WO2021109684A1 - 一种基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠及其制备方法和应用 - Google Patents

一种基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠及其制备方法和应用 Download PDF

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WO2021109684A1
WO2021109684A1 PCT/CN2020/116950 CN2020116950W WO2021109684A1 WO 2021109684 A1 WO2021109684 A1 WO 2021109684A1 CN 2020116950 W CN2020116950 W CN 2020116950W WO 2021109684 A1 WO2021109684 A1 WO 2021109684A1
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magnetic beads
photoaffinity
peg
capture
modified
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高涛
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南京师范大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • 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
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the technical field related to protein molecule interaction analysis, and specifically relates to a protein molecule weak interaction capture magnetic bead based on photoaffinity covalent connection, and a preparation method and application thereof.
  • PPIs Protein-protein interactions
  • the identification of PPIs is very important to biomedical research. It can realize the discovery of new signal pathways, the study of disease mechanisms, the identification of new drug targets, and the design of biomedical sensors. Protein molecules are the direct bearers of biological functions. Discovering unknown modes of action in signal transduction pathways through known protein molecules has become the most important method to explore molecular mechanisms. PPIs technology is an indispensable tool for biomedical research. Currently, the techniques used for PPIs analysis include pull-down assay, co-immunoprecipitation (Co-IP), tandem affinity purification mass spectrometry (TAP-MS), yeast two-hybrid system, bioinformatics analysis, etc. However, considering the complexity of PPIs, the analysis of PPIs still has greater technical challenges.
  • At least six interaction modes are known, (1) intra-domain action: the reaction interface is located in the same domain; (2) inter-domain action: the reaction interface is located in the same peptide In the two domains of the chain; (3) Homo-oligomer effect: the reaction interface is located in the same peptide chain that permanently interacts; (4) Homo-complex effect: the reaction interface is located in the same peptide chain that interacts transiently; (5) Hetero-oligomer action: the reaction interface is located in different peptide chains that interact permanently; (6) Hetero-complex action: the reaction interface is located in different peptide chains that interact transiently.
  • the dynamic changes of the mode of action and the comprehensive effects together affect and determine life activities.
  • Co-IP is the most commonly used method for identifying PPIs in biological research, which relies on the affinity of antibodies to bait proteins to capture candidate proteins or protein complexes, and then perform mass spectrometric identification or immunoblotting of the captured proteins.
  • the scope of application of the Co-IP method is limited to the screening of high-abundance/high-affinity binding proteins, and is not suitable for screening low-affinity binding proteins mediated by transient and weak PPIs.
  • the time-consuming and complicated operation steps between sample preparation and detection can cause uncertainty in the analysis results. At the same time, the instability of antibodies can also cause differences in results.
  • PTMs Post-translational modifications
  • More and more studies have shown that PTMs-mediated PPIs play an important role in the dynamic biochemical reactions of cells. effect. Therefore, screening PTMs sites and elucidating the functions of PTMs are crucial in biomedical research.
  • Many technologies can find PTMs sites on proteins, it is still challenging to identify the proteins that PTMs interact with, because the submolecular modifications on proteins often exhibit low abundance, dynamic changes, and mediate weak and transient interactions. effect.
  • the existing methods cannot meet the requirements of PTMs-mediated PPIs detection.
  • the present invention is based on photoaffinity covalently linked protein molecules weak interaction capture magnetic beads, referred to as photoaffinity magnetic beads (PAMBs), and the present invention is based on photoaffinity connection technology , Prepared photo-affinity magnetic beads (PAMBs) and applied them to the protein capture system for the first time, realizing the conversion of "protein-protein" weak interactions into covalent linkages, which are weak protein interactions (such as Post-translational modification (PTMs)-mediated PPIs analysis provides a capture magnetic bead and a new capture method; it can be effectively used in a universal assay to identify PTMs-mediated PPIs.
  • PTMs Post-translational modification
  • the invention also provides a preparation method and application of the weak interaction capturing magnetic beads based on the photoaffinity covalently connected protein molecules.
  • a photo-affinity covalently linked protein molecule weak interaction capture magnetic bead the capture magnetic bead is connected to two functional molecular layers from the inside to the outside: polymer Ethylene glycol PEG passivation layer and light affinity polypeptide probe layer.
  • the polyethylene glycol PEG passivation layer introduces the PEG passivation layer (PEG molecules) from the surface of the magnetic beads to form PEG-modified magnetic beads;
  • the polypeptide probe layer is a polypeptide with a thiol group and diazirine at the N-terminus.
  • the reactive groups of the polypeptide probe are the thiol group on the side chain of cysteine, and the photosensitive reactive group diaziridine modified on the lysine ⁇ -NH 2 respectively.
  • the PEG in the polyethylene glycol PEG passivation layer is a PEG molecule whose two ends are modified with amino groups and thiol groups respectively, that is, NH2-PEG-SH, which is used to form a passivation layer and is used as an intermediate linking molecule to connect respectively Magnetic beads and light affinity peptide probes.
  • the photoaffinity polypeptide probe is a synthetic polypeptide modified with two reactive groups at the N-terminus, and the reactive groups are the thiol group on the side chain of cysteine and the lysine ⁇ -NH 2 On the modified photoreactive group diaziridine.
  • the sequence of the photoaffinity polypeptide probe is: N-terminal modified cysteine Cys, followed by diaziridine modified lysine Lys, followed by a polypeptide capable of interacting with protein molecules, and its model sequence It is: CK (Diazirine)(X)n, where X represents an amino acid or a post-translationally modified amino acid, and n represents the number of amino acids.
  • sequence of the photoaffinity polypeptide probe is:
  • P2 C-K(Diazirine)-KAKTGAAGKFKR(Me2a)GK.
  • the method for preparing weak interaction capture magnetic beads based on photoaffinity covalently linked protein molecules of the present invention includes the following steps:
  • PEG passivation layer is introduced on the surface of magnetic beads to form PEG-modified magnetic beads:
  • the NH 2 -PEG5000-SH solution and NHS activated magnetic beads solution are equilibrated to room temperature. Specifically, 30 ⁇ L of magnetic beads are placed on the magnetic stand, the supernatant is discarded, and the pre-cooled 1 mM glacial acetic acid solution is added and vortexed gently for 15 seconds to collect the beads. Immediately add 300 ⁇ L of NH 2 -PEG5000-SH solution, and then vortex for 30 seconds. The solution was rotated and incubated for 2h at room temperature. Wash the beads twice with 1mL 0.1M glycine (pH 2.0) and ultrapure water, collect the magnetic beads, and store them at 4°C for later use.
  • the NH2-PEG5000-SH solution and the magnetic beads in step (1) are equilibrated to room temperature so that the magnetic beads are placed on the magnetic stand. The supernatant is discarded, and the glacial acetic acid solution is added and vortexed gently to collect the beads. Add the NH2-PEG5000-SH solution, then vortex and incubate the solution at room temperature with rotation, wash and collect the magnetic beads, and store them at 4°C to obtain PEG-modified magnetic beads.
  • the polymer resin in step (2) is Fmoc-Lys(Boc)-Wang resin.
  • step (2) Fmoc-Lys(Boc)-Wang resin (0.35mM g -1 ) was used as the starting material, and the peptide was synthesized from the C-terminus to the N-terminus by the Fmoc solid-phase method. Specifically, hexahydropyridine (dissolved in N,N'-dimethylformamide (DMF)) with a volume ratio of 25% was used to remove the N-terminal Fmoc protecting group to make the N-terminal a free amino group, and then use The amino acid material of 3 times the volume of the resin is introduced into the second end of the C terminal. In order to complete the synthesis of the entire peptide, each amino acid residue is connected in sequence.
  • DMF N,N'-dimethylformamide
  • Biaziridine-activated ester reacts directly with lysine side chain amino group at a specific site to obtain biaziridine-labeled peptide.
  • the resin is washed with N,N'-dimethylformamide (DMF) more than 6 times, and the reaction is controlled by Kaiser Test. If the condensation reaction of an amino acid is not complete, repeat the condensation reaction once until the desired target polypeptide is synthesized. Since bisaziridine is unstable under light, subsequent operations should be performed under dark conditions.
  • the target peptide was cleaved from the resin with a 90% trifluoroacetic acid (TFA) cleavage reagent by volume, the side chain protecting groups were removed at 30°C for 3 hours, a large amount of pre-cooled anhydrous ether was added to the filtrate, and the peptide was precipitated by centrifugation. Wash with anhydrous ether several times and dry to obtain crude peptide.
  • the crude peptide was purified by reversed-phase high performance liquid chromatography (HPLC).
  • the mobile phase A is an aqueous solution containing 0.05% TFA and 2% acetonitrile (CAN), and the mobile phase B is 90% acetonitrile/water, and the flow rate is 25 ml min -1 .
  • CAN aqueous solution containing 0.05% TFA and 2% acetonitrile
  • the mobile phase B is 90% acetonitrile/water
  • the flow rate is 25 ml min -1 .
  • the photoaffinity polypeptide probe solution uses dimethyl sulfoxide as the solvent, and the ratio of the volume of the polypeptide probe solution to the initial magnetic bead solution used to form the PEG-modified magnetic beads is 10:1.
  • Step (3) incubate the photo-affinity polypeptide probe solution with PEG-modified magnetic beads at room temperature, form disulfide bonds under reducing conditions, connect the photo-affinity polypeptide probe to the magnetic beads, and wash with phosphate buffer to obtain capture Magnetic beads, store at 4°C.
  • Step (3) peptide connection first prepare 0.5mg mL -1 photo-affinity peptide probe solution (40% DMSO aqueous solution as solvent), immediately add the PEG-modified magnetic beads of step (1) and mix it with Incubate at room temperature at 200 rpm for 4 hours on a shaker; add 1 mL of Storage Buffer and mix well, collect magnetic beads with a magnetic stand, discard the supernatant, and wash twice; add 300 ⁇ L of Storage Buffer and store at 4°C to obtain capture magnetic beads (PAMBs).
  • PAMBs capture magnetic beads
  • Wash Buffer A is 1mM glacial acetic acid
  • Coupling Buffer is 50mM borate pH 8.5 and 50mM PB
  • NH 2 -PEG5000-SH solution is a reagent with a concentration of 0.1M
  • Wash Buffer B is 0.1M glycine pH 2.0
  • Storage Buffer contains Coupling Buffer with a mass fraction of 0.05% sodium azide.
  • a protein sample is added to the magnetic beads, oscillated slowly at room temperature, and irradiated with an ultraviolet lamp at the same time to excite the biaziridine to form a covalent connection with the capture protein, and then the magnetic beads are collected, washed, and coupled to the magnetic beads.
  • the protein molecule is the captured protein molecule.
  • the captured protein molecules were separated and identified by amide gel electrophoresis.
  • the invention prepares protein molecules weak interaction capturing magnetic beads, which are used to capture and analyze the protein molecules of PPIs mediated by PTMs.
  • the present invention uses the photo-affinity covalent connection technology to propose a weak interaction between protein molecules based on the photo-affinity technology, and realizes the transformation of the weak interaction into a stable covalent connection, and prepares a capture magnet on this basis.
  • Bead PAMBs used to analyze and identify PTMs-mediated or other weak interaction-mediated PPIs. And the method is simple to operate, saves time, and has high specificity for the weak and transient PPIs detection mediated by PTMs.
  • PAMBs are composed of magnetic beads, polyethylene glycol (PEG) passivation layer and light affinity polypeptide probes, as shown in Figure 1.
  • Magnetic beads refer to NHS-activated magnetic beads;
  • the PEG passivation layer is PEG with modified mercaptan at the end;
  • the N-terminal of the photo-affinity polypeptide probe is modified with two reactive groups, namely the cysteine (C) side chain
  • the thiol group and lysine's ⁇ -NH2 modified photoreactive group bisaziridine
  • the thiol group is connected to the PEG terminal thiol group by forming a disulfide bond, and the bisaziridine is activated by light It can form a covalent link with interacting proteins to capture proteins that can interact with PTMs.
  • the PEG passivation layer can reduce the non-specific adsorption of protein molecules
  • the photo-affinity polypeptide probe can specifically recognize and capture the target protein molecule
  • the weak interaction between the photo-affinity polypeptide probe and the protein molecule is under ultraviolet irradiation conditions. It is transformed into a strong covalent connection, so as to realize the specific and efficient magnetic separation of weakly interacting protein molecules, and finally provide a capture magnetic bead and capture method for the study of protein-protein molecule weak interaction.
  • the formation and cleavage of the disulfide bond between the NH 2 -PEG-SH in the PEG-modified magnetic beads and the photo-affinity polypeptide probe simplifies the capture step of protein molecules, which is conducive to the integration of the photo-affinity probe and the magnetic bead. Connection, release of target protein molecules, and reuse of captured magnetic beads.
  • the preparation method and application of the protein molecule capturing magnetic beads based on the photoaffinity covalent connection technology of the present invention are respectively connected to two functional molecular layers from the inside to the outside, namely, a polyethylene glycol PEG passivation layer and a light affinity Polypeptide probe layer, PEG passivation layer can reduce non-specific adsorption of protein molecules, photo-affinity peptide probes can specifically identify and capture target protein molecules, and the weak interaction between photo-affinity peptide probes and protein molecules can be exposed to ultraviolet light Under the conditions, it is transformed into a strong covalent connection, so as to realize the specific and efficient capture and magnetic separation of weakly interacting protein molecules, and finally provide a capture magnetic bead and technical means for the study of protein-protein molecule weak interaction.
  • the present invention has the following advantages:
  • the present invention prepares a weak interaction capture magnetic bead based on photoaffinity covalently linked protein molecules, which can convert low-affinity PPIs into covalent linkages, and effectively realize the capture of weakly interacting protein molecules.
  • PAMBs photo-affinity magnetic beads
  • the photo-affinity magnetic beads (PAMBs) prepared by the present invention have fewer interacting proteins and more protein subclasses, indicating that the method has a high performance in the detection of PTMs-mediated PPIs. Specificity.
  • the photo-affinity magnetic beads (PAMBs) prepared by the present invention are helpful to the traditional method of studying the interaction between protein molecules, and are suitable for the capture of weakly interacting protein molecules.
  • the capture magnetic beads prepared by the present invention are simple to prepare and use It is convenient, saves time, and has good specificity and resolution for weak and transient protein molecular interaction detection.
  • the capture magnetic beads of the present invention can fill in the lack of technology for weakly interacting protein analysis. It is an urgently needed solution for many biomedical researches and will have good application and market prospects.
  • FIG. 1 Schematic diagram of the connection amount and connection efficiency analysis of PAMBs polypeptide probes
  • FIG. 3 Schematic diagram of the characterization of the stepwise preparation process of PAMBs
  • Figure 4 A schematic diagram of the feasibility analysis of PAMBs capturing interacting proteins
  • Figure 5 Schematic diagram of the relationship between the amount of PAMBs interacting protein captured and the protein concentration of the sample
  • Figure 6 Schematic diagram of the relationship between the amount of PAMBs interacting protein captured and the photo-affinity connection time
  • Figure 7 is a schematic diagram of comparing the performance difference between the photoaffinity system and the biotin-avidin system
  • Table 1 compares the performance of the photoaffinity system and the biotin-avidin system for capturing interacting proteins, and the mass spectrometry identification result table.
  • the raw materials in the present invention are all commercially available.
  • NH 2 -PEG5000-SH was purchased from Ponsure, model PS2-SN-5K, dissolved in a buffer containing 50 mM borate, pH 8.5;
  • the magnetic beads were purchased from ThermoFisher, model 88826, which was NHS activated magnetic bead solution; 1mL, the magnetic beads were dissolved in 10mg mL -1 N,N-dimethylacetamide DMAC.
  • the preparation method of PAMBs specifically includes the following steps:
  • Fmoc-Lys(Boc)-Wang resin (0.35mM g -1 ) is used as starting material, Fmoc solid-phase method is used to synthesize peptides from C-terminal to N-terminal; Use 25% hexahydropyridine (dissolved in N,N'-dimethylformamide DMF) to remove the N-terminal Fmoc protecting group to make the N-terminal a free amino group, and then use 3 times the volume of the amino acid of the resin The raw materials are introduced into the second end of the C-terminal.
  • each amino acid residue is connected in sequence; the biaziridin-activated ester reacts directly with the amino group of the lysine side chain at a specific site to obtain the biaziridin-labeled peptide .
  • the resin is washed with N,N'-dimethylformamide (DMF) more than 6 times, and the reaction is controlled by Kaiser Test. If the condensation reaction of an amino acid is not complete, repeat the condensation reaction once until the desired target polypeptide is synthesized. Since bisaziridine is unstable under light, subsequent operations should be performed under dark conditions.
  • the target peptide is cleaved from the resin with a 90% trifluoroacetic acid (TFA) lysis reagent by volume, and the side chain protecting groups are removed at 30°C for 3 hours.
  • TFA trifluoroacetic acid
  • a large amount of pre-cooled anhydrous ether is added to the filtrate, and the peptide is precipitated by centrifugation. Wash with anhydrous ether several times and dry to obtain crude peptide.
  • the crude peptide was purified by reversed-phase high performance liquid chromatography (HPLC).
  • the mobile phase A is an aqueous solution containing 0.05% TFA and 2% acetonitrile (CAN), and the mobile phase B is 90% acetonitrile/water, and the flow rate is 25 ml min -1 .
  • the photoaffinity polypeptide probe synthesized in this example is the terminal peptide of Flap endonuclease 1 protein (FEN1), and the sequence of the photoaffinity polypeptide probe is as follows:
  • P1 C-K(Diazirine)-KAKTGAAGKFKRGK;
  • P2 C-K(Diazirine)-KAKTGAAGKFKR(Me2a)GK.
  • Wash Buffer A is 1mM glacial acetic acid
  • Coupling Buffer is 50mM borate pH 8.5 and 50mM PB
  • NH 2 -PEG5000-SH solution is a reagent with a concentration of 0.1M
  • Wash Buffer B is 0.1M glycine pH 2.0
  • Storage Buffer contains Coupling Buffer with a mass fraction of 0.05% sodium azide.
  • the preparation process is shown in Figure 1.
  • the prepared PAMBs contain several elements, which together complete the function of protein capture.
  • a PEG passivation layer is introduced on the surface of the magnetic beads to avoid non-specific adsorption of proteins.
  • the photoaffinity polypeptide probe is introduced.
  • the thiol group is connected to the PEG layer by forming a disulfide bond.
  • the diaziridine can form a covalent connection with the PTMs-interacting protein to capture the protein that can interact with the PTMs.
  • connection amount and connection efficiency analysis of the peptide probes in PAMBs are shown in Figure 2.
  • the peptide probes attached to the magnetic beads increased from 0.8 ⁇ g to 31.1 ⁇ g per mg of magnetic beads, and the binding efficiency decreased to 51.9%, indicating that the peptide probes attached to the surface of the magnetic beads were close to saturation. Therefore, using 0.5 mg mL -1 polypeptide probe for preparation, the ligation efficiency is 55.3%.
  • CW0199S Nuclear and Cytoplasmic Extraction Kit
  • the protein molecules coupled on the magnetic beads are the captured protein molecules. Add 30 ⁇ L of 2 ⁇ loading buffer (Biyuntian) to stop the reaction and release the captured protein molecules.
  • Flap endonuclease 1 protein The terminal peptide of Flap endonuclease 1 protein (FEN1) was used as a polypeptide probe.
  • the preparation process of the capture magnetic beads uses unmethylated modified Flap endonuclease 1 protein (polypeptide probe P1) and methylated modified Flap endonuclease 1 protein (polypeptide probe P2) to prepare PAMBs respectively.
  • the experimental method of PAMBs capturing nuclear protein is as in Example 2. Add 30 ⁇ L of 2 ⁇ loading buffer (Biyuntian Lysis Solution) to stop the reaction and release the captured protein molecules. The connection of peptide probes and magnetic beads will cause changes in the size and charge of the magnetic beads. Therefore, DLS and Zeta potential (Delsa Nano C, USA) were used to characterize the prepared capture magnetic beads. As shown in Figure 3, the Zeta potential of the capture magnetic beads is 4.34mV.
  • P1 and P2 were used as polypeptide probes, and PTMs site-specific (Rme2a) antibodies were used as the model proteins that can be captured (primary antibody, antibody concentration of 1mg ml -1 , using 3% BSA diluted to 5 ⁇ g ml -1 ), fluorescently labeled antibody (FITC-labeled IgG, antibody concentration of 1 mg ml -1 , diluted to 5 ⁇ g ml -1 with 3% BSA) was used as a secondary antibody.
  • primary antibody antibody concentration of 1mg ml -1 , using 3% BSA diluted to 5 ⁇ g ml -1
  • fluorescently labeled antibody FITC-labeled IgG, antibody concentration of 1 mg ml -1 , diluted to 5 ⁇ g ml -1 with 3% BSA
  • Polypeptide probe P1 is not methylated, cannot capture the primary antibody of the model protein, and cannot bind to the fluorescently labeled antibody. Therefore, the polypeptide probe P1 hardly observes fluorescence, and the polypeptide probe P2 is methylated, and fluorescence can be observed.
  • Figure 4 shows the microscope images of PAMBs with and without the model protein captured, respectively. Fluorescence observation shows that the photo-affinity capture magnetic beads can capture the proteins interacting with PTMs.
  • the PAMBs capture protein experimental method is as in Example 2, using a microplate reader to detect the concentration of the capture protein at 560 nm, and test the protein loading capacity of PAMBs. As the cell protein concentration increases, the amount of the capture protein gradually increases, as shown in Figure 5.
  • PAMBs capture protein The experimental method of PAMBs capture protein is as in Example 2. Protein quantitative determination of capture protein concentration (microplate reader 560nm) As the UV irradiation time increases, the protein content on PAMBs increases, and the maximum amount can reach 0.46 ⁇ g protein per mg magnetic beads, indicating The load capacity of PAMBs is relatively high, as shown in Figure 6.
  • Polyacrylamide gel method The PAMBs capture protein experimental method is as in Example 2, and then the sample is heated at 95°C for 5 minutes, using P1 and P2 as polypeptide probes, and the captured protein is analyzed by the polyacrylamide gel method. Biotin-avidin technology and PAMBs were used for protein capture experiments. The polyacrylamide gel showed that the amount of protein captured by photoaffinity was significantly less than that of biotin-avidin, as shown in the upper diagram of Figure 7; biotin-avidin used the existing common method.
  • Protein quantitative determination protein quantitative determination of capture protein concentration (plate reader 560nm) shows that the amount of protein captured by photoaffinity is significantly reduced, P1 of photoaffinity capture magnetic beads is reduced to 81.7%, and P2 is reduced to 80.7%, such as Figure 7 is the bottom image.
  • the protein was separated by polyacrylamide gel, the experimental method was as in Example 3 (5), and the silver staining kit (24612, Thermo) was used for staining.
  • the gel was washed in ultrapure water for 5 minutes, and then fixed in a 30% ethanol solution containing 10% acetic acid for 15 minutes for a total of 2 times.
  • the gel was washed with 10% ethanol, and then washed twice in ultrapure water for 5 minutes each time.
  • Add sensitizer working solution 50 ⁇ L sensitizer and 25mL water). After 1 min, rinse with water twice, 1 min each time.
  • the gel was dyed in the dyeing working solution (0.5mL enhancer, 25mL dye) for 30min, washed twice with ultrapure water, 20s each time, and then developed in the developer solution (0.5mL enhancer, 25mL developer) until The band appears.
  • the color reaction was terminated by 5% acetic acid for 10 minutes.
  • the gel containing specific bands was cut into cubes with a width of 1 mm, and incubated with 50 mM ammonium bicarbonate and 50% acetonitrile for 1 hour until the color faded.
  • the faded gel was lyophilized and dehydrated, and the protein was digested with trypsin at 37°C overnight.
  • the peptides were collected, dried and dissolved in 0.5% acetic acid, and analyzed by LC/MS/MS.
  • the photo-affinity capture magnetic beads have fewer proteins and more types. In addition, approximately 60% of low-abundance proteins can be captured, much higher than the 16% in biotin-avidin. The results show that the photo-affinity capture magnetic beads are less interfered by the over-expressed protein of the sample. In addition, when compared with biotin-avidin, the photoaffinity capture magnetic beads show that the interaction is specific, indicating that the developed system is highly specific for PTMs-mediated PPIs, as shown in Table 1, further explanation The traditional method to identify protein-protein interactions has the problem of non-specific adsorption.
  • the capture magnetic beads can capture fewer proteins and reduce non-specific binding.

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Abstract

提供一种基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠及其制备方法和应用。所述捕获磁珠由内向外分别连接两层功能分子层，分别为聚乙二醇钝化层和光亲和多肽探针层，聚乙二醇钝化层由磁珠表面引入PEG分子形成修饰的磁珠；光亲和多肽探针为N-末端修饰有硫醇基和双吖丙啶的多肽。PEG钝化层可减少蛋白质分子的非特异吸附，光亲和多肽探针层可以特异识别并捕获目标蛋白质分子，光亲和多肽探针与蛋白质分子间的弱相互作用在紫外照射条件下转化为牢固的共价连接，实现弱相互作用蛋白质分子的特异高效的捕获和磁性分离。所述的捕获磁珠使用方便，对弱的和瞬时的蛋白质分子相互作用检测具有良好的特异性和分辨率。

Description

一种基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠及其制备方法和应用 技术领域
本发明属于蛋白质分子相互作用分析相关的技术领域，具体涉及一种基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠及其制备方法和应用。
背景技术
蛋白质-蛋白质相互作用(PPIs)在分子水平上介导几乎所有生命活动。PPIs的鉴定对生物医学研究至关重要，可实现新信号通路的发现、疾病发生机制的研究、新药物靶点的鉴定、生物医学传感器的设计等。蛋白质分子作为生物功能的直接承担者，通过已知蛋白质分子发现信号传导途径中的未知作用方式已经成为探究分子机制的最主要方法，PPIs技术是生物医学研究必不可少的工具。目前，用于PPIs分析的技术有下拉实验(Pull-down assay)、免疫共沉淀(Co-IP)、串联亲和纯化质谱法(TAP-MS)、酵母双杂交体系、生物信息学分析等。但是，考虑到PPIs复杂性，对PPIs的分析仍存在较大的技术挑战。
根据氨基酸的侧链和蛋白质的结构模体，已知至少存在六种相互作用方式，(1)结构域内作用：反应界面位于同一个结构域内；(2)结构域间作用：反应界面位于同一肽链的两个结构域内；(3)同源寡聚体作用：反应界面位于永久相互作用的相同肽链内；(4)同源复合物作用：反应界面位于瞬时相互作用的相同肽链内；(5)异源寡聚体作用：反应界面位于永久相互作用的不同肽链内；(6)异源复合物作用：反应界面位于瞬时相互作用的不同肽链内。作用方式的动态变化与综合效应共同影响和决定生命活动，除此之外，动力学、热力学、化学计量学和辅因子也会影响PPIs。因此，根据作用方式的不同选择合适的检测方法。例如，Co-IP是生物研究中最常用的鉴定PPIs的方法，其依赖于对诱饵蛋白的抗体亲和力，从而捕获候选蛋白或蛋白复合物，然后对捕获的蛋白进行质谱鉴定或免疫印记杂交。然而，Co-IP方法的适用范围局限于高丰度/高亲和力结合蛋白的筛选，不适用于筛选由瞬时的、弱的PPIs介导的低亲和力结合蛋白。而且，样品制备和检测之间耗时且复杂的操作步骤会造成分析结果的不确定性。与此同时，抗体的不稳定性也会造成结果的差异。
翻译后修饰(PTMs)即蛋白质在亚分子水平上的化学修饰，是介导和调控PPIs的主要机制，越来越多的研究表明，PTMs介导的PPIs在细胞的动态生化反应中发挥着重要作用。因此，筛选PTMs位点和阐明PTMs的功能在生物医学研究中至关重要。目前，虽然许多技术都能在蛋白质上找到PTMs位点，但鉴定 PTMs相互作用的蛋白质仍然具有挑战性，因为蛋白质上的亚分子修饰往往呈现低丰度、动态变化，介导微弱和短暂的相互作用。现有的方法不能满足PTMs介导的PPIs检测的要求。
发明内容
发明目的：针对现有技术存在的问题，本发明一种基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠，简称光亲和磁珠(PAMBs)，本发明基于光亲和连接技术，制备了光亲和磁珠(photo-affinity magnetic beads,PAMBs)，并首次应用到蛋白质捕获系统中，实现了“蛋白-蛋白”弱相互作用转化为共价连接，为蛋白质弱相互作用(如翻译后修饰，PTMs)介导的PPIs分析提供一种捕获磁珠和新的捕获方法；可以有效用于鉴定PTMs介导的PPIs的通用测定。
本发明还提供了一种基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠的制备方法和应用。
技术方案：为实现上述发明目的，如本发明所述一种基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠，所述捕获磁珠由内向外分别连接两层功能分子层：聚乙二醇PEG钝化层和光亲和多肽探针层，所述聚乙二醇PEG钝化层由磁珠表面引入PEG钝化层(PEG分子)，形成PEG修饰的磁珠；所述光亲和多肽探针层为N-末端有硫醇基和双吖丙啶(diazirine)的多肽。
其中，多肽探针的反应基团分别为半胱氨酸侧链上的硫醇基，以及赖氨酸ε-NH 2上修饰的光敏反应基团双吖丙啶。
其中，所述聚乙二醇PEG钝化层中的PEG为两端分别修饰氨基和硫醇基的PEG分子，即NH2-PEG-SH，用于形成钝化层，并作为中间连接分子分别连接磁珠和光亲和多肽探针。
其中，所述光亲和多肽探针为N-末端修饰有两个反应基团的合成多肽，反应基团分别为半胱氨酸侧链上的硫醇基，以及赖氨酸ε-NH 2上修饰的光敏反应基团双吖丙啶。
其所述光亲和多肽探针的序列为：N末端修饰半胱氨酸Cys，紧接着为双吖丙啶修饰的赖氨酸Lys，随后为能与蛋白质分子相互作用的多肽，其模式序列为：CK(Diazirine)(X)n，所述X代表氨基酸或翻译后修饰的氨基酸，n代表氨基酸的数目。
作为优选，所述光亲和多肽探针的序列为：
P2：C-K(Diazirine)-KAKTGAAGKFKR(Me2a)GK。
本发明所述的基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠的制备方法，包括如下步骤：
(1)磁珠表面引入PEG钝化层，形成PEG修饰的磁珠：
NH 2-PEG5000-SH溶液和NHS活化磁珠溶液平衡至室温，具体为取30μL磁珠被放置在磁力架上，弃上清液，加入预冷1mM冰乙酸溶液轻轻涡旋15s，收集珠子，立即加入300μL NH 2-PEG5000-SH溶液，然后30s涡旋振荡。溶液在室温旋转孵育2h。用1mL 0.1M甘氨酸(pH 2.0)和超纯水分别清洗2次，收集磁珠，4℃储存备用。
(2)光亲和多肽探针的合成：根据事先设计好的多肽序列(如P2序列)，先将所要合成的肽链的C端氨基酸的羧基以共价键固定到一个不溶性的高分子树脂上，然后从C端到N端重复去保护(除去要连接的氨基酸的α-氨基上的保护基团)，接着活化(激活前一个已和固相柱相连的氨基酸的羧基)，再偶联(让去保护的氨基与活化的羧基之间缩合，形成肽键)，最后洗脱过滤(去除没有反应完的各种试剂)，直至达到所要合成的多肽肽链；将琥珀酰亚胺酯活化的双吖丙啶分子与多肽肽链末端赖氨酸侧链氨基反应得到双吖丙啶标记肽；从树脂上裂解脱离得到光亲和多肽探针；(3)光亲和捕获磁珠的制备：配制光亲和多肽探针溶液，立即加入步骤(1)的PEG修饰的磁珠中混匀，用震荡室温孵育收集磁珠，存储备用得到捕获磁珠(PAMBs)。
作为优选，步骤(1)所述NH2-PEG5000-SH溶液和磁珠平衡至室温，为磁珠被放置在磁力架上，弃上清液，加入冰乙酸溶液轻轻涡旋，收集珠子，立即加入NH2-PEG5000-SH溶液，然后涡旋振荡，溶液在室温旋转孵育，洗涤收集磁珠，4℃储存备用得到PEG修饰的磁珠。
作为优选，步骤(2)所述高分子树脂为Fmoc-Lys(Boc)-Wang树脂。
进一步地，步骤(2)以Fmoc-Lys(Boc)-Wang树脂(0.35mM g -1)为起始原料，Fmoc固相法从C端到N端合成肽段。具体而言，用体积比为25％的六氢吡啶(溶于N，N'-二甲基甲酰胺(DMF))去除N端Fmoc保护基团，使N端成为游离氨基基团，然后用3倍体积树脂的氨基酸原料引入C端第二端，为了完成整个肽段的合成，各个氨基酸残基被依次连接。双吖丙啶活化的酯与特定位点赖氨酸侧链氨基直接反应得到双吖丙啶标记肽。上述反应每一步完成后，用N，N'-二甲基甲酰胺(DMF)洗涤树脂6次以上，Kaiser Test控制反应。如果一个氨基酸的缩合反应不完全，重复缩合反应一次，直到合成所需的目标多肽为止。由于双吖丙啶在光照下不稳定，后续的操作需在避光条件下进行。用体积比为90％ 的三氟乙酸(TFA)裂解试剂从树脂中裂解目标肽，30℃3h除去侧链保护基团，滤液中加入大量预冷的无水乙醚，离心使多肽沉淀。用无水乙醚洗涤多次，干燥得到粗肽。采用反相高效液相色谱法(HPLC)对粗肽进行纯化。流动相A为含0.05％TFA的水溶液和2％乙腈(CAN)，流动相B为90％乙腈/水，流速为25ml min -1。在溶剂冻干后，得到一种蓬松状态的纯肽即为光亲和多肽探针。采用MALDI-TOF测定其纯度。
其中，所述步骤(3)光亲和多肽探针溶液以二甲亚砜为溶剂，多肽探针溶液和形成PEG修饰的磁珠所用初始磁珠溶液体积之比为10:1。
其中步骤(3)将光亲和多肽探针溶液和PEG修饰的磁珠室温孵育，还原条件下形成二硫键，光亲和多肽探针连接到磁珠，用磷酸盐缓冲液洗涤，获得捕获磁珠，4℃储存。
具体方法：步骤(3)多肽的连接，先配制0.5mg mL -1光亲和多肽探针溶液(40％DMSO水溶液为溶剂)，立即加入步骤(1)的PEG修饰的磁珠混匀，用震荡仪200rpm室温孵育4h；加1mL Storage Buffer混匀，用磁力架收集磁珠，弃上清，洗两次；加300μL Storage Buffer，4℃存储备用得到捕获磁珠(PAMBs)。
上述Wash Buffer A为1mM冰乙酸；Coupling Buffer为50mM硼酸盐pH 8.5和50mM PB；NH 2-PEG5000-SH溶液为浓度0.1M的试剂；Wash Buffer B为0.1M甘氨酸pH 2.0；Storage Buffer为含质量分数0.05％叠氮钠的Coupling Buffer。
本发明所述的基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠在弱相互作用蛋白质的捕获中的应用。
本发明所述应用取蛋白样品加入磁珠中，室温缓慢振荡，同时用的紫外灯照射，激发双吖丙啶与捕获蛋白产生共价连接，随后用收集磁珠，清洗，磁珠上偶联的蛋白质分子即为所捕获蛋白质分子，加入还原试剂裂解NH 2-PEG-SH与光亲和多肽探针间形成的二硫键，释放结合有目标蛋白质分子的光亲和多肽探针，聚丙烯酰胺凝胶电泳分离鉴定捕获的蛋白质分子。
本发明制备了蛋白质分子弱相互作用捕获磁珠，用于捕获和分析PTMs介导的PPIs的蛋白质分子。本发明利用光亲和共价连接技术，提出了以光亲和技术为原理的蛋白质分子间弱相互作用，实现了弱相互作用转变为稳定的共价连接，并以此为基础制备了捕获磁珠PAMBs，用于分析和鉴定PTMs介导或其它弱相互作用介导的PPIs。并且方法操作简单，节省时间，对由PTMs介导的弱的、瞬时的PPIs检测具有较高的特异性。
本发明中：PAMBs由磁珠、聚乙二醇(PEG)钝化层和光亲和多肽探针组成，见图1。磁珠指NHS活化的磁珠；PEG钝化层为末端修饰硫醇的PEG；光亲和多肽探针N-末端修饰有两个反应基团，即半胱氨酸(C)侧链上的硫醇基和赖氨酸的ε-NH2上修饰的光敏反应基团(双吖丙啶)，硫醇基通过形成二硫键与PEG末端硫醇基连接，并且双吖丙啶被光照活化后可与互作蛋白形成共价连接，用以捕获能与PTMs相互作用的蛋白。本发明中PEG钝化层可减少蛋白质分子的非特异吸附，光亲和多肽探针可以特异识别并捕获目标蛋白质分子，光亲和多肽探针与蛋白质分子间的弱相互作用在紫外照射条件下转化为牢固的共价连接，从而实现弱相互作用蛋白质分子的特异高效的磁性分离，最终为研究蛋白质-蛋白质分子弱相互作用提供一种捕获磁珠和捕获方法。此外，PEG修饰的磁珠中的NH 2-PEG-SH与光亲和多肽探针间的二硫键的形成和裂解，简化蛋白质分子的捕获步骤，有利于光亲和探针与磁珠的连接，目标蛋白质分子的释放，以及捕获磁珠的重复利用。
本发明基于光亲和共价连接技术的蛋白质分子捕获磁珠的制备方法和应用，所述捕获磁珠由内向外分别连接两层功能分子层，即聚乙二醇PEG钝化层和光亲和多肽探针层，PEG钝化层可减少蛋白质分子的非特异吸附，光亲和多肽探针可以特异识别并捕获目标蛋白质分子，光亲和多肽探针与蛋白质分子间的弱相互作用在紫外照射条件下转化为牢固的共价连接，从而实现弱相互作用蛋白质分子的特异高效的捕获和磁性分离，最终为研究蛋白质-蛋白质分子弱相互作用提供一种捕获磁珠和技术手段。
有益效果：与现有技术相比，本发明具有如下优点：
(1)本发明制备了一种基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠，可以将低亲和作用的PPIs转化为共价连接，有效实现弱相互作用蛋白分子的捕获。
(2)本发明制备的光亲和磁珠(PAMBs)保证了高时空分辨率捕获弱相互作用的蛋白，非特异性相互作用降低81％。
(3)本发明制备的光亲和磁珠(PAMBs)与传统的蛋白捕获系统相比，相互作用蛋白更少，蛋白质亚类更多，表明该方法对PTMs介导的PPIs检测具有很高的特异性。
(4)本发明制备的光亲和磁珠(PAMBs)有助于传统研究蛋白质分子间相 互作用的方法，适用于弱相互作用蛋白质分子的捕获，同时本发明制备的捕获磁珠制备简单，使用方便，节省时间，对弱的和瞬时的蛋白质分子相互作用检测具有良好的特异性和分辨率。本发明的捕获磁珠可填补弱相互作用蛋白质分析的技术缺失，是目前很多生物医学研究急需的方案，将会有很好的应用和市场前景。
附图说明
图1 PAMBs的制备流程图。
图2 PAMBs多肽探针的连接量与连接效率分析示意图；
图3 PAMBs逐步制备过程的表征示意图；
图4 PAMBs捕获相互作用蛋白的可行性分析示意图；
图5 PAMBs相互作用蛋白捕获量与样品蛋白浓度的关系示意图；
图6 PAMBs相互作用蛋白捕获量与光亲和连接时间的关系示意图；
图7为比较光亲和系统与生物素-亲和素系统的性能差异示意图；
表1比较光亲和系统与生物素-亲和素系统捕获相互作用蛋白的性能，质谱鉴定结果表。
具体实施方式
下面结合附图和实施例对本发明作进一步说明。
实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明中的原料均市售可得。
其中，NH 2-PEG5000-SH购买于Ponsure，型号PS2-SN-5K，溶解于含有50mM硼酸盐的缓冲液中，pH 8.5；
磁珠购买于ThermoFisher，型号88826，为NHS活化的磁珠溶液；1mL，磁珠溶于10mg mL -1N，N-二甲基乙酰胺DMAC。
实施例1
PAMBs的制备方法，具体包括以下步骤：
(1)肽探针的合成及光敏部分的合成：以Fmoc-Lys(Boc)-Wang树脂(0.35mM g -1)为起始原料，Fmoc固相法从C端到N端合成肽段；用体积比为25％的六氢吡啶(溶于N，N'-二甲基甲酰胺DMF)去除N端Fmoc保护基团，使N端成为游离氨基基团，然后用3倍体积树脂的氨基酸原料引入C端第二端，为了完成整个肽段的合成，各个氨基酸残基被依次连接；双吖丙啶活化的酯与特定位点赖氨酸侧链氨基直接反应得到双吖丙啶标记肽。上述反应每一步完成后，用N，N'-二甲基甲酰胺(DMF)洗涤树脂6次以上，Kaiser Test控制反应。如果一个氨基酸的缩合反应不完全，重复缩合反应一次，直到合成所需的目标多肽为止。 由于双吖丙啶在光照下不稳定，后续的操作需在避光条件下进行。用体积比为90％的三氟乙酸(TFA)裂解试剂从树脂中裂解目标肽，30℃3h除去侧链保护基团，滤液中加入大量预冷的无水乙醚，离心使多肽沉淀。用无水乙醚洗涤多次，干燥得到粗肽。采用反相高效液相色谱法(HPLC)对粗肽进行纯化。流动相A为含0.05％TFA的水溶液和2％乙腈(CAN)，流动相B为90％乙腈/水，流速为25ml min -1。在溶剂冻干后，得到一种蓬松状态的纯肽即为光亲和多肽探针。采用MALDI-TOF测定其纯度。半胱氨酸是含有硫醇基的一个氨基酸，
本实施例合成的光亲和多肽探针为Flap核酸内切酶1蛋白(FEN1)的末端肽，光亲和多肽探针的序列如下所示：
P1：C-K(Diazirine)-KAKTGAAGKFKRGK；
P2：C-K(Diazirine)-KAKTGAAGKFKR(Me2a)GK。
(2)固定和封闭：具体方法：NH 2-PEG5000-SH溶液(PS2-SN-5K，pH 8.5)和磁珠(88826)平衡至室温，具体为30μL磁珠被放置在磁力架上，弃上清液，加入预冷的1mL Wash Buffer A溶液轻轻涡旋15s，收集珠子，立即加入300μL NH 2-PEG5000-SH溶液，然后30s涡旋振荡，溶液在室温旋转孵育2h。用1mL0.1M甘氨酸(pH 2.0)和超纯水分别清洗2次，收集磁珠得到PEG修饰的磁珠。
(3)多肽的连接：分别配制0.5mg mL -1光亲和多肽探针溶液P1和P2(40％DMSO水溶液为溶剂)，分别取300μL立即加入步骤(2)获得的全部PEG修饰的磁珠中混匀，用震荡仪200rpm室温孵育4h。加1mL Storage Buffer混匀，用磁力架收集磁珠，弃上清，洗两次。加300μL Storage Buffer，4℃存储备用得到捕获磁珠(PAMBs)。
上述Wash Buffer A为1mM冰乙酸；Coupling Buffer为50mM硼酸盐pH 8.5和50mM PB；NH 2-PEG5000-SH溶液为浓度0.1M的试剂；Wash Buffer B为0.1M甘氨酸pH 2.0；Storage Buffer为含质量分数0.05％叠氮钠的Coupling Buffer。
制备流程如图1所示，制备的PAMBs包含几个元件，它们一起完成蛋白捕获的功能。首先，在磁珠表面引入PEG钝化层以避免蛋白质的非特异性吸附。然后，引入光亲和多肽探针。在多肽探针上N-末端有两个反应基团，半胱氨酸侧链上的硫醇基，以及赖氨酸ε-NH2上修饰的光敏反应基团(双吖丙啶)。硫醇基团通过形成二硫键与PEG层连接，双吖丙啶被光活化后可与PTMs互作蛋白形成共价连接，用以捕获能与PTMs互作的蛋白。这两个基团共同起作用以促进 PAMBs在捕获系统中的功能。
PAMBs中的多肽探针的连接量与连接效率分析如图2。为了优化条件以改善PAMBs的性能，首先，使用浓度为0，0.05，0.1，0.25，0.5，1mg ml -1多肽来制备PAMBs。随着多肽浓度的增加，磁珠上附着的多肽探针从0.8μg增加到31.1μg每mg磁珠，并且结合效率降低至51.9％，说明磁珠表面附着的多肽探针接近饱和。因此，使用0.5mg mL -1的多肽探针进行制备，连接效率为55.3％。
实施例2
基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠捕获弱相互作用蛋白质分子
使用试剂盒提取细胞核蛋白(CW0199S,Nuclear and Cytoplasmic Extraction Kit)，以乳腺癌MCF-7细胞的细胞核蛋白为例，经蛋白纯化柱纯化，调节蛋白浓度至1mg mL -1。取300μL加入30μL 0.5mg mL -1PAMBs，室温振荡孵育30min，同时用365nm的紫外灯照射2h。用磁力架收集磁珠，弃上清液；随后使用PBST(0.05％Tween20)和超纯水分别清洗两次，，收集磁珠，磁珠上偶联的蛋白质分子即为所捕获蛋白质分子。加入30μL 2×上样缓冲液(碧云天)终止反应，释放捕获的蛋白分子。
实施例3
精氨酸甲基化作用蛋白的捕获与分析
PAMBs的制备方法同实施例1。
以Flap核酸内切酶1蛋白(FEN1)的末端肽作为多肽探针。
捕获磁珠制备过程使用未甲基化修饰的Flap核酸内切酶1蛋白(多肽探针P1)和甲基化修饰的Flap核酸内切酶1蛋白(多肽探针P2)，分别制备PAMBs。PAMBs捕获细胞核蛋白实验方法如实施例2。加入30μL 2×上样缓冲液(碧云天裂解液)终止反应，释放捕获的蛋白分子。多肽探针与磁珠连接会引起磁珠水化颗粒大小和电荷量的变化，因此，使用DLS和Zeta电位(DelsaNano C，USA)对制备的捕获磁珠进行表征。如图3，捕获磁珠Zeta电位为4.34mV，在使用PEG修饰后，电位显着地转变为-22.5mV，进一步修饰MB-PEG-P1和MB-PEG-P2，多肽探针的附着可将电位分别逆转至28.4mV和28.3mV。MB-PEG-P1和MB-PEG-P2之间的轻微zeta电位变化是由多肽探针P2上的甲基化修饰引起的。证明多肽成功连接在磁珠上。
(2)PAMBs捕获甲基化相互作用的蛋白。
为了测试捕获方法的可行性，分别以P1和P2为多肽探针，使用PTMs位点特异性(Rme2a)的抗体作为可捕获的模型蛋白(一抗，抗体浓度为1mg ml -1， 用3％BSA稀释至5μg ml -1)，荧光标记抗体(FITC标记的IgG，抗体浓度为1mg ml -1，用3％BSA稀释至5μg ml -1)作为二抗。首先将200μL PAMBs与1μL 5μg ml -1一抗摇床室温孵育2h，置于磁力架上，去上清，用PBST摇床清洗3次，每次5min。在光亲和力捕获反应后，加入200μL 5μg ml -1二抗室温孵育1h，置于磁力架上，去上清，用PBST置于摇床洗3次，每次10min，去上清。加入超纯水重悬磁珠，取10μL置于载玻片，显微镜观察荧光。多肽探针P1未甲基化，捕获不到模型蛋白一抗，不能与荧光标记的抗体结合，因此多肽探针P1几乎观察不到荧光，多肽探针P2甲基化，可观察到荧光。图4分别显示捕获和未捕获模型蛋白的PAMBs的显微镜图像。荧光观察表明光亲和捕获磁珠可以捕获PTMs相互作用的蛋白质。
(3)PAMBs相互作用蛋白捕获量与样品蛋白浓度的关系。
PAMBs捕获蛋白实验方法如实施例2，使用酶标仪560nm检测捕获蛋白浓度，测试PAMBs的蛋白质负载能力，随着细胞蛋白浓度的增加，捕获蛋白的量逐渐增多，如图5。
(4)PAMBs相互作用蛋白捕获量与光亲和连接时间的关系。
PAMBs捕获蛋白实验方法如实施例2，蛋白质定量测定捕获蛋白浓度(酶标仪560nm)随着紫外线照射时间的延长，PAMBs上蛋白质的含量增加，最大量可达到0.46μg蛋白质每mg磁珠，表明PAMBs的负载能力相对较高，如图6。
(5)比较光亲和捕获磁珠与生物素-亲和素技术的性能差异。
1)聚丙烯酰胺凝胶法：PAMBs捕获蛋白实验方法如实施例2，然后样品95℃加热5min，以P1和P2为多肽探针，聚丙烯酰胺凝胶法分析捕获的蛋白。生物素-亲和素技术和PAMBs分别用于蛋白的捕获实验。聚丙烯酰胺凝胶显示光亲和捕获的蛋白质的量显著少于生物素-亲和素，如图7上图；生物素-亲和素采用现有常用方法。
2)蛋白质定量测定：蛋白质定量测定捕获蛋白浓度(酶标仪560nm)显示光亲和捕获的蛋白质的量显著减少，光亲和捕获磁珠的P1减少为81.7％，P2减少为80.7％，如图7下图。
(6)比较光亲和捕获磁珠与生物素-亲和素捕获相互作用蛋白的质谱分析
聚丙烯酰胺凝胶分离蛋白质，实验方法如实施例3的(5)，，使用银染试剂盒(24612，Thermo)染色。凝胶在超纯水中洗涤5min，然后在含10％乙酸的30％乙醇溶液中固定15min，共2次。用10％乙醇洗涤凝胶，然后在超纯水中洗涤2次，每次5min。加入敏化剂工作液(50μL敏化剂与25mL水)。1min后，用水冲洗两次，每次1min。凝胶在染色工作液(0.5mL增强剂，25mL染色剂) 中染色30min，用超纯水冲洗2次，每次20s，然后在显影液(0.5mL增强剂，25mL显影剂)中显影，直到条带出现。5％乙酸终止显色反应10min将含有特异性条带的凝胶切成宽1mm的立方体，50mM碳酸氢铵和50％乙腈孵育1h至褪色。使褪色凝胶冻干脱水，用胰蛋白酶对蛋白质进行消化，37℃过夜。收集肽段，干燥后用0.5％乙酸溶解，LC/MS/MS进行分析。光亲和捕获磁珠的蛋白质更少且种类更多。此外，大约60％的低丰度蛋白质可以被捕获，远高于生物素-亲和素中的16％。结果表明，光亲和捕获磁珠受样品过表达的蛋白质干扰较小。此外，当与生物素-亲和素相比时，光亲和捕获磁珠显示相互作用是特异性的，表明所开发的系统对PTMs介导的PPIs具有高度特异性，如表1，进一步说明传统方法鉴定蛋白质间相互作用，会有非特异性吸附的问题，捕获磁珠能捕获的蛋白质少，减少了非特异性结合。
表1
Figure PCTCN2020116950-appb-000001

Claims (8)

  1. 一种基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠，其特征在于，所述捕获磁珠由内向外分别连接两层功能分子层：聚乙二醇PEG钝化层和光亲和多肽探针层，所述聚乙二醇PEG钝化层由磁珠表面引入PEG钝化层，形成PEG修饰的磁珠；所述光亲和多肽探针层为N-末端修饰有硫醇基和双吖丙啶的多肽。
  2. 根据权利要求1所述的基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠，其特征在于，所述聚乙二醇PEG钝化层中的PEG为两端分别修饰氨基和硫醇基的PEG分子，即NH2-PEG-SH，用于形成钝化层，并作为中间连接分子分别连接磁珠和光亲和多肽探针。
  3. 根据权利要求1所述的基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠，其特征在于，所述光亲和多肽探针为N-末端修饰有两个反应基团的合成多肽，反应基团分别为半胱氨酸侧链上的硫醇基，以及赖氨酸ε-NH 2上修饰的光敏反应基团双吖丙啶分子(diazirine)。
  4. 根据权利要求1-3任意所述的基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠，其所述光亲和多肽探针的序列为：N末端修饰半胱氨酸Cys，紧接着为双吖丙啶修饰的赖氨酸Lys，随后为能与蛋白质分子相互作用的多肽，其模式序列为：CK(Diazirine)(X)n，所述X代表氨基酸或翻译后修饰的氨基酸，n代表氨基酸的数目。
  5. 一种权利要求1所述的基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠的制备方法，其特征在于，包括如下步骤：
    (1)磁珠表面引入PEG钝化层，形成PEG修饰的磁珠：NH 2-PEG5000-SH溶液和磁珠平衡至室温，收集磁珠，储存备用；
    (2)光亲和多肽探针的合成：将所要合成的肽链的C端氨基酸的羧基以共价键固定到一个不溶性的高分子树脂上，然后从C端到N端重复去保护(除去要连接的氨基酸的α-氨基上的保护基团)，接着活化，再偶联，最后洗脱过滤，直至达到所要合成的多肽肽链；将琥珀酰亚胺酯活化的双吖丙啶分子与多肽肽链末端赖氨酸侧链氨基反应得到双吖丙啶标记肽；从树脂上裂解脱离得到光亲和多肽探针；
    (3)光亲和捕获磁珠的制备：将光亲和多肽探针溶液和PEG修饰的磁珠室温孵育，二甲亚砜还原条件下形成二硫键，光亲和多肽探针连接到磁珠，用磷酸盐缓冲液洗涤，获得捕获磁珠。
  6. 根据权利要求5所述的制备方法，其特征在于，步骤(1)所述NH2-PEG5000-SH溶液和磁珠平衡至室温，优选为磁珠被放置在磁力架上，弃上 清液，加入冰乙酸溶液轻轻涡旋，收集珠子，立即加入NH2-PEG5000-SH溶液，然后涡旋振荡，溶液在室温旋转孵育，洗涤收集磁珠，4℃储存备用得到PEG修饰的磁珠。
  7. 一种权利要求1所述的基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠在弱相互作用蛋白质的捕获中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述应用取蛋白样品加入磁珠中，室温缓慢振荡，同时用紫外灯照射，激发双吖丙啶与捕获蛋白产生共价连接，随后用收集磁珠，清洗，磁珠上偶联的蛋白质分子即为所捕获蛋白质分子，加入还原试剂裂解NH2-PEG-SH与光亲和多肽探针间形成的二硫键，释放结合有目标蛋白质分子的光亲和多肽探针，聚丙烯酰胺凝胶电泳分离，质谱鉴定捕获的蛋白质分子。
PCT/CN2020/116950 2019-12-06 2020-09-23 一种基于光亲和共价连接的蛋白质分子弱相互作用捕获磁珠及其制备方法和应用 WO2021109684A1 (zh)

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