WO2010082743A2 - Method for checking and quantifying protein-ligand interaction by combining a crosslinking method and accelerator mass spectroscopy - Google Patents

Abstract

The present invention provides a novel method for quantifying the production of a protein-ligand composite. Further, the present invention provides a method for checking and quantifying in vitro and in vivo protein-DNA interaction by combining a crosslinking method and accelerator mass spectroscopy. Further, the present invention provides a method for defining a nucleotide excision repair (NER) pathway.

Description

Methods for Identification and Quantification of Protein-ligand Interactions by Combination of Crosslinking and Accelerated Mass Spectrometry

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.

About 30,000 human genes have been discovered so far, and genomics research focuses on identifying the mechanism by which these genes form the complexity of life. One area of increasing interest is understanding the components and kinetics of molecular complexes formed to drive dynamic metabolic processes. Main topics of interest are the identification of binding partners, the definition of specific recognition sites and the quantification of the occupied levels of molecules involved.

To date, various in vivo and in vitro methods have been used to reveal protein-ligand interactions. One of the simplest and direct ways to study these interactions is to use crosslinking methods, for example using ultraviolet light or formaldehyde to covalently attach proteins to their target molecules. While these methods have made progress in the identification of molecular complexes, not all molecules or interaction sites are identifiable by such techniques and the quantification of binding kinetics and speed has been limited by the lack of detection sensitivity and accuracy.

Methods that allow greater sensitivity and accuracy in the quantification of crosslinked products will allow for a more detailed assessment of binding and inhibition rates, and importantly allow them to be studied using lower concentrations of molecules in small samples. And provide a more quantitative assessment of the kinetics of molecular complex formation and rate at biologically relevant concentrations. This presents new opportunities for the study of metabolic networks and for studying the role of epigenetic factors in cellular function.

On the other hand, in vitro measurements of the affinity and specificity of protein-ligand interactions have long been available for many proteins. However, these interactions are almost certainly modified by the complex intracellular environment and other molecules that compete with the ligand. If we can measure specific protein-ligand interactions in vivo, we can not only get a more accurate description of how the protein behaves, but also compare the binding in vitro and in vivo. It will be understood how the intrinsic binding specificity of is modified in the cell.

However, quantifying interactions in vivo has proved significantly more difficult than in vitro. However, over the last decade, methods for covalently crosslinking proteins to DNA in vivo (conventional crosslinking methods) have been developed to measure the interaction of sequence-specific DNA-binding proteins with genomic DNA. have. In brief, the crosslinking method is used to induce covalent crosslinking between protein and DNA using a crosslinking agent such as UV and formaldehyde, and then purify the crosslinked protein-DNA complex and cleavage using a restriction enzyme. do. Subsequently, after immunoprecipitation of the protein-DNA complex, immunoprecipitated DNA-protein complexes are identified using Southern blot or PCR amplification. Early work merely studied the interaction of a few gene fragments with proteins. More recent studies have combined DNA microarray technology with in vivo crosslinking to measure the interaction of several sequence-specific transcription factors in yeast with thousands of potential binding sites throughout the yeast genome. However, conventional crosslinking strategies have some drawbacks: 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. will be.

Accelerated mass spectrometry, meanwhile, 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". Labels Modern tracks up to 97.8 amol ¹⁴ C per milligram, corresponding to 6.11 femtocury (fCi) per milligram or 0.0136 dpm per milligram carbon (standard reference material-4990). ± 0.25% for modern materials and ± 10% for Pleistocene samples Accelerated mass spectrometry is a very simple analysis that produces very sensitive and precise quantification Accelerated mass spectrometry has become a common tool for pharmacokinetic analysis, where Small (μg) doses containing labeled portions can be chromatographed without relying on prior knowledge of secondary standards or metabolic pathways. It is quantified directly on the re-accelerating mass spectrometry without introducing a pre-specified or molecular modification or internal standard, is specific for only the label compound in any biological or chemical media. ¹⁴ C is relatively low, almost constant natural background is Accelerated mass spectrometry provides higher quantitative sensitivity to stable isotopes or other probes (TRACER) Some fluorescence quantifies at amol levels, while they require derivatization processes that are not suitable for in vivo tracing. This creates a probe that is not chemically equivalent, and is generally difficult to apply to many biochemical systems.

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.

We have invented a new method for the identification and quantification of protein-ligand complex formation after crosslinking using accelerated mass spectrometry detection. This method also provides increased sensitivity and potentially higher accuracy in the quantification of binding rates. By combining accelerated mass spectrometry with well-established crosslinking methods, not only protein-ligand interactions can be observed, but also higher sensitivity than conventional dye-based staining methods.

Accordingly, it is an object of the present invention to provide a new method for the quantification of protein-ligand complex formation. More specifically, 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.

It is also an object of the present invention to provide a method for identifying and quantifying protein-DNA interaction in vitro and in vivo by a combination of crosslinking methods and accelerated mass spectrometry.

It is also an object of the present invention to provide a method for identifying a nucleotide excision repair (NER) pathway by a combination of crosslinking methods and accelerated mass spectrometry.

In one aspect, the invention provides a method of identifying and quantifying protein-ligand interactions.

Specifically, 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. Provided are methods for identifying and quantifying protein-ligand interactions, including comparing signals from and signals from accelerated 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.

In the first step of the method, 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. At this time, an example of 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. Preferably 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.

In vitro, 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. For the separation of the protein-ligand complex, polyacrylamide gel electrophoresis (PAGE), which is commonly used for the separation of proteins and the like, High performance liquid chromatography (HPLC), high speed protein liquid chromatography (FPLC) and the like can be used.

Polyacrylamide gel electrophoresis, high performance liquid chromatography, high speed protein liquid chromatography, and the like for the separation of proteins or DNA are well known in the art, for example, see Molecular Cloning: A Laboratory Manual. . Cold Spring Harbor Laboratory Press].

The fourth step of the method is a step of measuring the protein-ligand complex separated by electrophoresis, etc. by accelerated mass spectrometry.

In the case of separating protein-ligand complexes by polyacrylamide gel electrophoresis, after electrophoresis, the gel is cut to a constant size and the gel pieces are each measured by accelerated mass spectrometry.

In order to measure the gel 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.

In the case of separating protein-ligand complexes by high performance liquid chromatography or high-speed protein liquid chromatography, after collecting each fraction obtained over time by chromatography, the solvent is removed and the organic substance remaining in each fraction is dried. After oxidizing, copper oxide (CuO) is used to oxidize and 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.

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).

In the third step of the method, in the case of using a method such as high performance liquid chromatography or high speed protein liquid chromatography to separate the protein-ligand complex, the signals obtained by the accelerated mass spectrometry are chromatographed for each fraction obtained by chromatography. Compare with gram.

As such, 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.

In the present invention, a liquid scintillation counter (LSC) may be used instead of accelerated mass spectrometry for radioisotope analysis.

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.

When the protein-ligand complexes are separated by high performance liquid chromatography or high speed protein chromatography, a uniform solution can be obtained. Thus, radioisotopes can be analyzed by a liquid scintillation counter without additional processes such as blotting.

In another aspect, 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.

Specifically, 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.

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. At this time, 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. Preferably 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. (PNAS, 104, 11203-11208 (2007), DOI: 10. 1073 / pnas.0701733104, Chem. Res. Toxicol. 20, 1745-1751 (2007), DOI: 10.1021 / tx700376a, and Chem.Res.Toxicol. 19, 622-626 (2006), DOI: 10.1021 / tx060058c).

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. In order to crosslink the radioisotope labeled DNA with the protein, UV light, form 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. For the extraction of the DNA crosslinked with the protein, 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. For the degradation of DNA, a variety of 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. In order to locate the complex after separation of the protein-DNA complex by gel electrophoresis, a staining method using a Coomassie ^® dye solution (BioRad) may be used.

When using high performance liquid chromatography or high protein liquid chromatography to separate the protein-DNA complex, each fraction obtained by chromatography is collected and then used for accelerated mass spectrometry.

In the fifth step of the method, the protein-DNA complex is measured by accelerated mass spectrometry. In the case where the protein-DNA complex is separated by gel electrophoresis, the gel is measured in a small amount to measure the gel by accelerated mass spectrometry. After cutting into pieces of size and converting these gel pieces into graphite, 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.

In addition, in the case of using high performance liquid chromatography or high-speed protein liquid chromatography instead of gel electrophoresis to separate protein-DNA complexes, 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.

In addition, the 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.

In another aspect, the present invention provides a method of identifying binding proteins for specific DNA sequences using shuttle vectors and accelerated mass spectrometry.

Specifically, 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. Iii) ligation of the duplex DNA into a plasmid, iv) introducing the plasmid containing the duplex DNA into an in vivo or ex vivo system, v) covalently binding the protein with the plasmid vi) isolating and digesting the plasmid DNA-protein complexes; vii) isolating the plasmid DNA-protein complexes; viii) measuring the isolated DNA-protein complexes by accelerated mass spectrometry.

By the above process, a novel binding protein that binds to an oligonucleotide having a specific sequence can be identified.

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.

Methods for preparing oligonucleotides containing radioisotopes at a given position using radiolabeled nucleotides, forming duplex DNA by annealing with complementary oligonucleotides, ligating duplex DNA to plasmids, and the like are described herein. It is well known as a common technical method in the art, and these methods are described, for example, in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press.

Any radioisotope measurable by an accelerated mass spectrometer can be used in the method, preferably ¹⁴ C.

In the fourth step of the method, 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.

By comparing the signal obtained by the accelerated mass spectrometry with the staining picture of the electrophoretic gel obtained in the seventh step, it is possible to identify and quantify the binding protein binding to the DNA of the specific sequence.

In addition, the 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.

In another aspect, the present invention provides a method for identifying a nucleotide excision repair (NER) pathway.

Specifically, 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.

By checking the radioactivity after treatment with the NER enzyme of DNA containing radioisotopes at different positions by the above method, it is possible to confirm how many nucleotides are removed during the nucleotide removal repair pathway.

As described above, methods for preparing oligonucleotides containing radioisotopes at a given position using radiolabeled nucleotides, methods for forming duplex DNA by annealing with complementary oligonucleotides, and the like are common in the art. It is well known as a technical method and these methods are described, for example, in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press.

Any radioisotope measurable by the accelerated mass spectrometer and introduced into the nucleotides can be used in the method, preferably ¹⁴ C.

Materials and methods for introducing damage to the duplex DNA in the third step of the method are known in the art, for example, 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.

In addition, the 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. In addition, new proteins that specifically bind to DNA of specific sequences can be efficiently identified and quantified.

In addition, 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-α (ER-α) 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.

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.

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.

Through the following examples will be described the present invention in more detail. This embodiment is intended to illustrate the present invention in more detail and is not intended to limit the scope of the present invention to these examples.

Example

Example 1 Accelerated Mass Spectrometry-Based Crosslinking Assay for In Vitro Protein-ligand Interactions

Due to the high binding affinity (~ 0.2 nM) between estrogen receptor-α (ER-α) and 17β-estradiol (E2) and the well-defined biochemical properties of this complex, it reveals in vitro protein-molecular interactions Estrogen receptor-α and radiocarbon-labeled 17β-estradiol were selected for this purpose.

Since 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.

2.1 fmol E2 (American radiolabel) containing 21.4 pmol of ER-α (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.

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-α without any treatment, (c) 135 pmol ER without any treatment. -α, (d) 135 pmol (1.35 μM) of ER-α 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). (E) 21.4 pmol of ER-α in binding buffer was incubated with 2.1 fmol of E2 containing 0.91 fmol of [ ¹⁴ C] E2, followed by formaldehyde and lysine ((0.09% w / v and 125 mM, respectively). Final concentration), (f) 21.4 pmol of ER-α in binding buffer was treated with 2.1 fmol of E2 containing 0.91 fmol of [ ¹⁴ C] E2 and only after incubation with lysine (125 mM final concentration).

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.

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-α-E2 crosslinking protein (lane (d)) and the original ER-α 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-α protein and the E2 ligand.

On the other hand, as a control experiment, the results of the experiment under the same conditions except that no formaldehyde was added to the sample (lane (f) of FIG. 1A), and no radiocarbon was observed in the gel fragments from the lane (f). No [ ¹⁴ C] E2 eluted from the gel and did not stay in the region associated with the 66 kD ER-α protein.

In addition, lane (d) of FIG. 1A treated with formaldehyde after incubation of unlabeled E2 and ER-α showed a higher molecular weight complex compared to ER-α (lane (c)) without any treatment. Confirmed the formation (ie, lower mobility).

These results show that 21.4 pmol (˜1.4 μg) of ER-α is detectable by AMS but not by Coomassie staining (lanes (e) or (f) in FIG. 1A). The detection limit using dye binding in this experiment was 135 pmol (˜9.0 μg) of ER-α (lane (d)).

More importantly, the ER-α used in this experiment was partially purified ER-α (80% purity guaranteed by the manufacturer), while the radiocarbons contained the covalently bound ER-α- [ 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-α from nonspecific binding.

Example 2 Accelerated Mass Spectrometry-Based Crosslinking Assay for Protein-DNA Interaction

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).

In addition, 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 experimental procedure of this example is summarized in FIG. 4C and as follows, and the results are shown in FIG. 5.

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). With 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. In FIG. 5A, lanes I and II are control experiments without photo-crosslinking, lanes III and IV are HMGB1 full-length proteins, and lanes V and VI are HeLa nuclear extracts. After the obtained gel was cut to a constant size (right of FIG. 5A), the radiocarbon content of the cut gel pieces was measured using AMS (FIG. 5B). The results in 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.

Example 3 Pathway Analysis of Nucleotide Excision Repair (NER) Enzymes for Damaged DNA

In a similar manner as in Example 2, 61 base pair 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.

As shown in 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). These results show that the damaged part of DNA is removed by Nucleotide Excision Repair (NER), and it can be confirmed to what extent the damaged part around the damaged part is repaired by the method of the present invention.

Claims (26)

1. i) introducing the radiolabeled ligand molecule into an in vitro, ev vivo or in vivo system,

   ii) covalently crosslinking the ligand molecule to the target protein,

   iii) separating the protein-ligand complex,

   iv) measuring the isolated protein-ligand complex by Accelerator Mass Spectrometry (AMS), and

   v) A method for identifying and quantifying protein-ligand interactions, comprising comparing signals from AMS with signals from conventional detection methods.
2. The method of claim 1, wherein the radiolabeled molecule is selected from the group consisting of drugs, hormones, metabolites, nucleotides in DNA or RNA, and proteins labeled with radioisotopes.
3. The method of claim 2, wherein the radioisotope is ¹⁴ C. 4.
4. The method of claim 1, wherein the in vitro, ex vivo or in vivo system is selected from the group consisting of purified proteins, cell extracts, nuclear extracts, prokaryotic cells, and eukaryotic cells.
5. The method of claim 1, wherein the crosslinking of the ligand and the protein is made by a crosslinking agent selected from the group consisting of UV light, formaldehyde, glutaraldehyde, and photoactivating linkers.
6. The method of claim 1, wherein the separation of the ligand-protein complex is accomplished by a method selected from the group consisting of polyacrylamide gel electrophoresis, high performance liquid chromatography, and fast protein liquid chromatography.
7. The method of claim 6, wherein the separation of the ligand-protein complex is by polyacrylamide gel electrophoresis.
8. i) introducing a radioisotope-labeled molecule into genomic or cellular DNA,

   ii) incubating the genomic or cellular DNA containing the radioisotope-labeled molecule with a cell extract or nuclear extract and crosslinking the DNA with the protein,

   iii) extracting and digesting the protein-DNA complex,

   iv) separating 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.
9. The method of claim 8, wherein the radioisotope-labeled molecule is selected from normal nucleosides, oxidized nucleosides, or DNA-damaging agents labeled with radioisotopes.
10. The method of claim 9, wherein the DNA-damaging agent is a platinum-based anticancer drug, nitrogen mustard, cyclophosphamide, melphalan, chlorambucil, bis-chloroethylnitrosourea, streptozosin, laying The method is selected from the group consisting of plates and thiotepa.
11. The method of claim 8, wherein the crosslinking of the DNA and the protein is made by a crosslinking agent selected from the group consisting of UV light, formaldehyde, glutaraldehyde, and photoactivating linkers.
12. The method of claim 8, wherein the separation of the DNA-protein complex is accomplished by a method selected from the group consisting of polyacrylamide gel electrophoresis, high performance liquid chromatography, and fast protein liquid chromatography.
13. The method of claim 12, wherein the separation of the DNA-protein complex is by polyacrylamide gel electrophoresis.
14. The method of claim 8, wherein the radioisotope is ¹⁴ C. ¹⁰ .
15. i) preparing an oligonucleotide containing a radioisotope at a given position using radiolabeled nucleotides,

    ii) annealing the oligonucleotides to complementary oligonucleotides to form duplex DNA,

    iii) ligation of said duplex DNA into a plasmid,

    iv) introducing the plasmid containing the duplex DNA into an in vivo or ex vivo system,

    v) covalently binding the protein with the plasmid,

    vi) separating and digesting the plasmid DNA-protein complex,

    vii) separating the plasmid DNA-protein complex digest, and

    viii) identifying and quantifying binding proteins for specific DNA sequences, comprising measuring the isolated DNA-protein complexes by accelerated mass spectrometry.
16. 16. The method of claim 15, wherein a crosslinker is selected from the group consisting of UV light, formaldehyde, glutaraldehyde, and photoactivated linkers for crosslinking DNA and protein.
17. The method of claim 15, wherein the plasmid containing the duplex DNA is introduced into the in vivo system by electroporation into cells.
18. 16. The method of claim 15, wherein said duplex DNA is incubated with a nuclear extract or a cell extract and introduced into the ex vivo system.
19. 16. The method of claim 15, wherein the separation of the plasmid DNA-protein complex digest is by a method selected from the group consisting of polyacrylamide gel electrophoresis, high performance liquid chromatography, and high speed protein liquid chromatography.
20. The method of claim 19, wherein the separation of the plasmid DNA-protein complex digest is by polyacrylamide gel electrophoresis.
21. The method of claim 15, wherein the radioisotope is ¹⁴ C. ¹⁶ .
22. i) preparing a set of oligonucleotides in which radioisotope labels are introduced sequentially at different positions,

    ii) preparing a duplex DNA set using oligonucleotides complementary to each oligonucleotide of the oligonucleotide set,

    iii) introducing damage to the duplex DNA,

    iv) incubating the duplex DNA with NER enzyme or nuclear extract and dNTPs, and

    v) A method for identifying a nucleotide excision repair (NER) pathway comprising the steps of isolating the resulting DNA and measuring it by accelerated mass spectrometry to confirm radioactivity.
23. The method of claim 22, wherein the separation of the resulting DNA is performed by a method selected from the group consisting of polyacrylamide gel electrophoresis, high performance liquid chromatography, and fast protein liquid chromatography. .
24. The method of claim 23, wherein the separation of DNA is by polyacrylamide gel electrophoresis.
25. 23. The method of claim 22, wherein the radioisotope is ¹⁴ C.
26. 23. The method of any one of claims 1, 8, 15 and 22, wherein the measurement of radioactivity is made by liquid scintillation counter instead of accelerated mass spectrometry.

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