。
该炔基化磁珠的制备方法如下：
第一步，羧基磁珠悬液进行磁性分离，除去上清液；
第二步，在分离产物中加入有机溶剂，使用移液器缓慢吹打多次，以重悬磁珠，再次磁性分离，去除上清液，重复洗涤多次分离产物，重悬磁珠在有机溶剂中，获得磁珠悬液；
第三步，配置(N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐)第一溶液，用于活化羧基；
第四步，配置(NH 3-PEG4-Alkyne)第二溶液；
第五步，将第三步获得的第一溶液加入第二步获得的磁珠悬液中；
第六步，将第二溶液加入第五步获得的磁珠悬液中；
第七步，将第六步获得的混合液置于37℃摇床中，摇晃孵育；
第八步，磁性分离，去除上清液；
第九步，加入乙醇进行吹打重悬，磁性分离，去除上清液，对分离产物重复洗涤，最后重悬磁珠在乙醇水溶液中；
第十步，在4℃的条件下保存。
该制备方法进一步具体为：
第一步，羧基磁珠悬液置于离心管中，放在磁力架上等待1-3min，进一步优选2min，进行磁性分离，用移液器吸走并丢弃上清液；
第二步，在分离产物中加入有机溶剂，有机溶剂优选DMF，每1ml第一步使用的羧基磁珠悬液使用1ml有机溶剂，使用移液器缓慢吹打8-12次，优选10次，以重悬磁珠，再次磁性分离1-3min，优选2min，去除上清液，重复洗涤3-5次分离产物，重悬磁珠在有机溶剂中，有机溶剂优选DMF，获得磁珠悬液；
第三步，配置(N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐)第一溶液，浓度为5-15mg/mL，进一步优选为10mg/mL；
第四步，配置(NH3-PEG4-Alkyne)第二溶液，浓度为150mg/mL-250mg/mL，进一步优选200mg/mL；
第五步，将第三步获得的第一溶液加入第二步获得的磁珠悬液中,添加量为1ml磁珠悬液对应5ml第一溶液；
第六步，将第二溶液加入第五步获得的磁珠悬液中，添加量为1ml磁珠悬液对应10μl第二溶液；
第七步，将第六步获得的混合液置于37℃摇床中，摇晃孵育1-3小时，优选2小时；
第八步，磁性分离1-3min，优选2min，去除上清液；
第九步，加入乙醇浓度10％-30％，优选20％的乙醇溶液，进行吹打重悬，磁性分离1-3min，进一步优选2min，去除上清液，对分离产物重复洗涤4-6次，最后重悬磁珠在醇浓度10％-30％，优选20％的乙醇水溶液中；
第十步，该磁珠悬液保存于4℃，避免冻结。
该炔基化磁珠的制备方法如下：
第一步，羧基磁珠悬液进行磁性分离，除去上清液；
第二步，在分离产物中加入有机溶剂，使用移液器缓慢吹打多次，以重悬磁珠，再次磁性分离，去除上清液，重复洗涤多次分离产物，重悬磁珠在有机溶剂中，获得磁珠悬液；
第三步，配置(N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐)第一溶液，用于活化羧基；
第四步，配置叠氮基丙胺第二溶液；
第五步，将第三步获得的第一溶液加入第二步获得的磁珠悬液中；
第六步，将第二溶液加入第五步获得的磁珠悬液中；
第七步，将第六步获得的混合液置于37℃摇床中，摇晃孵育；
第八步，磁性分离，去除上清液；
第九步，加入乙醇进行吹打重悬，磁性分离，去除上清液，对分离产物重复洗涤，最后重悬磁珠在乙醇水溶液中；
第十步，在4℃的条件下保存。
该制备方法进一步具体为：
第一步，羧基磁珠悬液置于离心管中，放在磁力架上等待1-3min，进一步优选2min，进行磁性分离，用移液器吸走并丢弃上清液；
第二步，在分离产物中加入有机溶剂，有机溶剂优选DMF，每1ml第一步使用的羧基磁珠悬液使用1ml有机溶剂，使用移液器缓慢吹打8-12次，优选10次，以重悬磁珠，再次磁性分离1-3min，优选2min，去除上清液，重复洗涤3-5次分离产物，重悬磁珠在有机溶剂中，有机溶剂优选DMF，获得磁珠悬液；
第三步，配置(N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐)第一溶液，浓度为5-15mg/mL，进一步优选为10mg/mL；
第四步，配置叠氮基丙胺第二溶液，浓度为150mg/mL-250mg/mL，进一步优选200mg/mL；
第五步，将第三步获得的第一溶液加入第二步获得的磁珠悬液中,添加量为1ml磁珠悬液对应5ml第一溶液；
第六步，将第二溶液加入第五步获得的磁珠悬液中，添加量为1ml磁珠悬液对应10μl第二溶液；
第七步，将第六步获得的混合液置于37℃摇床中，摇晃孵育1-3小时，优选2小时；
第八步，磁性分离1-3min，优选2min，去除上清液；
第九步，加入乙醇浓度10％-30％，优选20％的乙醇溶液，进行吹打重悬，磁性分离1-3min，进一步优选2min，去除上清液，对分离产物重复洗涤4-6次，最后重悬磁珠在醇浓度10％-30％，优选20％的乙醇水溶液中；
第十步，该磁珠悬液保存于4℃，避免冻结。
本发明还提供一种生物正交化学的大分子一步捕获方法，其采用功能化磁珠与生物大分子通过点击化学反应获得。
所述功能化磁珠可以为上述制备的叠氮化磁珠或炔基化磁珠。
所述生物大分子可以为嵌入了炔基或叠氮非天然氨基酸的蛋白质，以及嵌入了炔基或叠氮非天然核苷酸的核酸。
所述点击化学反应为叠氮炔环加成反应。
所述一步捕获方法具体包括：
第一步，配置硫酸铜水溶液、抗坏血酸钠水溶液、(三(3-羟丙基三氮基甲基)胺)水溶液、十二烷基硫酸钠水溶液、4-羟乙基哌嗪乙磺酸水溶液、尿素水溶液、乙腈水溶液；
第二步，取上面制备的炔基磁珠悬液或叠氮磁珠悬液，使用蛋白或核酸缓冲液置换，重悬于蛋白缓冲液中；
第三步，按顺序依次加入硫酸铜水溶液、抗坏血酸钠水溶液、(三(3-羟丙基三氮基甲基)胺)水溶液、待捕获的蛋白样品、第二步制备的磁珠悬液和超纯水；
第四步，配置好反应混合液后，放在恒温混匀仪上，孵育；
第五步，磁性分离；
第六步，还原烷基化；
第七步，洗涤磁珠；
第八步，磁珠上的蛋白经胰蛋白酶消化，用于LC-MS分析。
本发明提供的上述捕获方法，进一步具体包括：
第一步，配置300-600mmol/L，进一步优选500mmol/L硫酸铜水溶液、0.5mol/L-1.5mol/L，进一步优选1mol/L抗坏血酸钠水溶液、50mmol/L-150mmol/L优选100mmol/L(三(3-羟丙基三氮基甲基)胺)水溶液、0.5％-1.5％,进一步优选1％的十二烷基硫酸钠水溶液、30mmol/L-60mmol/L，进一步优选50mmol/L 4-羟乙基哌嗪乙磺酸水溶液(pH值为8)、6mol/L-10mol/L，进一步优选8mol/L尿素溶液、20％-40％,进一步优选30％的乙腈水溶液；
第二步，取上面制备的炔基磁珠悬液或叠氮磁珠悬液，使用蛋白或核酸 缓冲液置换3-5次，重悬于蛋白缓冲液中；
第三步，按顺序依次加入硫酸铜水溶液、抗坏血酸钠水溶液、(三(3-羟丙基三氮基甲基)胺)水溶液、待捕获的蛋白样品、第二步制备的磁珠悬液和超纯水，体积比优选为0.1-0.3:1:0.3-0.5:1-1.5:15-25：20-25,进一步优选0.2:1:0.5：1.25:20：23.8；
第四步，配置好反应混合液后，放在恒温混匀仪上，温度设置为25℃，转速1200rpm-2000rpm，优选1600rpm，孵育0.5-1.5小时，优选1小时；
第五步，将反应混合液置于磁力架上进行磁性分离，1-3min，优选2min，去除上清液；
第六步，向样品中加入还原烷基化试剂，包括：500mM三(2-羧乙基)膦，1M氯乙酰胺，10％SDS，50mM 4-羟乙基哌嗪乙磺酸，在37℃孵育30min，随后磁性分离去除上清液；
第七步，依次采用十二烷基硫酸钠水溶液洗涤3-5次，尿素溶液洗涤3-5次，乙腈洗涤3-5次，4-羟乙基哌嗪乙磺酸水溶液洗涤3-5次磁珠；
第八步，磁珠上的蛋白经过胰蛋白酶消化，用于LC-MS分析。
所述第一步中，尿素溶液是采用50mmol/L 4-羟乙基哌嗪乙磺酸水溶液(pH值为8)作为缓冲液，溶解尿素获得的。
所述第二步中，蛋白缓冲液优选为RIPA蛋白缓冲液：25mM Tris·HCl pH＝7.6,150mM NaCl,1％NP-40,1％sodium deoxycholate,0.1％SDS。
核酸缓冲液优选为10mM Tris-HCl pH＝7.5,1mM EDTA,1M NaCl,0.1％SDS。
炔基化磁珠的合成与蛋白质的捕获流程如下所示：
Figure PCTCN2021138134-appb-000001
叠氮化磁珠的合成与蛋白质的捕获流程如图所示：
Figure PCTCN2021138134-appb-000002
以下采用实施例来详细说明本发明的实施方式，借此对本发明如何应用技术手段来解决技术问题，并达成技术效果的实现过程能充分理解并据以实施。
实施例1炔基化磁珠
第一步，取1mL Beaverbeads TM公司生产的羧基磁珠，型号70102-5悬液于2.0mL离心管，放在磁力架上等待2min，磁性分离后去除上清液；第二步，加入1mL DMF(N,N-二甲基甲酰胺)，使用移液器缓慢反复吹吸10次，使磁珠悬浮并均匀分散于溶剂中(重悬)，磁性分离2min后去除上清液，重复洗涤3次，最后使磁珠重悬在1mL DMF中；第三步，配制第一溶液：10mg/mL EDC(N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐)的DMF溶液；第四步，配制第二溶液：200mg/mL NPA(NH3-PEG4-Alkyne)的DMF溶液；第五步，向第二步的1mL磁珠悬液中加入5mL第一溶液；第六步，向第五步的6mL混合液中加入10μL第二溶液；加入第二溶液后颠倒混匀，置于37℃摇床中，摇晃孵育2小时；反应时间结束后，磁性分离2min，去除上清液；加入1mL20％乙醇水溶液进行吹打重悬，磁性分离2min后去除上清液，重复洗涤4次，最后重悬磁珠在1mL 20％乙醇水溶液中；该磁珠悬液保存于4℃。
实施例2叠氮化磁珠
采用与实施例1相同的制备方法，区别在于采用200mg/mL叠氮基丙胺的DMF溶液替代200mg/mL NPA(NH3-PEG4-Alkyne)的DMF溶液。
实施例3叠氮修饰的蛋白质
培养细胞至覆盖率到达80％；去除细胞中旧的培养基，加入5mL PBS缓冲液(来自biological industries，货号02-023-1A)，轻轻摇晃，去除上清液，重复清洗2次；加入10mL无甲硫氨酸的培养基，放入37℃的细胞培养箱，培养30min；去除前面的细胞培养基，加入10mL无甲硫氨酸的培养 基；向实验细胞中加入10μL的100mM L-叠氮基高丙氨酸的DMSO溶液(叠氮化甲硫氨酸，来自thermo scientific，货号C10102)对蛋白质进行叠氮化标记；轻摇均匀，放入37℃的细胞培养箱，培养6小时；对培养皿中的细胞用5mL PBS缓冲液洗涤一次；用细胞刮刀轻轻地把细胞刮下，收集于1.5ml离心管中，通过200rcf离心5min去除上清液；向细胞沉淀加入200μL RIPA细胞裂解液(来自thermo scientific，货号89900)，重悬细胞，冰浴30min；将细胞悬液放在冰水浴中，使用超声破碎仪，破碎细胞；将样品，在4℃下，用20000rcf离心10min；转移上清液至新的离心管，此为提取的细胞蛋白溶液，含有叠氮修饰的蛋白；使用BCA蛋白检测试剂盒(来自thermo scientific，货号23227)测定总蛋白浓度；取1mg的蛋白溶液，进行下面的点击化学反应。
比较例1
采用文献3中的方法制备炔基化树脂悬液。
采用本发明实施例1制备的炔基化磁珠和比较例1提供的炔基化树脂进行生物正交化学的大分子一步捕获，如下表1所示。
表1蛋白捕获方法。
Figure PCTCN2021138134-appb-000003
Figure PCTCN2021138134-appb-000004
Figure PCTCN2021138134-appb-000005
反应后的磁珠用流式细胞技术(FCM)对磁珠上的荧光进行定量分析，实验流程如图1。检测结果如图2，图2A和2B表示了炔基化磁珠(红)与结合了488nm荧光基团的炔基化磁珠(绿)在分别在荧光通道(FITC)响应情况，图2C表明超过99％的磁珠都结合了荧光。说明完成了捕获。
所有上述的首要实施这一知识产权，并没有设定限制其他形式的实施这种新产品和/或新方法。本领域技术人员将利用这一重要信息，上述内容修改，以实现类似的执行情况。但是，所有修改或改造基于本发明新产品属于保留的权利。
以上所述，仅是本发明的较佳实施例而已，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。

Claims (8)

  1. 一种功能化磁珠，其特征在于：所述功能化磁珠为炔基化磁珠，其采用NH 3-PEG4-Alkyne与羧基磁珠偶联合成。
  2. 权利要求1所述功能化磁珠的制备方法，其特征在于，包括：
    第一步，羧基磁珠悬液进行磁性分离，除去上清液；
    第二步，在分离产物中加入有机溶剂，使用移液器缓慢吹打多次，以重悬磁珠，再次磁性分离，去除上清液，重复洗涤多次分离产物，重悬磁珠在有机溶剂中，获得磁珠悬液；
    第三步，配置(N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐)第一溶液，用于活化羧基；
    第四步，配置(NH 3-PEG4-Alkyne)第二溶液；
    第五步，将第三步获得的第一溶液加入第二步获得的磁珠悬液中；
    第六步，将第二溶液加入第五步获得的磁珠悬液中；
    第七步，将第六步获得的混合液置于37℃摇床中，摇晃孵育；
    第八步，磁性分离，去除上清液；
    第九步，加入乙醇进行吹打重悬，磁性分离，去除上清液，对分离产物重复洗涤，最后重悬磁珠在乙醇水溶液中；
    第十步，在4℃的条件下保存。
  3. 一种功能化磁珠，其特征在于：所述功能化磁珠为叠氮化磁珠，其采用叠氮基丙胺与羧基磁珠偶联合成。
  4. 如权利要求3所述功能化磁珠的制备方法，其特征在于，包括：
    第一步，羧基磁珠悬液进行磁性分离，除去上清液；
    第二步，在分离产物中加入有机溶剂，使用移液器缓慢吹打多次，以重悬磁珠，再次磁性分离，去除上清液，重复洗涤多次分离产物，重悬磁珠在有机溶剂中，获得磁珠悬液；
    第三步，配置(N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐)第一溶液，用于活化羧基；
    第四步，配置叠氮基丙胺第二溶液；
    第五步，将第三步获得的第一溶液加入第二步获得的磁珠悬液中；
    第六步，将第二溶液加入第五步获得的磁珠悬液中；
    第七步，将第六步获得的混合液置于37℃摇床中，摇晃孵育；
    第八步，磁性分离，去除上清液；
    第九步，加入乙醇进行吹打重悬，磁性分离，去除上清液，对分离产物重复洗涤，最后重悬磁珠在乙醇水溶液中；
    第十步，在4℃的条件下保存。
  5. 一种生物正交化学的大分子一步捕获方法，其特征在于：采用功能化磁珠与生物大分子通过点击化学反应获得，所述功能化磁珠为权利要求1所述的炔基化磁珠或权利要求3所述的叠氮化磁珠。
  6. 如权利要求5所述的生物正交化学的大分子一步捕获方法，其特征在于：所述生物大分子可以为进行了炔基化或叠氮化的蛋白质或核酸。
  7. 如权利要求5或6所述的生物正交化学的大分子一步捕获方法，其特征在于：所述点击化学反应为叠氮炔环加成反应。
  8. 权利要求5-7任一项所述的生物正交化学的大分子一步捕获方法，其特征在于，包括：
    第一步，配置硫酸铜水溶液、抗坏血酸钠水溶液、(三(3-羟丙基三氮基甲基)胺)水溶液、十二烷基硫酸钠水溶液、4-羟乙基哌嗪乙磺酸水溶液、尿素水溶液、乙腈水溶液；
    第二步，取权利要求1或2制备的磁珠悬液，使用蛋白或核酸缓冲液置换，重悬于蛋白缓冲液中；
    第三步，按顺序依次加入硫酸铜水溶液、抗坏血酸钠水溶液、(三(3-羟丙基三氮基甲基)胺)水溶液、待捕获的蛋白样品、第二步制备的磁珠悬液和超纯水；
    第四步，配置好反应混合液后，放在恒温混匀仪上，孵育；
    第五步，磁性分离；
    第六步，还原烷基化；
    第七步，洗涤磁珠；
    第八步，磁珠上的蛋白经胰蛋白酶消化，用于LC-MS分析。
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