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WO2010082743A2 - Méthode de vérification et de quantification d'une interaction protéine-ligand par combinaison d'une méthode de réticulation et de la spectrométrie de masse accélérée - Google Patents

Méthode de vérification et de quantification d'une interaction protéine-ligand par combinaison d'une méthode de réticulation et de la spectrométrie de masse accélérée Download PDF

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WO2010082743A2
WO2010082743A2 PCT/KR2010/000147 KR2010000147W WO2010082743A2 WO 2010082743 A2 WO2010082743 A2 WO 2010082743A2 KR 2010000147 W KR2010000147 W KR 2010000147W WO 2010082743 A2 WO2010082743 A2 WO 2010082743A2
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dna
protein
complex
ligand
crosslinking
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WO2010082743A3 (fr
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하상수
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Kyung Hee University
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Kyung Hee University
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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

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  • the present invention relates to methods for the identification and quantification of protein-ligand complex formation. More specifically, the present invention relates to a method for identifying and quantifying protein-ligand complex formation by a combination of accelerator mass spectrometry and crosslinking.
  • Accelerated mass spectrometry was first developed for geological and archaeological studies. Accelerated mass spectrometry is an isotope ratio mass spectrometry for accurately determining the concentration of very rare ( ⁇ 1:10 9 ) isotopes in isolated samples. Accelerated mass spectrometry is therefore applied to determine the concentration of long-lived radioisotopes, such as 14 C, where decay counting is an inefficient method for quantification.
  • the cosmic ray interaction maintains the atmosphere at a nearly constant radiocarbon concentration of 1.2: 10 12 , which is the same concentration that approximates all the plants and animals that eat them to the unit "modern".
  • the high sensitivity of accelerated mass spectrometry can affect experimental design in several ways: 1) the radioactive isotopic dose can be reduced to insignificant levels of radiolysis, harmful waste streams, and human exposure; and 2) biological systems.
  • the chemical dose of ethanol is minimized below the physiological and toxic levels, enabling realistic analysis of the effects resulting from low chemical doses, and 3) even if the sampled material must be fractionated into specific biomolecules prior to quantification. Sample size can be reduced to an amount that can be obtained from a limited, often non-invasive process.
  • the present invention aims to provide higher accuracy and sensitivity than conventional methods by combining crosslinking methods and accelerated mass spectrometry in the quantification of protein-ligand interactions.
  • NER nucleotide excision repair
  • the invention provides a method of identifying and quantifying protein-ligand interactions.
  • the present invention provides a process for i) introducing a radiolabeled ligand molecule into an in vitro, ev vivo or in vivo system, ii) sharing the ligand molecule to a target protein.
  • Crosslinking iii) separating the protein-ligand complex, iv) measuring the isolated protein-ligand complex by Accelerator Mass Spectrometry (AMS), and v) from a conventional detection method.
  • AMS Accelerator Mass Spectrometry
  • the first step of the method is the introduction of the radiolabeled ligand molecule in vitro, in an ex vivo or in vivo system.
  • the radiolabeled molecule is a ligand molecule for which the binding protein is to be identified, which may be a drug, hormone, metabolite, nucleotide in DNA or RNA, or the like labeled with a radioisotope.
  • a radioisotope that can be used for labeling a ligand is 14 C, but is not limited thereto, and other isotopes may be used depending on the type of ligand to be labeled.
  • a ligand labeled 14 C is used.
  • Ligands labeled with radioactive isotopes include Sigma-Aldrich, Cambridge Isotope Laboratories, American Radiolabeled Chemical, Inc., Moravec Biochemical. It can be purchased from companies such as Moravek Biochemicals and can also be obtained through chemical synthesis.
  • ex vivo or in vivo systems used in step 1 of the method contain candidate proteins capable of binding to labeled ligand molecules, such as purified proteins, cell extracts, nuclear extracts, prokaryotic cells, eukaryotic cells, and the like. May be any system.
  • the second step of the method is a step of covalently crosslinking the ligand molecule to the target protein, wherein the crosslinking agent for covalently binding the radiolabeled ligand molecule to the target protein is UV light, formaldehyde, glutaraldehyde, or light.
  • Crosslinking agents commonly used for inducing covalent crosslinking between proteins and ligands, such as activating linkers, can be used.
  • the third step of the method is to separate the ligand-protein complex formed by crosslinking.
  • polyacrylamide gel electrophoresis PAGE
  • HPLC High performance liquid chromatography
  • FPLC high speed protein liquid chromatography
  • the fourth step of the method is a step of measuring the protein-ligand complex separated by electrophoresis, etc. by accelerated mass spectrometry.
  • the gel is dried, oxidized using copper oxide (CuO), and the generated carbon dioxide is collected.
  • the obtained carbon dioxide is then reduced with titanium hydride to obtain graphite in solid form, which is then measured by an accelerated mass spectrometer according to the manufacturer's instructions.
  • the fifth step of the method is a step of comparing and analyzing the signal measured by the accelerated mass spectrometry and the signal by the conventional signal detection method, and the conventional detection method of the signal for comparison with the signal measured by the accelerated mass spectrometry Dyeing method using dyes most commonly used in gel electrophoresis. Dyes that may be used in such dyeing methods include Coomassie ® dye solutions (Bio-Rad).
  • the signals obtained by the accelerated mass spectrometry are chromatographed for each fraction obtained by chromatography. Compare with gram.
  • the signal by the accelerated mass spectrometry enables the identification and quantification of a very small amount of protein-ligand complex, thereby quantifying the interaction between the protein and the ligand more accurately and sensitively than the conventional method.
  • a liquid scintillation counter may be used instead of accelerated mass spectrometry for radioisotope analysis.
  • the radioactive isotope-containing material In order to use the liquid scintillation counter, the radioactive isotope-containing material must be uniformly dissolved in a universal solvent. However, since the gel is a polymer, it cannot be dissolved. After blotting it is dissolved in a solvent such as water and analyzed by liquid scintillation counter.
  • the present invention provides a method for identifying and quantifying the interaction of protein with DNA by a combination of crosslinking methods and accelerated mass spectrometry.
  • the present invention relates to i) introducing radioisotope-labeled molecules into genomic or cellular DNA, ii) incubating genomic or cellular DNA and cell extracts or nuclear extracts containing the radioisotope-labeled molecules. Cross-linking DNA and protein, iii) extracting and digesting the protein-DNA complex, iv) isolating the protein-DNA complex, v) measuring the protein-DNA complex by accelerated mass spectrometry And vi) comparing the signal by accelerated mass spectrometry with the signal by conventional methods for identifying DNA-protein complexes.
  • the first step of the method is the step of introducing a radioisotope-labeled molecule into genomic or cellular DNA, wherein the radioisotope-labeled molecule is a normal nucleoside, oxidized nucleoside or DNA-damaging agents and the like.
  • DNA-damaging agents that can be used in the method include platinum-based anticancer drugs, nitrogen mustard, cyclophosphamide, melphalan, chlorambucil, bis-chloroethylnitrosourea, streptozosin, DNA-damaged anticancer drugs, including but not limited to busulfan and thiotepa.
  • Radioisotopes normal nucleosides, oxidized nucleosides, or DNA-damaging agents labeled with radioisotopes are purchased from various companies selling chemicals labeled with radioisotopes as described above or synthesized. It can be commissioned and can also be obtained through chemical synthesis.
  • an example of a radioisotope that can be used for labeling is 14 C, but is not limited thereto, and other isotopes may be used depending on the molecule to be labeled.
  • 14 C is used for labeling.
  • Incorporation of radioisotope labeled nucleosides, anticancer drugs, etc. into genomic or cellular DNA can be accomplished by culturing the cells in a culture medium containing such radioisotope labeled molecules, as known in the art.
  • the second step of the method is incubating the radioisotope labeled DNA with a cell extract or nuclear extract and crosslinking the DNA with the protein.
  • crosslinking agents commonly used in the art may be used, including but not limited to aldehydes, glutaraldehydes, photoactivated linkers, and the like.
  • the third step of the method is the step of extracting and digesting the DNA bound to the protein from the reactant.
  • a technique known in the art may be used, for example, nitro DNA-protein complexes can be extracted by blotting with cellulose (nitrocellulose) or nylon or PVDF.
  • restriction enzymes known in the art for specifically cleaving a site of a specific sequence may be used, and an appropriate restriction enzyme may be selected and used in consideration of the DNA sequence.
  • the fourth step of the method is a step of separating the degraded protein-DNA complex as described above, gel electrophoresis or high performance liquid chromatography or high-speed protein liquid chromatography can be used.
  • Gel electrophoresis for separation of protein-DNA complexes may utilize 1D or 2D polyacrylamide gel electrophoresis.
  • Gel electrophoresis is well known in the art and is described, for example, in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press.
  • a staining method using a Coomassie ® dye solution (BioRad) may be used.
  • each fraction obtained by chromatography is collected and then used for accelerated mass spectrometry.
  • the protein-DNA complex is measured by accelerated mass spectrometry.
  • the gel is measured in a small amount to measure the gel by accelerated mass spectrometry.
  • radioisotope levels are measured by an accelerated mass spectrometer. The method of converting the gel pieces to graphite and measuring the radioisotope levels by an accelerated mass spectrometer is as described above.
  • Binding proteins binding to DNA are identified in vitro and in vivo by comparing the signal identified by the accelerated mass spectrometer with the location of the DNA-protein complex identified by gel staining after gel electrophoresis and determining the radioisotope content. Can be quantified.
  • each fraction obtained over time by chromatography is collected as described above.
  • the organic material remaining in each fraction is dried, and then oxidized using copper oxide (CuO), and only carbon dioxide generated is collected.
  • the obtained carbon dioxide is then reduced with titanium hydride to give graphite in solid form, which is then measured by an accelerated mass spectrometer.
  • CuO copper oxide
  • liquid scintillation counter may be used instead of the accelerated mass spectrometry for radioisotope analysis in the method of the present invention as described above.
  • the present invention provides a method of identifying binding proteins for specific DNA sequences using shuttle vectors and accelerated mass spectrometry.
  • the present invention provides a method for producing a duplex DNA by i) preparing an oligonucleotide containing a radioisotope at a given position using radiolabeled nucleotides, and ii) annealing the oligonucleotide to a complementary oligonucleotide.
  • Shuttle vectors are plasmids designed to grow in two different host species, and DNA inserted into the shuttle vector can be tested or manipulated in two different cell types.
  • radioisotope measurable by an accelerated mass spectrometer can be used in the method, preferably 14 C.
  • the plasmid DNA can be introduced into the cell by, for example, electroporation to introduce the plasmid DNA into the in vivo system. Or by incubating with the cell extract.
  • Crosslinking of the DNA and the protein in the fifth step of the method is commonly used in the art as described above, including but not limited to UV light, formaldehyde, glutaraldehyde, or photoactivating linkers, and the like. Crosslinkers can be used.
  • Separation and digestion of the plasmid in the sixth step of the method can be carried out using techniques well known in the art, such techniques are described in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press] and the like, the decomposition of the plasmid can be made using a variety of known restriction enzymes can be used by selecting an appropriate restriction enzyme in consideration of the oligonucleotide sequence.
  • Separation of the plasmid DNA-protein complex digest in the seventh step of the method may be performed by polyacrylamide gel electrophoresis, high performance liquid chromatography, high-speed protein liquid chromatography, etc., after separation by electrophoresis, etc.
  • the dye can be photographed by dyeing the gel with the dye and then used for comparison with signals obtained by accelerated mass spectrometry of the gel.
  • Accelerated mass spectrometry of the DNA-protein complex in the eighth step of the method is performed by converting the fraction obtained by gel or chromatography into graphite in the same manner as described for the other aspects of the present invention, followed by an accelerated mass spectrometer. It can measure by.
  • liquid scintillation counter may be used instead of the accelerated mass spectrometry for radioisotope analysis in the method of the present invention as described above.
  • the present invention provides a method for identifying a nucleotide excision repair (NER) pathway.
  • NER nucleotide excision repair
  • the present invention provides a method for preparing a duplex DNA set using i) preparing oligonucleotide sets sequentially introduced at different positions of radiolabels, ii) oligonucleotides complementary to each oligonucleotide of the oligonucleotide set. Step, iii) introducing damage to the duplex DNA, iv) incubating the duplex DNA with a NER enzyme or nuclear extract and dNTPs, and v) separating the resulting DNA and subjecting it to accelerated mass spectrometry. Measuring to confirm radioactivity.
  • radioisotope measurable by the accelerated mass spectrometer and introduced into the nucleotides can be used in the method, preferably 14 C.
  • cisplatin can be used, see Chem. Res. Toxicol. 20, 1745. -1751 (2007), DOI: 10.1021 / tx700376a, and Chem. Res. Toxicol. 19, 622-626 (2006), DOI: 10.1021 / tx060058c, and the like.
  • Separation of DNA in the fifth step of the method may be performed by gel electrophoresis, high performance liquid chromatography, high-speed protein liquid chromatography, etc. as described above, and the method of measuring the separated DNA by accelerated mass spectrometry is described above. As shown.
  • liquid scintillation counter may be used instead of the accelerated mass spectrometry for radioisotope analysis in the method of the present invention as described above.
  • the method of the present invention combines crosslinking methods with accelerated mass spectrometry to identify and quantify protein-ligand interactions, as well as provide higher accuracy and sensitivity than conventional dye-based staining methods.
  • new proteins that specifically bind to DNA of specific sequences can be efficiently identified and quantified.
  • the present invention requires that in order to confirm the interaction of the ligand with the protein, 1) the target protein must be specified in advance to examine its binding partner, and 2) specific antibodies for the target protein must be available for the immunoprecipitation step. To overcome the significant disadvantages of conventional crosslinking methods.
  • 1A is a gel photograph after crosslinking E2 containing Estrogen Receptor- ⁇ (ER- ⁇ ) and [ 14 C] E2 and separated by gel electrophoresis, and gels cut for measurement by accelerated mass spectrometry It is a figure which shows a part.
  • FIG. 1B is a graph showing the radiocarbon content of each gel piece obtained by measuring the gel pieces cut out from the gel of FIG. 1A by accelerated mass spectrometry.
  • FIG. 2 is a schematic summarizing a method according to one aspect of the present invention for identifying sequence specific binding proteins by crosslinking and accelerated mass spectrometry using genomic DNA or cellular DNA into which molecules labeled with radioisotopes have been introduced to be.
  • FIG. 3 is a schematic summarizing a method according to one aspect of the present invention for identifying sequence specific binding proteins by crosslinking and accelerated mass spectrometry using plasmids containing radiolabeled oligonucleotides.
  • 4A shows molecules labeled with carbon isotopes.
  • FIG. 4B depicts 25 base pair oligonucleotides for reaction with the molecules of FIG. 4A.
  • 4C summarizes the experimental procedure for determining the interaction between protein and DNA.
  • 5A shows the SDS-PAGE results (left) and cleaved gel pieces (right) of covalently bound protein-DNA conjugates.
  • 5B shows the radiocarbon content of gel pieces measured by AMS.
  • 6A shows oligonucleotides to be labeled with carbon isotopes.
  • 6B depicts an experimental procedure for the analysis of pathways of nucleotide removal repair enzymes for damaged DNA and sets of oligonucleotides labeled with carbon isotopes at different positions.
  • 6C depicts the radiocarbon content of oligonucleotides from which damaged DNA has been repaired, as measured by AMS.
  • Estrogen receptor- ⁇ and radiocarbon-labeled 17 ⁇ -estradiol were selected for this purpose.
  • E2 has two hydroxyl groups, these moieties were expected to react with formaldehyde to form a complex covalently bound to the reactive site on the estrogen receptor-a protein binding site.
  • E2 (American radiolabel) containing 21.4 pmol of ER- ⁇ (molecular weight 66.4 kD, 214 nM final concentration) (Sigma Aldrich, St. Louis, MO), [ 14 C] E2 0.91 fmol Binding Buffer [40 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10% (v / v) Glycerol, 10 mM with Dechemistry, Inc., St. Louis, MO Na 2 MoO 4 10 mM DTT] was incubated at 4 ° C. for 16 h.
  • Formaldehyde (0.09% w / v final concentration) was then added to the solution. After incubation and sometimes mixed for 15 minutes at room temperature, the lysine solution was added (125 mM final concentration) to quench the reaction and the resulting mixture NuPAGE No Bex (Novex) ® Bis-Tris gels (Invitrogen (Invitrogen Gel gel electrophoresis according to the manufacturer's instructions. After completion of the electrophoretic separation, the gel was stained with Bio-Rad Coomassie ® staining solution and photographed.
  • FIG. 1A The results are shown in Figure 1A.
  • the lanes in the gel photograph of FIG. 1A were as follows: (a) protein ladder, (b) 270 pmol ER- ⁇ without any treatment, (c) 135 pmol ER without any treatment. - ⁇ , (d) 135 pmol (1.35 ⁇ M) of ER- ⁇ in binding buffer was incubated with 2.1 fmol of unlabeled E2 followed by formaldehyde and lysine (0.09% w / v and 125 mM final concentration, respectively).
  • Lanes (e) and (f) of the gel were then cut into 0.8 x 0.4 cm 2 sized pieces as shown in FIG. 1A and the gel pieces were dried under vacuum and then converted to graphite for AMS measurement. Each obtained gel piece was measured by AMS for radiocarbon content.
  • FIG. 1B The radiocarbon content measurement results of the cut gel pieces are shown in FIG. 1B.
  • 1B shows that the radiocarbon levels are consistent with the background in all gel pieces except gel piece # 2 from lane (e).
  • Gel piece # 2 from lane (e) contained 83% of the added radiocarbons derived from [ 14 C] E 2.
  • the radiocarbon level in gel piece # 2 was estimated to be about 83% of the total radiocarbon in the system, based on the assumption that each gel piece weighs 3.5 mg.
  • This band corresponds to a molecular weight of 66 kD and is relative to the Coomassie-stained ER- ⁇ -E2 crosslinking protein (lane (d)) and the original ER- ⁇ protein standard shown in lanes (b) and (c). Co-eluted at the location. Since gel electrophoresis used denaturing conditions, this data shows a covalent bond between the ER- ⁇ protein and the E2 ligand.
  • lane (d) of FIG. 1A treated with formaldehyde after incubation of unlabeled E2 and ER- ⁇ showed a higher molecular weight complex compared to ER- ⁇ (lane (c)) without any treatment. Confirmed the formation (ie, lower mobility).
  • the ER- ⁇ used in this experiment was partially purified ER- ⁇ (80% purity guaranteed by the manufacturer), while the radiocarbons contained the covalently bound ER- ⁇ - [ 14 C] E2 complex. Since only detected gel fragments were detected, the AMS-based assay of the present invention can distinguish covalent binding of E2 to ER- ⁇ from nonspecific binding.
  • a molecule labeled with a carbon isotope such as FIG. 4A was prepared to examine the interaction of the protein with the DNA (American Radiolabeled Chemicals, Inc., St. Louis, MO).
  • Figure 4B is a 25 base pair oligonucleotide for reacting with the molecule of Figure 4A, synthesized by a phosphoramidite method using an Applied Biosystems 392 DNA / RNA synthesizer, One d (GpG) for platination (relatively large sized GG in FIG. 4B) and 3 'biotin group on the complementary strand.
  • This biotin group is a DNA-protein crosslinked addition using a solid support (streptavidin-coated magnetic beads (Promega)) having a streptavidin group after the reaction of FIG. 4A molecule with 25 base pair oligonucleotides. Introduced for easy separation of adducts.
  • the molecules labeled with the carbon isotopes of FIG. 4A are two orientational isomers (Pt isomers I and II) because the coordination sphere around platinum is asymmetric.
  • the isomers were separated using ion exchange HPLC and then each reacted with the prepared oligonucleotides (lanes I, III and V in FIG. 5A using Pt isomer I and lanes II, IV and VI using Pt isomer II).
  • high-mobility group B1 HMGB1 full-length proteins (lanes I, II, III and IV) known to selectively bind to plated DNA and all proteins in HeLa cells. After binding to HeLa nuclear extract (lanes V and VI) and cross-linked by photo-crosslinking method.
  • Photo-crosslinking was achieved by splitting 365 nm light at 0 ° C. for 2 hours using a UV Stratalinker. Covalently bound protein-DNA conjugates obtained by photo-crosslinking were separated using streptavidin-coated magnetic beads, then separated from the beads according to the manufacturer's manual, and separated by 10% SDS-PAGE. Gel photographs of SDS-PAGE are shown on the left side of FIG. 5A.
  • lanes I and II are control experiments without photo-crosslinking
  • lanes III and IV are HMGB1 full-length proteins
  • lanes V and VI are HeLa nuclear extracts. After the obtained gel was cut to a constant size (right of FIG.
  • FIG. 5B show that proteins that selectively bind to a given DNA or damaged DNA (eg, HMGB1 full-length protein) can be identified by the methods of the present invention with very high sensitivity and specificity.
  • oligonucleotides were prepared (by a step-by-step phosphoramidite method using an Applied Biosystems 392 DNA / RNA Synthesizer to position carbon isotopes at desired positions). Synthesized, FIG. 6A). The asterisks in FIG. 6B show the position of the carbon isotopes, representing the 15th and 56th adenine of the upper strand of DNA of FIG.
  • Prepared oligonucleotides containing carbon isotopes have one d (GpG) for platinum and the 3 'biotin group on the complementary strand. As in Example 2, this biotin group was introduced for easy separation of the final product using a solid support (streptavidin-coated magnetic beads, promega) with streptavidin groups.
  • FIG. 6B two prepared oligonucleotides labeled at different positions with carbon isotopes were reacted with cisplatin, respectively, and then reacted with HeLa nuclear extract in the presence of dNTPs to remove the damaged portion.
  • I was.
  • the resulting DNA was isolated using streptavidin-coated magnetic beads, then separated from the beads according to the manufacturer's manual, and its radiocarbon content was measured using AMS (FIG. 6C).
  • the amount of radiocarbon of DNA I in FIG. 6C is not zero because not all DNA is damaged by cisplatin (see FIG. 5A).

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Abstract

La présente invention concerne une nouvelle méthode de quantification de la production d'un composite protéine-ligand. En outre, la présente invention concerne une méthode de vérification et de quantification in vitro et in vivo de l'interaction protéine-ADN par combinaison d'une méthode de réticulation et de la spectrométrie de masse accélérée. En outre, la présente invention concerne une méthode de définition d'une voie de réparation par excision de nucléotides (NER).
PCT/KR2010/000147 2009-01-14 2010-01-11 Méthode de vérification et de quantification d'une interaction protéine-ligand par combinaison d'une méthode de réticulation et de la spectrométrie de masse accélérée Ceased WO2010082743A2 (fr)

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KR10-2009-0002900 2009-01-14
KR20090002900 2009-01-14
KR1020090095903A KR101137502B1 (ko) 2009-01-14 2009-10-09 가교결합과 가속 질량 분석법의 조합에 의한 단백질-리간드 상호작용의 확인 및 정량화 방법
KR10-2009-0095903 2009-10-09

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CN112920174A (zh) * 2021-02-02 2021-06-08 上海交通大学 一种光敏感型化合物及其制备方法和应用

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AU2004257191B2 (en) * 2003-07-03 2009-05-07 The Regents Of The University Of California Genome mapping of functional DNA elements and cellular proteins
US20070140967A1 (en) * 2005-12-19 2007-06-21 Wood Nichole L Agents and methods for analyzing protein interactions

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Publication number Priority date Publication date Assignee Title
CN112920174A (zh) * 2021-02-02 2021-06-08 上海交通大学 一种光敏感型化合物及其制备方法和应用

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