KR20180091345A - Methods for identification of proteins using phenolic compounds for protein labeling - Google Patents

Abstract

The present invention relates to a phenol compound and to a manufacturing method thereof, and specifically, to desthiobiotin-phenol (DBP), a manufacturing method thereof, a protein labeling method using the DBP, and a use for detecting a protein. The present invention relates to the method for detecting the protein using the phenol compound. More particularly, the present invention relates to the method for detecting the protein using the DBP, which can identify the labeled protein.

Description

Methods for identifying proteins using phenolic compounds for protein labeling [

The present invention relates to a composition for detecting a protein comprising a phenol compound for protein labeling, and a method for detecting a protein using the phenol compound. Specifically, the present invention relates to a method for detecting proteins by labeling proteins using Desthiobiotin-Phenol (DBP).

Over 18,000 proteins, known to be present in human cells, function in their own unique structures in the appropriate organelle.

Proteins form a tertiary structure through hydrogen bonding, ionic bonding, hydrophobic bonding, etc. through a secondary structure that changes from a linear primary structure to a spiral or screening structure, The three tertiary structures are gathered to form a quaternary structure. Because the structure of proteins is very complex and the structure changes fluidly depending on the surrounding environment in which the proteins exist, it is difficult to know the exact structure of the proteins.

Historically, biologists have used reactive biotin probes to identify membrane protein topology or to find solvent accessible residues on protein surfaces. However, biotin-ester or biotin maleimide has a long life span in aqueous solution (t1 / 2 = 1 hr) and can be applied only to purified proteins or cell membranes, thus there are limitations in application.

Recently, peroxidase was expressed in the mitochondrial matrix and intermembrane space, and the surrounding proteins were labeled with biotin phenol to map the space between the mitochondrial matrix and the membrane. However, since this method analyzes peptides that are not labeled with phenol groups, there is a possibility of obtaining false positive results. In addition, in the conventional case, due to the oxidizing action of the sulfur atom of biotin, not a single final product but various kinds of final products are mixed, and thus there is a problem that mass spectrometry of the peptide directly labeled with a phenol group can not be performed.

In order to solve the above problems, a probe for protein labeling using peroxidase has been developed and the present invention has been completed.

It is an object of the present invention to provide a method for mass spectrometric analysis of a labeled protein or peptide in which only one labeled protein or peptide exists because no sulfur is present in the compound, A phenolic compound having the following formula (1), and a method for producing the same.

[Chemical Formula 1]

Still another object of the present invention is to provide a composition for labeling a protein or peptide comprising the phenolic compound of formula (I), a stereoisomer thereof, a tautomer thereof, or a salt thereof.

Still another object of the present invention relates to a method for labeling a protein or peptide comprising a phenol compound of the above formula (1), a stereoisomer thereof, a tautomer thereof, or a salt thereof.

In addition, the object of the present invention is to provide a method for mass spectrometric analysis of a labeled protein or a peptide, wherein only one labeled protein or peptide is present because no sulfur element is present in the compound, A method for detecting a protein by labeling the protein using the phenol compound having the formula (1).

Still another object of the present invention is to provide a composition for detecting a protein comprising a phenol compound having the formula (1).

In order to solve the above problems, a probe for protein labeling using peroxidase has been developed and the present invention has been completed.

It is an object of the present invention to provide a method for mass spectrometric analysis of a labeled protein or peptide in which only one labeled protein or peptide exists because no sulfur is present in the compound, A phenolic compound having the following formula (1), and a method for producing the same.

[Chemical Formula 1]

Still another object of the present invention is to provide a composition for labeling a protein or peptide comprising the phenolic compound of formula (I), a stereoisomer thereof, a tautomer thereof, or a salt thereof.

Still another object of the present invention relates to a method for labeling a protein or peptide comprising a phenol compound of the above formula (1), a stereoisomer thereof, a tautomer thereof, or a salt thereof.

In addition, the object of the present invention is to provide a method for mass spectrometric analysis of a labeled protein or a peptide, wherein only one labeled protein or peptide is present because no sulfur element is present in the compound, A method for detecting a protein by labeling the protein using the phenol compound having the formula (1).

Still another object of the present invention is to provide a composition for detecting a protein comprising a phenol compound having the formula (1).

Since Desthiobiotin-Phenol (DBP), which is a phenol compound according to the present invention, does not contain a sulfur atom, only one kind of final product exists, and when a protein is expressed using the resultant product, It can detect. In detail, the desiobiotin-phenol according to the present invention binds to and labels the tyrosine residue of the protein, and information on the structure of the protein can also be confirmed.

An example of the present invention is to provide a phenol compound having the structure of the following formula (1), its stereoisomers (Sautomers), tautomers (Tautomers) or salts thereof.

[Chemical Formula 1]

An example of the present invention is to provide a method for preparing the compound of formula (1), which comprises coupling a compound of formula (2) with tyramine by an amide bond.

 (2)

An example of the present invention is to provide a composition for labeling a protein or peptide comprising the phenol compound of Formula 1, a stereoisomer thereof, a tautomer thereof, or a salt thereof.

In one embodiment of the present invention, there is provided a method for producing a target organism, the method comprising: introducing a polynucleotide comprising a gene encoding a targeting sequence and a gene encoding a peroxidase into a cell organelle where a target protein in the cell exists for labeling;

Treating the cell with the compound and hydrogen peroxide;

Disrupting the cell to obtain a cell lysate; And

And then labeling the cell lysate with streptavidin (SA) beads to obtain a labeled protein.

The present inventors have designed a new chemical probe. The novel chemical probe of the present invention can serve to label proteins with peroxidase.

In the present invention, when cells are treated with a peroxidase and the above-described chemical probe (phenol compound with debiotin) and hydrogen peroxide are treated, the action of the APEX expressed in the cells causes the deoxyribonucleotide deoxyribose The phenoxy radical to which the desistiobiotin is bound forms a covalent bond with a tyrosine group present in proteins present within a radius of about 5 to 50 nm, Resulting in binding of these proteins to destiobiotin. In addition, since the phenoxy radical to which the desoxybiotin is bound is present only for a short time not to cross the phospholipid membrane, when biotin-bound phenoxy radicals are generated in each of the cell organelles surrounded by the membrane, Biotin binds only to the intrinsic proteins in the organelle.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a novel phenol compound, its stereoisomers, tautomers or salts thereof, having the structure of the following formula (1).

 [Chemical Formula 1]

The novel compound having the structure of Formula 1 is Desthiobiotin-Phenol (DBP).

In the present specification, the term "stereoisomer" refers to a molecule having a different spatial arrangement between atoms and members, but it is divided into a diastereomer and an enantiomer depending on the difference in the bonding method.

As used herein, the term "tautomer " refers to a structural isomer that is readily convertible to an organic material, and the conversion between two tautomers is generally known to occur by altering the bonding position of the hydrogen atom.

Salts for the novel compounds, stereoisomers, or tautomers thereof include both acid addition salts of bases and stereochemically isomeric forms thereof, for example, addition salts of organic acids or inorganic acids. The salt is not particularly limited as long as it contains any salt that maintains the activity of the parent compound in the subject to be administered and does not cause undesirable effects.

Such salts include inorganic and organic salts and include, for example, acetic, nitric, aspartic, sulfonic, sulfuric, maleic, glutamic, formic, succinic, phosphoric, phthalic, tannic, tartaric, hydrobromic , Propionic acid, benzenesulfonic acid, benzoic acid, stearic acid, lactic acid, bicarboxylic acid, unsulfuric acid, non-tartaric acid, oxalic acid, butyric acid, calcium adduct, carbonic acid, chlorobenzoic acid, citric acid, But are not limited to, sulfonic acid, fumaric acid, gluceptic acid, eicosylic acid, pamoic acid, gluconic acid, methylnitric acid, malonic acid, hydrochloric acid, hydroiodo acids, hydroxynaphthol acid, isethionic acid, But are not limited to, hydrochloric acid, hydrobromic acid, hydrobromic acid, hydrobromic acid, hydrobromic acid, hydrobromic acid, hydrobromic acid, hydrobromic acid, menthyl acid, menthyl acid, mucilic acid, naphthylic acid, muconic acid, p-nitromethanesulfonic acid, hexamic acid, pantothenic acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, have.

Examples of the salts include salts of alkali and alkaline earth metals such as ammonium salts, lithium salts, sodium salts, potassium salts, magnesium salts and calcium salts such as benzathine, N-methyl-D-glucamine, Salts with organic bases such as arabinic salts, and salts with amino acids such as arginine, lysine. The salt form may also be converted to the free form by treatment with an appropriate base or acid.

The phenol compound generates radicals by peroxidase, and radicals can also be produced in the peroxidase itself. The phenol compound or the radical generated in the peroxidase may be propagated. The position where radicals are generated in the phenol compound and the process of propagating the radicals are shown in FIG.

The generated radical may form a covalent bond with a tyrosine group present in a protein or a peptide to bind to the protein or peptide. The distance that the radical binds to the protein or peptide may be 5 to 50 nm, But is not limited thereto.

The peroxidase may be selected from the group consisting of soybean ascorbate peroxidase, Arabidopsis ascorbate peroxidase, spinach ascorbate peroxidase, saccharomyces cerevisiae cytochrome c peroxide The enzymes Saccharomyces cerevisiae cytochrome c peroxidase, Phanerochaete chrysosporium PCL1, Phanerochaete chrysosporium PCM, Coprinopsis cinereosporium peroxidase, An enzyme consisting of the amino acid sequence of SEQ ID NO: 1, an enzyme consisting of the amino acid sequence of SEQ ID NO: 3, and an amino acid sequence of SEQ ID NO: 5 An enzyme selected from the group consisting of Soil can be, but is not limited to this.

The ascorbate peroxidase comprising the amino acid sequence of SEQ ID NO: 1 is encoded by the nucleotide sequence of SEQ ID NO: 2, and the ascorbate peroxidase comprising the amino acid sequence of SEQ ID NO: 3 is encoded by the nucleotide sequence of SEQ ID NO: Lt; / RTI >

In the present invention, the enzyme consisting of the amino acid sequence of SEQ ID NO: 5 is Horseradish Peroxidase (HRP). The hosadradish peroxidase exhibits activity in a secretory pathway with an oxidizing environment. In the present invention, in the intracellular space, such as an endoplasmic reticulum or Golgi, in the secretory pathway naturally oxidized, The same effect as the ascorbate peroxidase of No. 3 can be obtained. The horseradish peroxidase comprising the amino acid sequence of SEQ ID NO: 5 may be encoded by the nucleotide sequence of SEQ ID NO:

The present invention provides a process for preparing a compound of formula (1).

In the present invention, the compound of the formula (1) can be prepared by the reaction of the reaction formula (1) using the compound of the following formula (2) as a starting material.

[Chemical Formula 1]

(2)

[Reaction Scheme 1]

The amide bond may be formed by dissolving the compound of Formula 2 in an organic solvent, adding a coupling agent and tyramine, and the coupling agent may be formed by coupling the compound of Formula 2 with tyramine, But are not limited to, 2- (7-aza-1H-benzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and N, N-diisopropylethylamine (DIPEA).

The present invention provides a protein or peptide labeling composition comprising a phenol compound represented by the following general formula (1).

In the present invention, the protein labeling composition may include, but is not limited to, a phenol compound represented by the following formula (1), a stereoisomer thereof, a tautomer thereof, or a salt thereof:

[Chemical Formula 1]

The phenol compound generates radicals by peroxidase, and radicals can also be produced in the peroxidase itself. The phenol compound or the radical generated in the peroxidase may be propagated. The position where radicals are generated in the phenol compound and the process of propagating the radicals are shown in FIG.

The generated radical may form a covalent bond with a tyrosine group present in a protein or a peptide to bind to the protein or peptide. The distance that the radical binds to the protein or peptide may be 5 to 50 nm, But is not limited thereto.

The peroxidase may be selected from the group consisting of soybean ascorbate peroxidase, Arabidopsis ascorbate peroxidase, spinach ascorbate peroxidase, saccharomyces cerevisiae cytochrome c peroxide The enzymes Saccharomyces cerevisiae cytochrome c peroxidase, Phanerochaete chrysosporium PCL1, Phanerochaete chrysosporium PCM, Coprinopsis cinereosporium peroxidase, An enzyme consisting of the amino acid sequence of SEQ ID NO: 1, an enzyme consisting of the amino acid sequence of SEQ ID NO: 3, and an amino acid sequence of SEQ ID NO: 5 An enzyme selected from the group consisting of Soil can be, but is not limited to this.

In the present invention, the protein labeling composition can be used to label target proteins in cells for labeling.

The present invention provides a method for labeling a protein using a phenol compound represented by the following formula (1).

The method of labeling the protein can be performed by the following method, but is not limited thereto.

Specifically, the present invention provides a method for detecting a target organism, the method comprising: introducing a polynucleotide including a gene encoding a targeting sequence and a gene encoding a peroxidase into a cell organelle where a target protein in the cell exists for a label; And

Treating the cell with a phenol compound represented by the following formula (1) and hydrogen peroxide to label the protein in the cell with the phenol compound.

[Chemical Formula 1]

And the step of preparing the polynucleotide before the step of introducing the polynucleotide into the cell.

The process for producing the polynucleotide may include preparing a polynucleotide fused so that a gene encoding a targeting sequence and a gene encoding a peroxidase are linked to a cell organelle in which the target protein exists, no.

In the process of preparing the polynucleotide, the cell organelles may be selected from the group consisting of mitochondria, endoplasmic reticulum, nucleus, cytoplasm, and Golgi. The gene coding for the targeting sequence in the cell organelle may include a mitochondrial substrate expression gene, But are not limited to, a gene, a space expression gene between mitochondrial double membranes, a nuclear expression gene, an extracellular membrane expression gene, an endoplasmic reticulum expression gene, an endoplasmic reticulum expression gene, or an intracellular expression gene. The genes encoding the targeting sequences into the organelles are shown in Table 1 below.

Plasmid Characteristic Promoter / Vector Detail mito-APEX NotI-mito-BamHI-V5-APEX-Stop-XhoI
Mitochondrial substrate expression CMV / pCDNA3 mito matrix targeting sequence: MLATRVFSLVGKRAISTSVCVRAH
V5: GKPIPNPLLGLDST APEX-NES
NotI-Flag-APEX-NES-Stop-XhoI
Cytoplasmic expression CMV / pCDNA3 NES: LQLPPLERLTLD IMS-APEX NotI-LACTB (1-68) -BamHI-V5-APEX-Stop-XhoI
Expression of space between mitochondrial membrane CMV / pCDNA3 LACTB (1-68): MYRLLSSVTARAAATAGPAWDGGRRGAHRRPGLPVLGLGWAGGLGLGLGLALGAKLVVGLRGAVPIQS
APEX-NLS NotI-Flag-APEX-EcoRI-NLS-Stop-XhoI
Nuclear manifestation CMV / pCDNA3 NLS: PKKKRKVDPKKKRKVDPKKKRKV APEX-TM EcoRI-ss-HA-ApaI-APEX-SacII-myc-TM-Stop-NotI
Extracellular membrane expression CMV / pDisplay ss: Igk chain signal sequence in pDisplay (Invitrogen).
HA: YPYDVPDYA
myc: EQKLISEEDL
TM: transmembrane domain of PDGF receptor from pDisplay (Invitrogen) APEX-KDEL EcoRI-ss-BglII-APEX-V5-KDEL-Stop-NotI
Expression of endoplasmic reticulum CMV / pDisplay KDEL: ER retention motif
C1 (1-29) -APEX HindIII-C1 (1-29) -NotI-APEX-V5-Stop-XhoI
Expression of endoplasmic reticulum outer membrane CMV / pCDNA3 C1 (1-29): MDPVVVLGLCLSCLLLLSLWKQSYGGGKL (endoplasmic reticulum membrane anchor) W41AAPX-CAAX NotI-Flag-W41AAPX-EcoRI-CAAX-Stop-XhoI
Intracellular membrane expression CMV / pCDNA3 CAAX: RSKLNPPDESGPGCMSCKCVLS HRP-TM EcoRI-ss-HA-ApaI-HRP-SacII-myc-TM-Stop-NotI
Extracellular membrane expression CMV / pDisplay The HRP gene was a gift from Frances Arnold (Caltech) HRP-KDEL EcoRI-ss-BglII-HRP-V5-KDEL-Stop-NotI
Expression of endoplasmic reticulum CMV / pDisplay

The step of introducing the polynucleotide into a cell may be mediated through, but not limited to, transfection or transduction. The polynucleotide of APEX2 (the amino acid of SEQ ID NO: 3 and the salt sequence of SEQ ID NO: 4) which further enhances the peroxidase activity in the above APEX polynucleotide site can be used but is not limited thereto.

In the step of treating the phenol compound and the hydrogen peroxide, the treatment of the compound may be performed by treating the compound having the structure of Formula 1 at a concentration of 0.1 uM to 1 mM for 10 to 60 minutes, but is not limited thereto .

In the step of treating the phenol compound and the hydrogen peroxide, the peroxidase is selected from the group consisting of soybean ascorbate peroxidase, Arabidopsis ascorbate peroxidase, spinach ascorbate peroxidase, , Saccharomyces cerevisiae cytochrome c peroxidase, Phanerochaete chrysosporium PCL1, Phanerochaete chrysosporium PCL1, Phanerochaete chrysosporium PCL1, PCM), Coprinopsis cinerea peroxidase, Peanut Peroxidase, Arabidopsis ATPA2, an enzyme consisting of the amino acid sequence of SEQ ID NO: 1, an amino acid sequence of SEQ ID NO: 3 And an amino acid sequence of SEQ ID NO: 5 May be at least one enzyme selected from the group consisting of binary luer enzyme, the process of the hydrogen peroxide is not intended to be to process for 1 to 10 minutes of hydrogen peroxide at a concentration of 0.1 to 2 mM, but limited.

The cell is not limited and may include all animal cells, plant cells, microorganisms, and the like. For example, it may be a mammalian cell capable of transfection. Specific examples thereof include kidney cells, cancer cells, skin cells, ovarian cells, synovial cells, peripheral blood mononuclear cells, fibroblasts, fibroblasts, neurons, epithelial cells, keratinocytes, hematopoietic cells, melanocytes, cartilage cells, macrophages, And may include muscle cells, blood cells, bone marrow cells, lymphocyte cells, mononuclear cells, lung cells, pancreatic cells, liver cells, gastric cells, intestinal cells, cardiac cells, bladder cells, urethral cells, embryonic germ cells or cumulus cells But is not limited thereto. More specifically, the animal cells can be cultured in an animal such as kidney cells (HEK293T cells, HEK293 cells), cancer cells (HeLa cells, etc.), monkey kidney cells (COS-7 cells, etc.), osteosarcoma cells (U2OS cells, etc.) Cells, etc.), ovary cells (CHO cells, etc.), and the like.

In the present invention, when a peroxidase is injected into a cell and a phenol compound and hydrogen peroxide are treated, the phenol is converted into a phenoxy radical by the action of the enzyme expressed in the cell, and the phenoxy radical is a protein having a radius of about 5 to 50 nm And binds to the proteins by forming a covalent bond with the tyrosine group present in the ss. In addition, since the produced phenoxy radicals exist only for a short time so as not to be able to cross the phospholipid membrane, when phenoxy radicals are generated in the membrane-enclosed cell organelles, only phenolic compounds Lt; / RTI >

The compound produces a radical by a peroxidase enzyme and the produced radical forms a covalent bond with a tyrosine group present in a protein or peptide to bind to the protein or peptide, Or the distance to the peptide may be 5 to 50 nm, but is not limited thereto.

Since each intracellular organelle has an intrinsic protein body, the proteins to which deschiobiotin is bound are different for each organelle and the proteins present in the space in which the peroxidase is expressed are labeled. After the labeled cells are dissolved, the labeled proteins can be stuck to streptavidin (SA) -beads capable of binding to destiobiotin, and trypsin treatment can be performed to remove unlabeled peptides. Then, the formamide is treated with the labeled protein bound to the beads and heated to obtain the labeled peptide, which is then mass analyzed to identify the protein and confirm its structural information.

The organelle refers to a structural unit that is present in the interior of a cell and that divides various functions of cells. Preferably, in the present invention, a cell organelle is a structure surrounded by an intracellular membrane, and forms an intracellular compartment that is separated from another space and means an organelle having a unique microenvironment. These organelles have their own functions and may be, for example, mitochondria, endoplasmic reticulum, nucleus, cytoplasm, Golgi, lysosomes, pagosomes, peroxisomes and the like. In the case of plant cells, . ≪ / RTI >

The step of labeling the intracellular protein with the phenol compound is followed by disruption of the cell to obtain a cell lysate. Streptavidin (SA) beads are added to the cell lysate to form a label attached to the streptavidin bead And a step of obtaining a protein.

When a streptavidin bead is added to the cell lysate in the step of obtaining the labeled protein bound to the streptavidin bead, a phenol compound, specifically, a labeled protein to which DBP is bound can bind to the streptavidin bead.

The present invention provides a method for labeling a protein using the phenol compound and a method for identifying the labeled protein to detect the protein.

Specifically, introducing into a cell a polynucleotide comprising a gene encoding a targeting sequence and a gene encoding a peroxidase into a cell organelle in which an intracellular target protein exists for detection;

Treating the cell with a phenol compound having a structure represented by the following formula (1) and hydrogen peroxide to label the intracellular protein with the phenol compound;

Adding a Streptavidin (SA) bead to the cell lysate to obtain a labeled protein bound to the streptavidin bead; And

And detecting the target protein by detecting the target protein in the obtained product.

[Chemical Formula 1]

And the step of preparing the polynucleotide before the step of introducing the polynucleotide into the cell.

The process for producing the polynucleotide may include preparing a polynucleotide fused so that a gene encoding a targeting sequence and a gene encoding a peroxidase are linked to a cell organelle in which the target protein exists, no.

In the step of introducing the polynucleotide into the cell, the cell organelle may be selected from the group consisting of mitochondria, an endoplasmic reticulum, a nucleus, a cytoplasm and a Golgi apparatus. The gene coding for the targeting sequence to the cell organelle may include a mitochondrial substrate- But are not limited to, a cytoplasmic expression gene, a mitochondrial bilayer membrane expression gene, a nuclear expression gene, an extracellular membrane expression gene, an endoplasmic reticulum expression gene, an endoplasmic reticulum expression gene, or an intracellular membrane expression gene. The genes encoding the targeting sequences into the organelles are shown in Table 1 above.

The step of introducing the polynucleotide into a cell may be mediated through, but not limited to, transfection or transduction.

In the treatment of the phenolic compound and hydrogen peroxide, the treatment of the phenolic compound may be, but not limited to, treating the phenolic compound at a concentration of 0.1 to 1 mM for 10 to 60 minutes.

The compound produces a radical by a peroxidase enzyme and the produced radical forms a covalent bond with a tyrosine group present in a protein or peptide to bind to the protein or peptide, Or the distance to the peptide may be 5 to 50 nm, but is not limited thereto.

The peroxidase may be selected from the group consisting of soybean ascorbate peroxidase, Arabidopsis ascorbate peroxidase, spinach ascorbate peroxidase, saccharomyces cerevisiae cytochrome c peroxide The enzymes Saccharomyces cerevisiae cytochrome c peroxidase, Phanerochaete chrysosporium PCL1, Phanerochaete chrysosporium PCM, Coprinopsis cinereosporium peroxidase, An enzyme consisting of the amino acid sequence of SEQ ID NO: 1, an enzyme consisting of the amino acid sequence of SEQ ID NO: 3, and an amino acid sequence of SEQ ID NO: 5 An enzyme selected from the group consisting of Soil can be, but is not limited to this.

The organelles may be selected from the group consisting of mitochondria, endoplasmic reticulum, nucleus, cytoplasm and Golgi apparatus, but the present invention is not limited thereto.

After labeling the intracellular protein with the phenol compound, cells are disrupted to obtain a cell lysate, and streptavidin (SA) beads are added to the cell lysate to obtain a labeled protein.

When a streptavidin bead is added to the cell lysate in the step of obtaining the labeled protein bound to the streptavidin bead, a phenol compound, specifically, a labeled protein to which DBP is bound can bind to the streptavidin bead.

The step of identifying the target protein in the product may include treating the labeled protein bound to the streptavidin bead with an enzyme to cleave the protein, separating the cleaved fragment bound to the streptavidin bead, And analyzing the separated cleavage fragments. However, the present invention is not limited thereto.

Separation of the cleaved fragments may be performed by desalting, but is not limited thereto.

The enzyme may be, but is not limited to, trypsin.

The step of analyzing the intracellular position of the target protein may be performed prior to the step of introducing the polynucleotide into the cell.

In the step of analyzing the intracellular location of the target protein, a method of confirming the intracellular protein position using a peroxidase enzyme can be used, which is described in detail in Korean Patent Laid-Open Publication No. 2016-0071960.

The streptavidin beads may be streptavidin-coated magnetic beads, but are not limited thereto.

The method of identifying a target protein in the step of identifying a target protein in the above-mentioned product may be mass analysis, Western blot, fluorescence microscopy, dot blot, or ELISA, but is not limited thereto.

The present invention relates to a method for detecting a protein using a phenol compound. More particularly, the present invention relates to a method for detecting a protein using desthiobiotin-phenol (DBP) You can identify the labeled protein by way of detection.

1 is a reaction formula for preparing Desthiobiotin-Phenol (DBP) according to an embodiment of the present invention.
FIG. 2 is a graph showing the results of biotin-phenol biotin-phenol biosynthesis assay in which biotin-phenol was detected when peptide was detected by biotin-phenol and dextobiotin-phenol according to an embodiment of the present invention, Fig.
The left side of FIG. 3 shows the place labeled with desistiobiotin-phenol according to an embodiment of the present invention in various colors according to the mass spectrometry signal intensity. On the right side, the protein structure and the mass analysis signal intensity, In the graph of FIG.
FIG. 4 is a graph showing MS / MS spectral results of biotin-phenol, oxidized biotin-phenol and peptide labeled with dextobiotin-phenol according to an embodiment of the present invention.
FIG. 5 is a table showing the peptide score (Sequest X corr value) of a labeled peptide according to an embodiment of the present invention.
6 is a photograph showing Streptavidin (SA) -HRP Western Blot (WB) results of biotin-phenol-labeled APEX transfected whole cell lysate according to an embodiment of the present invention.
Figure 7 is a photograph showing Streptavidin (SA) -HRP Western blot (WB) results of whole cell lysates transfected with desipio biotin-phenol labeled APEX according to one embodiment of the present invention.
FIG. 8 shows SA-bead enrichment using Sco1-APEX2 of a protein labeled with DBP or BP according to an embodiment of the present invention.
FIG. 9 is a result of an imaging assay using Sco1-APEX2 of a protein labeled with DBP or BP according to an embodiment of the present invention.
FIG. 10 is a graph showing the positions labeled with 215 DBPs in mitochondrial matrix-APEX2, Lactb-APEX2 or Sco1-APEX2 transfected cells and untransfected cells according to an embodiment of the present invention.
Figure 11 is a graph showing the mitochondrial specificity of Groups I through V according to one embodiment of the present invention.
Figure 12 shows the number of identified proteins and known proteins according to one embodiment of the present invention.
FIG. 13 is a graph showing proteins labeled by Lactb-APEX2 or Sco1-APEX2 according to an embodiment of the present invention.
Figure 14 is a diagram showing the crystal structure of an embodiment CYP51A1 of the present invention and the tyrosine residues (green) and heme (red) labeled by its DBP.
FIG. 15 shows the results of labeling of cells transfected with TMEM261-V5-APEX2 according to an embodiment of the present invention by labeling with DBP.
16 is a view showing a membrane topology of TMEM 261 in mitochondria according to an embodiment of the present invention.
17 is a view showing the mitofilin (IMMT) protein phase according to an embodiment of the present invention.
18 is a view showing the phase of the IMM-TM protein according to an embodiment of the present invention.
FIG. 19 is a diagram illustrating the IMM-TM phase determined according to a labeled site result according to an embodiment of the present invention. FIG.
FIG. 20 is a diagram showing the membrane phases of LETM1 and HIGD2A determined according to the labeled site results according to an embodiment of the present invention. FIG. DBP-labeled sites were labeled with a green circle by matrix-APEX2, and DBP labeled sites by IMS-APEX2 were indicated by a blue circle.
FIG. 21 is a view showing a peripheral membrane protein binding face according to a result of an embodiment of the present invention. FIG. The DBP-labeled spot by matrix-APEX2 is indicated by a green circle, and the DBP labeled spot by IMS-APEX2 is indicated by a blue circle.
Figure 22 is a diagram showing the membrane binding faces of some surface membrane proteins based on the labeled sites in an IMM according to an embodiment of the present invention. The DBP-labeled spot by matrix-APEX2 is indicated by a green circle, and the DBP labeled spot by IMS-APEX2 is indicated by a blue circle.
23 is a map mapping a DBP-labeled site to a mitochondrial ATP synthase complex (PDB ID: 2WSS) according to an embodiment of the present invention. labeled with DBP-labeled tyrosine residues (green) by matrix-APEX2 and DBP labeled sites (blue) by IMS-APEX2.
24 is a map mapping DBP-labeled sites to mitochondrial complex II (PDB ID: 1BCC) (left) and complex IV (PDB ID 1OCC) (right) according to an embodiment of the present invention. labeled DBP-labeled tyrosine residues (green) by matrix-APEX2 and DBP labeled sites (blue) by IMS-APEX2.
25 is a map mapping a DBP-labeled site to a mitochondrial chaperone complex (right: PDB ID: 4PJ1) according to an embodiment of the present invention. The tyrosine residue (green) labeled with DBP by matrix-APEX2 was displayed.
26 shows the results of ¹ H-NMR of DBP synthesized according to an embodiment of the present invention.
FIG. 27 shows the results of ¹³ C-NMR of DBP synthesized according to an embodiment of the present invention.
28 is a schematic diagram showing a method of protein detection using BP (Biotin-Phenol).
FIG. 29 is a schematic diagram showing a method of detecting a protein using DBP, and the structure of a protein can be searched using the above method.
30 is a schematic diagram illustrating a position where radicals are generated in DBP and a process of propagating the radicals according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  One. Desthiobiotin -Phenol synthesis and purification

A solution of 0.233 M was prepared by dissolving 100 mg of desthiobiotin (Sigma, Catalog No. D1411) in 2 mL of DMSO (dimethyl sulfoxide, Sigma-Aldrich, USA) at room temperature (25 ° C) (0.257 M) and 2.0 equivalents of DIPEA (N, N-diisopropylethylamine, 0.467 M) were added at room temperature For 10 minutes to prepare a mixed solution. Then, 2.0 equivalents of tyramine (0.467 M) was added to the mixed solution, and the reaction was completed by stirring at room temperature overnight. The synthesis method is illustrated in FIG.

The prepared Desthiobiotin-phenol was purified by HPLC (Yeonlin Semi-prep HPLC YL9100) using a C18 reverse phase column (Agilent Dynamax 250 X 21.4 mm, microsorb 300-5 C18) in a 5-95% gradient acetonitrile / Minute. The HLPC operation was performed according to the manufacturer's protocol, and the resulting compound eluted near 20 minutes with a yield of about 70%.

Purified by HPLC, Desthiobiotin-phenol was subjected to NMR according to the manufacturer's protocol using a Bruker 400 MHz NMR machine.

^The results for ¹ H-NMR are shown in FIG. 26 and those for ¹³ C-NMR are shown in FIG. From the ¹ H-NMR and ¹³ C-NMR results shown in Figs. 26 and 27, it was found that the substance purified by HPLC was Desthiobiotin-phenol.

^{1 H-NMR (400 MHz,} d4-Methanol): δ 1.10 (3H, d, J = 6.5 Hz), 1.30-1.46 (8H, m), 1.57 (2H, m), 2.14 (2H, t, J = M), 6.70 (2H, d, J = 8 Hz), 7.02 (2H, (2H, d, J = 8 Hz).

¹³ C-NMR (100 MHz, d6-DMSO): δ 15.508, 25.212, 25.591, 28.710, 29.522, 34.439, 35.371, 40.433, 50.259, 55.016, 115.049, 129.450, 129.564, 155.581, 162.865, 171.963, HMRS m / z (C ₁₈ H ₂₈ N ₃ O ₃ ): theoretical: 334.2131, observed: 334.2136.

Example  2. Protein labeling

2-1. Transformation

HEK293T cells purchased from ATCC were cultured in MEM (Minimum Essential Medium, Gibco) medium containing 10% (v / v) fetal bovine serum, 50 units / mL penicillin and 50 ug / mL streptomycin at 37 ° C For 16-24 hours in a 5% CO2 incubator. Cells were cultured in two T75 flasks.

It is known that different organelles (for example, mitochondria matrix, intermembrane space of mitochondrial (IMS), outer mitochondrial membrane (OMM), endoplasmic reticulum, nucleus, cytoplasm, ), A vector into which a polynucleotide inserted with a gene coding for an amino acid sequence targeted to each cell organelle and a gene sequence of APEX2 was inserted was transfected with Lipofectamine 2000 (Thermo Fisher Scientific) transfection reagent Lt; RTI ID = 0.0 > HEK293T < / RTI > The above vectors used different kinds of organelles depending on the organelle to be targeted, and the structures and characteristics of the vectors used in the present invention are summarized in Table 2 below.

Item denomination Characteristic Promotor /
Vector Detail One Mito-V5-APEX2
(unprocessed: 32 kDa, processed: 29 kDa) NotI-Mito-BamHI-V5-APEX2-Stop-XhoI CMV /
pCDNA3 Mito-: MLATRVFSLVGKRAISTSVCVRAH (matrix targeting sequence, Fornuskova et al., 2010) 2 ScoI-V5-APEX2
(unprocessed: 62 kDa,
processed: 59 kDa) KpnI-ScoI-BamHI-
V5-APEX2-Stop-
XhoI CMV /
pCDNA3 Sco1 (NM_004589) 3 ScoI-V5-APEX2
(unprocessed: 62 kDa,
processed: 59 kDa) HindIII-ScoI-
BamHI-V5-
APEX2-Stop-XhoI CMV /
pCDNA5 Sco1 (NM_004589) 4 Tom20-V5-APEX2
(45.7 kDa) NotI-Tom20-10aa
linker-NheI-V5-
APEX2-Stop-XhoI CMV /
pCDNA3 10aa linker: GGSGDPPVAT
Tom20 (NM_014765.2) 5 TRMT61B-V5-APEX2
(81.5 kDa) NotI-TRMT61B-
NheI-V5-APEX2-
Stop-XhoI CMV /
pCDNA3 TRMT61B (NM_017910) 6 ATP5J-V5-APEX2
(unprocessed: 41.2 kDa,
processed: 37.2 kDa) NotI-ATP5J-
BamHI-V5-
APEX2-Stop-XhoI CMV /
pCDNA3 ATP5J (NM_001685) 7 MGST3-V5-APEX2
(45.1 kDa) NotI-MGST3-
BamHI-V5-
APEX2-Stop-XhoI CMV /
pCDNA3 MGST3 (NM_004528) 8 V5-APEX2-NES
(29.8 kDa) NotI-V5-APEX2-
NES-Stop-XhoI CMV /
pCDNA3 NES: LQLPPLERLTLD
(nuclear exclusion signal) 9 LACTB-V5-APEX2
(unprocessed: 35 kDa,
processed: 29 kDa) NotI-LACTB-
BamHI-V5-
APEX2-Stop-XhoI CMV /
pCDNA3 LACTB (1-68): MYRLLSSVTARAAATAGPAWDGGRRGAHRRPGLPVLGLGWAGGLGLGLGLALGAKLVVGLRGAVPIQS
(IMS targeting signal, Polianskyte et al., 2009)
Flag: DYKDDDDK 10 TMEM261-V5-APEX2
(unprocessed: 42.8 kDa) HindIII-TMEM261-
BamHI-V5-
APEX2-Stop-XhoI CMV /
pCDNA3 TMEM261 (NM_033428.1) 11 Pdk1-V5-APEX2 *
(unprocessed, 81.5 kDa, processed: 76.0 kDa) NotI-Pdk1-10aa linker-NheI-V5-APEX2-Stop-XhoI CMV /
pCDNA3 Pdk1 (NM_002610)

2-2. Intracellular  Protein labeling

HEK293T cells were cultured in two T75 flasks and APEX2 was transfected into HEK293T cells. APEX2 was transfected into HEK293T cells, and after 12 to 24 hours, Desthiobiotin-phenol was treated to a concentration of 250 uM and incubated at 37 DEG C for 30 minutes. Then, H ₂ O ₂ was added to a concentration of 1 mM, and after 1 minute, 5 mM Trolox (Sigma Aldrich, product number 238813), 10 mM sodium azide, 10 mM sodium ascorbate And then washed three times with DPBS (Dulbecco's Phosphate Buffered Saline, Thermo Fisher Scientific). Cells are pipetted into 20 mL DPBS, transferred to a 50 mL tube, and centrifuged at 1500 g for 5 minutes to make cell pellet and discard the supernatant. The cells were transferred to a 1.5-mL microtube by pipetting with DBPS, centrifuged at a speed of 1500 g for 5 minutes, and the cell pellet was discarded. (Biosesang, R2002) with 1X protease inhibitor cocktail (Sigma, P8849), PMSF 1 mM (phenylmethylsulfonylfluoride, Biosesang, P1021) and RIPA lysis buffer supplemented with a minerals solution Were all dissolved (Lysis).

2-3. Labeled Of peptide  purchase

The cell lysate was centrifuged at a temperature of 4 캜 for 10 minutes at a speed of 20,000 rpm using a centrifuge to obtain only supernatant. The supernatant was placed in an Amicon filter (10K centrifugal filter, Merck Milipore, Amicon Ultra-15) and centrifuged at 7500 g for 20 min at 4 ° C. After that, PBS was added and centrifugation was performed three more times. The remaining solution was transferred to a 1.5-mL microtube, and PBS was added to make 1 mL. Then, 300 μL of streptavidin (SA) -coated magnetic beads was added and slowly returned at room temperature (25 ° C.) for 1 hour. The supernatant was placed on a magnetic stand and stored separately for use as a test for whether the target protein was fully enrichment-free. The magnetic beads were then washed three times with PBS. PBS was removed and suspended in 100 uL of 6 M Urea, 2 M Thiourea, 10 mM Hepes. 20 uL of 100 mM DTT in 50 mM Ammonium bicarbonate (ABC) was added and centrifuged at 56 ° C for 1 hour at 800 rpm. 35 uL of 300 mM iodoacetamide in 50 mM ABC was added and centrifuged at 800 rpm for 30 minutes. Add 840 uL of 50 mM ABC. And 8 uL of 1 ug / uL trypsin (Promega, V5280). And centrifuged at 37 ° C for 6-18 hours at 800 rpm. The supernatant was collected by putting it on a magnetic stand and the magnetic beads were washed with PBS 4 times. 250 uL of 95% (v / v) formamide, 10 mM EDTA, pH 8.2 solution was added and heated at 95 캜 for 10 minutes. Then, immediately put it on the magnetic stand, put it on ice, and it cooled down. 750 uL of 3% (v / v) Acetonitrile / 0.1% (v / v) formic acid solution was added.

2-4. Peptides  Isolation of Cleaved Fragments

Desatting was performed using a Varian Bond ELUT (Agilent, 12109301) in the following manner in order to isolate the peptide cleavage fragment bound to the streptavidin.

Add 1 mL of 3% (v / v) Acetonitrile / 0.1% (v / v) Formic acid, 1 mL of 100% acetonitrile and 2 mL of 3% v / v Acetonitrile / To activate the column. The sample was slowly poured into the column to attach the peptide cleavage fragment to the column. The column was washed with 2 mL of 3% (v / v) Acetonitrile / 0.1% (v / v) formic acid solution. The peptide was eluted with 1.4 mL of 70% (v / v) Acetonitrile / 0.1% (v / v) Formic acid solution and 500 uL of 100% acetonitrile.

For mass analysis, the extracted solution was completely dried and then re-dissolved in 0.1% (v / v) formic acid.

Comparative Example  1. Treatment with biotin-phenol

Experiments were carried out in the same manner as in Examples 1 and 2 except that APEX2 was transfected into HEK293T cells in Example 2 and treated at a phenol compound treatment time of 12 to 24 hours in a concentration of 500 uM instead of destiobiotin- - phenol.

Experimental Example  1. Mass spectrometry

The peptide cleavage fragments obtained by carrying out Examples 1 and 2 and Comparative Example 1 were obtained by confirming that the peptide labeled with desistiobiotin-phenol synthesized in the present invention had no oxidized form One peptide cleavage fragment was subjected to mass spectrometry in the following manner.

(150 mm x 75 [mu] m) for 90 minutes at a flow rate of 300 nL / min of a 10 to 24% (v / v) Acetonitrile / 0.1% (v / v) Formic acid gradient to separate the peptide mixture, Was used.

Then, an Orbitrap spectrometer (Thermo Fisher Scientific, Germany) was used for MS / MS analysis of precursor ion scan MS spectra (m / z 400-2000). The resolution of the Orbitrap was set at a resolution of 60,000 at 400 m / z and utilized an internal internal lock mass function. Twenty strongest ions were separated and fragmented by CID (collisionally induced dissociation) mode.

MS / MS analysis was performed using Sequest (Thermo Fisher Scientific, San Jose, CA, USA; version 1.4.1.14) Tandem (The GPM, thegpm.org; version CYCLONE (Dec. 1, 2010)).

Specifically, Sequest and X! Tandem set the homo sapiens protein sequence database (89113 entries, UniProt ( www.uniprot.org) ) to assume the digestion by trypsin enzyme. Sequest and X! Tandem searched for a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 10.0 PPM.

Carbamidomethyl cysteine is a fixed modification of Sequest and X! Tandem and was performed under the following conditions:

Glu-> pyro-Glu of n-terminus,

ammonia-loss of n-terminus, and

gln- > pyro-Glu of n-terminus.

Methionine oxidation and BP (delta monoisotopic mass: 361.146), oxidized BP (delta monoisotopic mass: 377.1409), and tyrosine DBP (delta monoisotopic mass: 331.1896) Tandem and Sequest were used as variable modifications.

As shown in Figs. 4 and 5, when peptides were labeled with des-biotin-phenol by the methods of Examples 1 and 2, des-thiobiotin-phenol (Y + DBP) was covalently bonded to the tyrosine group of the peptide It was possible to detect only the mass value M + 331 Da of the probe. However, when the biotin-phenol was treated in place of the desiobiotin-phenol in the method of Comparative Example 1, biotin-phenol (Y + BP) was covalently bonded to the tyrosine group of the peptide M + 361 Da, which is the mass value of one probe, and M + 377 Da, which is the mass value of the probe of the oxidized form of the biotin phenol (Y + oxidized BP). The results are illustrated in FIG.

3 shows the correlation between the mass spectrometry signal intensity and the solvent access area of the protein based on the mass analysis results. As a result, it was confirmed that the mass analyzing signal intensity increases positively as the solvent access area increases.

Experimental Example  2. Identification of labeled proteins

In order to confirm the peptide labeled with dextobiotin-phenol synthesized and used in the present invention, the cell lysate obtained in Examples 2-1 to 2-2 and the cell lysate obtained in Comparative Example 1 Western blotting was performed in the following manner.

Specifically, the cell lysate obtained by carrying out Example 2-2 and Comparative Example 1 was sampled for SDS-PAGE and electrophoresed on an SDS-PAGE gel at 150V for about 1 hour. Then, electrophoresed proteins on SDS-PAGE gels were transferred to a nitrocellulose membrane, visualized by ponceau staining, and analyzed with TBST solution (Tris-Buffered Saline and Tween 20), and the mixture was stirred at room temperature for 1 hour with a 2% (w / v) dialyzed BSA solution. SA-HRP was diluted to 10000: 1 with 2% (w / v) dialyzed BSA solution and stirred at room temperature for 1 hour. Membrane was then washed with TBST four times for 5 minutes each time. Membranes washed with TBST were developed with a developer and images were obtained using a LAS 4000 instrument (GE, USA, LAS 4000) according to the manufacturer's protocol.

6 and 7 are 1 (MitoMatrix-APEX2) in Table 2, 2 is LACTB-APEX2 (IMS) in Table 2, 3 is Tom20-APEX2 (OMM) in Table 2, APEX2 (NMS) in Table 2, 11 (Pdk1-APEX2 (matrix) in Table 2), No. 3 (SCO1-APEX2 (TRMT61B-APEX2), 8 is 6 (ATP5J-APEX2) in Table 2, and 9 is 7 (MGST3-APEX2) in Table 2.

As shown in FIG. 7, the western blot pattern of the protein labeled by desthiobiotin-phenol was confirmed in each organelle, and the western blot pattern of the labeled protein was labeled with biotin-phenol as shown in FIG. 6 Similar to the western blot pattern of the protein.

Experimental Example  3. Obtained label Of peptide  Confirm

In order to identify the labeled peptides obtained by binding to streptavidin in Example 2-3, the labeled peptides obtained by carrying out Example 2-3 and Comparative Example 1 were subjected to Western blotting in the same manner as in Experimental Example 2 .

DBP was the method of Example 1 and Example 2, and BP was Comparative Example 1. The input is the sample before binding to streptavidin, the flow-through is the supernatant after binding to streptavidin and immersed in the magnetic stand, and SA-Enriched is the sample bound to streptavidin.

As shown in FIG. 8, it was confirmed that the protein labeled with DBP existing in the input specifically binds to streptavidin beads, and the result shows that the BP-labeled protein specifically binds to streptavidin beads Similar to the Western blot pattern.

Experimental Example  4. Fluorescence Microscopy Analysis

Cells labeled with DBP by the methods of Examples 1 and 2 and cells labeled with BP by the method of Comparative Example 1 were analyzed by fluorescence microscopy in the following manner.

에서 보관한 메탄올을 -20℃에서 5분 동안 처리하여 세포를 투과시켰다 (Permeabilized).For fluorescence microscopic analysis, the cells labeled with DBP by the methods of Examples 1 and 2 and the cells labeled with BP by the method of Comparative Example 1 were treated with 4% (v / v) paraformaldehyde With DPBS solution at room temperature for 15 min. The cells were then washed three times with DPBS (Dulbecco's Phosphate-Buffered Saline, Gibco), and 4

Was treated at -20 ° C for 5 minutes to permeabilize the cells (Permeabilized).

 After the cells were permeated as above, the cells were washed three times with DPBS and blocked with DPBS (blocking buffer) containing 2% (w / v) BSA (Bovine serum albumin) for 1 hour at room temperature.

To detect the expression of the APEX2-fusion protein, cells were incubated with mouse anti-V5 antibody (Invitrogen, R960-25, 1: 5000 dilution) for 1 hour at room temperature. Cells were then washed four times for 5 minutes each using TBST and then simultaneously injected with secondary Alexa Fluor 488-goat anti-mouse IgG (Invitrogen, A-11001, 1: 1000 dilution) and streptavidin-Alexa Fluor 568 IgG (Invitrogen, , 1: 1000 dilution) for 30 minutes at room temperature. The cells were then washed four times for 5 minutes each using TBST and placed on ice-stored DPBS for imaging using an EVOS Epi-fluorescence microscope (Life Technology). Imaging using a fluorescence microscope was performed according to the manufacturer's protocol. The results are shown in Figs. 9 and 15.

As can be seen from FIG. 9, the fluorescence imaging results of the cells labeled with DBP by the methods of Examples 1 and 2 and the cells labeled with BP by the method of Comparative Example 1 were the same without any significant difference.

15 shows the expression of Mito-BFP and TMEM261-V5-APEX2 in U2OS cells in the same manner as in Examples 2-1 and 2-2, followed by labeling with DBP. Green was the observation of the expression pattern of TMEM261-V5-APEX2 with anti-V5, red was the pattern of SA-568, that is, the pattern of DBP-labeled protein, and blue was Mito-BFP, that is, the mitochondrial pattern . Since the green and blue overlap well, TMEM261-V5-APEX2 is expressed well in the mitochondria, and the red does not overlap well because the DBP label is not limited space but OMM (Outer mitochondrial membrane or IMS (Intermembrane space). Unlike the IMM (Inner Mitochondrial Membrane), OMM and IMS are unconstrained spaces because they have pores in the OMM.

Experimental Example  5. DBP labeled specific Cell organelle  Protein detection

Experiments were conducted using the methods of Examples 1 and 2 in order to detect proteins present in specific cell cell organelles by the method of the present invention, and the obtained peptide cleavage was subjected to mass spectrometry in the same manner as in Experimental Example 1, The results were analyzed.

As a result, as can be seen from FIGS. 10 to 14, it was confirmed that the ENO1 protein present in the cytoplasm existing in proximity to the LACTB protein present in the intermembrane space (IMS) of mitochondrial organelles was labeled, and mitochondria It was confirmed that HIGD1A protein present in IMS which is close to SCO1 protein, which is dependent on space between cell organelles, is marked. Since the OMM is permeable and the IMM is impermeable, LACTB is located close to the OMM and can be labeled with DBP as a protein present in the cytoplasm, whereas SCO1 is either an IMM or a matrix ), It is judged that the protein present in the matrix can not be labeled with DBP.

That is, since LACTB and SCO1 are both proteins present in IMS, LACTB is located closer to the cytoplasmic side in IMS space than SCO1, and SCO1 is located closer to IMM in IMS space than LACTB, LACTB-APEX2 can label proteins present in the cytoplasm, but SCO1-APEX2 can not label proteins present in the cytoplasm.

Experimental Example  6. Specific to DBP Cell organelle  Protein is labeled Membrane orientation  Structure identification

The method of detecting labeled proteins using biotin-phenol (BP) of Comparative Example 1 (FIG. 28) does not know which tyrosine residues on the protein reacted with biotin-phenol, but the method of Examples 1 and 2 of the present invention (Fig. 29), the protein is labeled with desthiobiotin-phenol (DBP), and the tyrosine residue on the protein is identified by mass spectrometry. Thus, the protein labeled with DBP And also to confirm the structural information of the user.

Specifically, when APEX was expressed in the space between mitochondria (IMS) by the same method as in Examples 1 and 2 and subjected to DBP treatment and mass analysis in the same manner as Experimental Example 1, IMMT In the case of the mitochondrial membrane protein, it was confirmed that only the protein domain exposed in the intermembrane space was labeled with DBP.

In addition, when APEX was expressed in the mitochondrial matrix space, it was confirmed that the domains exposed to the mitochondrial matrix space were labeled differently from those expressed in the IMS as shown in FIG.

The membrane orientation was determined using SCO1-APEX2 (blue color) expressed in the space between the mitochondrial bilayers and Matrix-APEX2 (green color) expressed in the mitochondrial matrix space in the same manner as described above. 18-22.

The membrane orientation of various mitochondrial membrane proteins, for which the membrane orientation of mitochondrial membrane proteins has not yet been established, can be clearly identified through the labeling of proteins present in specific cell cell organelles using the DBP of the present invention and the identification method of the proteins.

The result of mapping the mitochondrial ATP synthase complex (PDB ID: 2WSS) labeled with DBP in the same manner as above was mapped to the mitochondrial complex II (PDB ID: 1BCC) and complex IV (PDB ID 1OCC) 24, and the mitochondrial chaperone complex (right: PDB ID: 4PJ1).

<110> UNIST (ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY) <120> Methods for identification of proteins using phenolic compounds          for protein labeling <130> DPP20160206KR <160> 6 <170> KoPatentin 3.0 <210> 1 <211> 692 <212> PRT <213> Artificial Sequence <220> <223> Engineered ascorbate peroxidase (APEX) <400> 1 Met Glu Thr Gly Leu Tyr Leu Tyr Ser Ser Glu Arg Thr Tyr Arg Pro   1 5 10 15 Arg Thr His Arg Val Ala Leu Ser Glu Arg Ala Leu Ala Ala Ser Pro              20 25 30 Thr Tyr Arg Gly Leu Asn Ala Ser Pro Ala Leu Ala Val Ala Leu Gly          35 40 45 Leu Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Leu Tyr Ser Leu Tyr Ser      50 55 60 Leu Glu Ala Arg Gly Gly Leu Tyr Pro His Glu Ile Leu Glu Ala Leu  65 70 75 80 Ala Gly Leu Leu Tyr Ser Ala Arg Gly Cys Tyr Ser Ala Leu Ala Pro                  85 90 95 Arg Leu Glu Met Glu Thr Leu Glu Ala Arg Gly Leu Glu Ala Leu Ala             100 105 110 Pro His Glu His Ile Ser Ser Glu Arg Ala Leu Ala Gly Leu Tyr Thr         115 120 125 His Arg Pro His Glu Ala Ser Pro Leu Tyr Ser Gly Leu Tyr Thr His     130 135 140 Arg Leu Tyr Ser Thr His Arg Gly Leu Tyr Gly Leu Tyr Pro Arg Pro 145 150 155 160 His Glu Gly Leu Tyr Thr His Arg Ile Leu Glu Leu Tyr Ser His Ile                 165 170 175 Ser Pro Arg Ala Leu Ala Gly Leu Leu Glu Ala Leu Ala His Ile Ser             180 185 190 Ser Glu Arg Ala Leu Ala Ala Ser Asn Ala Ser Asn Gly Leu Tyr Leu         195 200 205 Glu Ala Ser Pro Ile Leu Glu Ala Leu Ala Val Ala Leu Ala Arg Gly     210 215 220 Leu Glu Leu Glu Gly Leu Pro Arg Leu Glu Leu Tyr Ser Ala Leu Ala 225 230 235 240 Gly Leu Pro His Glu Pro Arg Ile Leu Glu Leu Glu Ser Glu Arg Thr                 245 250 255 Tyr Arg Ala Leu Ala Ala Ser Pro Pro His Glu Thr Tyr Arg Gly Leu             260 265 270 Asn Leu Glu Ala Leu Ala Gly Leu Tyr Val Ala Leu Val Ala Leu Ala         275 280 285 Leu Ala Val Ala Leu Gly Leu Val Ala Leu Thr His Arg Gly Leu Tyr     290 295 300 Gly Leu Tyr Pro Arg Leu Tyr Ser Val Ala Leu Pro Arg Pro His Glu 305 310 315 320 His Ile Ser Pro Arg Gly Leu Tyr Ala Arg Gly Gly Leu Ala Ser Pro                 325 330 335 Leu Tyr Ser Pro Arg Gly Leu Pro Arg Pro Arg Pro Gly Leu Gly             340 345 350 Leu Tyr Ala Arg Gly Leu Glu Pro Arg Ala Ser Pro Ala Leu Ala Thr         355 360 365 His Arg Leu Tyr Ser Gly Leu Tyr Ser Glu Arg Ala Ser Pro His Ile     370 375 380 Ser Leu Glu Ala Arg Gly Ala Ser Pro Val Ala Leu Pro His Glu Gly 385 390 395 400 Leu Tyr Leu Tyr Ser Ala Leu Ala Met Glu Thr Gly Leu Tyr Leu Glu                 405 410 415 Thr His Arg Ala Ser Pro Gly Leu Asn Ala Ser Pro Ile Leu Glu Val             420 425 430 Ala Leu Ala Leu Ala Leu Glu Ser Glu Arg Gly Leu Tyr Gly Leu Tyr         435 440 445 His Ile Ser Thr His Arg Ile Leu Glu Gly Leu Tyr Ala Leu Ala Ala     450 455 460 Leu Ala His Ile Ser Leu Tyr Ser Gly Leu Ala Arg Gly Ser Glu Arg 465 470 475 480 Gly Leu Tyr Pro His Glu Gly Leu Gly Leu Tyr Pro Arg Thr Arg Pro                 485 490 495 Thr His Arg Ser Glu Arg Ala Ser Asn Pro Arg Leu Glu Ile Leu Glu             500 505 510 Pro His Glu Ala Ser Pro Ala Ser Asn Ser Glu Arg Thr Tyr Arg Pro         515 520 525 His Glu Thr His Arg Gly Leu Leu Glu Leu Glu Ser Glu Arg Gly Leu     530 535 540 Tyr Gly Leu Leu Tyr Ser Gly Leu Gly Leu Tyr Leu Glu Leu Glu Gly 545 550 555 560 Leu Asn Leu Glu Pro Arg Ser Glu Arg Ala Ser Pro Leu Tyr Ser Ala                 565 570 575 Leu Ala Leu Glu Leu Glu Ser Glu Arg Ala Ser Pro Pro Arg Val Ala             580 585 590 Leu Pro His Glu Ala Arg Gly Pro Arg Leu Glu Val Ala Leu Ala Ser         595 600 605 Pro Leu Tyr Ser Thr Tyr Arg Ala Leu Ala Ala Leu Ala Ala Ser Pro     610 615 620 Gly Leu Ala Ser Pro Ala Leu Ala Pro His Glu Pro His Glu Ala Leu 625 630 635 640 Ala Ala Ser Pro Thr Tyr Arg Ala Leu Ala Gly Leu Ala Ala Leu Ala                 645 650 655 His Ile Ser Gly Leu Asn Leu Tyr Ser Leu Glu Ala Ser Glu Arg Gly             660 665 670 Leu Ala Leu Glu Ala Gly Leu Tyr Pro His Glu Ala Leu Ala Ala Ser         675 680 685 Pro Ala Leu Ala     690 <210> 2 <211> 750 <212> DNA <213> Artificial Sequence <220> <223> gene of Engineered ascorbate peroxidase (APEX) <400> 2 atgggaaagt cttacccaac tgtgagtgct gattaccagg acgccgttga gaaggcgaag 60 cgctgagaag ttccactctg ctggaacctt tgacaagggc acgaagaccg gtggaccctt cggaaccatc 180 aagcaccctg ccgaactggc tcacagcgct aacaacggtc ttgacatcgc tgttaggctt 240 ttggagccac tcaaggcgga gttccctatt ttgagctacg ccgatttcta ccagttggct 300 ggcgttgttg ccgttgaggt cacgggtgga cctaaggttc cattccaccc tggaagagag 360 gacaagcctg agccaccacc agagggtcgc ttgcccgatg ccactaaggg ttctgaccat 420 ttgagagatg tgtttggcaa agctatgggg cttactgacc aagatatcgt tgctctatct 480 gggggtcaca ctattggagc tgcacacaag gagcgttctg gatttgaggg tccctggacc 540 tctaatcctc ttattttcga caactcatac ttcacggagt tgttgagtgg tgagaaggaa 600 ggtctccttc agctaccttc tgacaaggct cttttgtctg accctgtatt ccgccctctc 660 gttgataaat atgcagcgga cgaagatgcc ttctttgctg attacgctga ggctcaccaa 720 aagctttccg agcttgggtt tgctgatgcc 750 <210> 3 <211> 691 <212> PRT <213> Artificial Sequence <220> <223> Engineered ascorbate peroxidase 2 (APEX2) <400> 3 Met Glu Thr Gly Leu Tyr Leu Tyr Ser Ser Glu Arg Thr Tyr Arg Pro   1 5 10 15 Arg Thr His Arg Val Ala Leu Ser Glu Arg Ala Leu Ala Ala Ser Pro              20 25 30 Thr Tyr Arg Gly Leu Asn Ala Ser Pro Ala Leu Ala Val Ala Leu Gly          35 40 45 Leu Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Leu Tyr Ser Leu Tyr Ser      50 55 60 Leu Glu Ala Arg Gly Gly Leu Tyr Pro His Glu Ile Leu Glu Ala Leu  65 70 75 80 Ala Gly Leu Leu Tyr Ser Ala Arg Gly Cys Tyr Ser Ala Leu Ala Pro                  85 90 95 Arg Leu Glu Met Glu Thr Leu Glu Ala Arg Gly Leu Glu Ala Leu Ala             100 105 110 Pro His Glu His Ile Ser Ser Glu Arg Ala Leu Ala Gly Leu Tyr Thr         115 120 125 His Arg Pro His Glu Ala Ser Pro Leu Tyr Ser Gly Leu Tyr Thr His     130 135 140 Arg Leu Tyr Ser Thr His Arg Gly Leu Tyr Gly Leu Tyr Pro Arg Pro 145 150 155 160 His Glu Gly Leu Tyr Thr His Arg Ile Leu Glu Leu Tyr Ser His Ile                 165 170 175 Ser Pro Arg Ala Leu Ala Gly Leu Leu Glu Ala Leu Ala His Ile Ser             180 185 190 Ser Glu Arg Ala Leu Ala Ala Ser Asn Ala Ser Asn Gly Leu Tyr Leu         195 200 205 Glu Ala Ser Pro Ile Leu Glu Ala Leu Ala Val Ala Leu Ala Arg Gly     210 215 220 Leu Glu Leu Glu Gly Leu Pro Arg Leu Glu Leu Tyr Ser Ala Leu Ala 225 230 235 240 Gly Leu Pro His Glu Pro Arg Ile Leu Glu Leu Glu Ser Glu Arg Thr                 245 250 255 Tyr Arg Ala Leu Ala Ala Ser Pro Pro His Glu Thr Tyr Arg Gly Leu             260 265 270 Asn Leu Glu Ala Leu Ala Gly Leu Tyr Val Ala Leu Val Ala Leu Ala         275 280 285 Leu Ala Val Ala Leu Gly Leu Val Ala Leu Thr His Arg Gly Leu Tyr     290 295 300 Gly Leu Tyr Pro Arg Leu Tyr Ser Val Ala Leu Pro Arg Pro His Glu 305 310 315 320 His Ile Ser Pro Arg Gly Leu Tyr Ala Arg Gly Gly Leu Ala Ser Pro                 325 330 335 Leu Tyr Ser Pro Arg Gly Leu Pro Arg Pro Arg Pro Gly Leu Gly             340 345 350 Leu Tyr Ala Arg Gly Leu Glu Pro Arg Ala Ser Pro Pro Arg Thr His         355 360 365 Arg Leu Tyr Ser Gly Leu Tyr Ser Glu Arg Ala Ser Pro His Ile Ser     370 375 380 Leu Glu Ala Arg Gly Ala Ser Pro Val Ala Leu Pro His Glu Gly Leu 385 390 395 400 Tyr Leu Tyr Ser Ala Leu Ala Met Glu Thr Gly Leu Tyr Leu Glu Thr                 405 410 415 His Arg Ala Ser Pro Gly Leu Asn Ala Ser Pro Ile Leu Glu Val Ala             420 425 430 Leu Ala Leu Ala Leu Glu Ser Glu Arg Gly Leu Tyr Gly Leu Tyr His         435 440 445 Ile Ser Thr His Arg Ile Leu Glu Gly Leu Tyr Ala Leu Ala Ala Leu     450 455 460 Ala His Ile Ser Leu Tyr Ser Gly Leu Ala Arg Gly Ser Glu Arg Gly 465 470 475 480 Leu Tyr Pro His Glu Gly Leu Gly Leu Tyr Pro Arg Thr Arg Pro Thr                 485 490 495 His Arg Ser Glu Arg Ala Ser Asn Pro Arg Leu Glu Ile Leu Glu Pro             500 505 510 His Glu Ala Ser Pro Ala Ser Asn Ser Glu Arg Thr Tyr Arg Pro His         515 520 525 Glu Thr His Arg Gly Leu Leu Glu Leu Glu Ser Glu Arg Gly Leu Tyr     530 535 540 Gly Leu Leu Tyr Ser Gly Leu Gly Leu Tyr Leu Glu Leu Glu Gly Leu 545 550 555 560 Asn Leu Glu Pro Arg Ser Glu Arg Ala Ser Pro Leu Tyr Ser Ala Leu                 565 570 575 Ala Leu Glu Leu Glu Ser Glu Arg Ala Ser Pro Pro Arg Ala Leu             580 585 590 Pro His Glu Ala Arg Gly Pro Arg Leu Glu Val Ala Leu Ala Ser Pro         595 600 605 Leu Tyr Ser Thr Tyr Arg Ala Leu Ala Ala Leu Ala Ala Ser Pro Gly     610 615 620 Leu Ala Ser Pro Ala Leu Ala Pro His Glu Pro His Glu Ala Leu Ala 625 630 635 640 Ala Ser Pro Thr Tyr Arg Ala Leu Ala Gly Leu Ala Ala Leu Ala His                 645 650 655 Ile Ser Gly Leu Asn Leu Tyr Ser Leu Glu Ala Ser Glu Arg Gly Leu             660 665 670 Ala Leu Glu Ala Gly Leu Tyr Pro His Glu Ala Leu Ala Ala Ser Pro         675 680 685 Ala Leu Ala     690 <210> 4 <211> 750 <212> DNA <213> Artificial Sequence <220> <223> gene of Engineered ascorbate peroxidase 2 (APEX2) <400> 4 atgggaaagt cttacccaac tgtgagtgct gattaccagg acgccgttga gaaggcgaag 60 cgctgagaag ttccactctg ctggaacctt tgacaagggc acgaagaccg gtggaccctt cggaaccatc 180 aagcaccctg ccgaactggc tcacagcgct aacaacggtc ttgacatcgc tgttaggctt 240 ttggagccac tcaaggcgga gttccctatt ttgagctacg ccgatttcta ccagttggct 300 ggcgttgttg ccgttgaggt cacgggtgga cctaaggttc cattccaccc tggaagagag 360 gacaagcctg agccaccacc agagggtcgc ttgcccgatc ccactaaggg ttctgaccat 420 ttgagagatg tgtttggcaa agctatgggg cttactgacc aagatatcgt tgctctatct 480 gggggtcaca ctattggagc tgcacacaag gagcgttctg gatttgaggg tccctggacc 540 tctaatcctc ttattttcga caactcatac ttcacggagt tgttgagtgg tgagaaggaa 600 ggtctccttc agctaccttc tgacaaggct cttttgtctg accctgtatt ccgccctctc 660 gttgataaat atgcagcgga cgaagatgcc ttctttgctg attacgctga ggctcaccaa 720 aagctttccg agcttgggtt tgctgatgcc 750 <210> 5 <211> 870 <212> PRT <213> Artificial Sequence <220> <223> Horseradish peroxidase <400> 5 Met Glu Thr Gly Leu Asn Leu Glu Thr His Arg Pro Arg Thr His Arg   1 5 10 15 Pro His Glu Thr Tyr Arg Ala Ser Pro Ala Ser Asn Ser Glu Arg Cys              20 25 30 Tyr Ser Pro Arg Ala Ser Asn Val Ala Leu Ser Glu Arg Ala Ser Asn          35 40 45 Ile Leu Glu Val Ala Leu Ala Arg Gly Ala Ser Pro Thr His Arg Ile      50 55 60 Leu Glu Val Ala Leu Ala Ser Asn Gly Leu Leu Glu Ala Arg Gly Ser  65 70 75 80 Glu Arg Ala Ser Pro Pro Arg Ala Arg Gly Ile Leu Glu Ala Leu Ala                  85 90 95 Ala Leu Ala Ser Glu Arg Ile Leu Glu Leu Glu Ala Arg Gly Leu Glu             100 105 110 His Ile Ser Pro His Glu His Ile Ser Ala Ser Pro Cys Tyr Ser Pro         115 120 125 His Glu Val Ala Leu Ala Ser Asn Gly Leu Tyr Cys Tyr Ser Ala Ser     130 135 140 Pro Ala Leu Ala Ser Glu Arg Ile Leu Glu Leu Glu Leu Glu Ala Ser 145 150 155 160 Pro Ala Ser Asn Thr His Arg Thr His Arg Ser Glu Arg Pro His Glu                 165 170 175 Ala Arg Gly Thr His Arg Gly Leu Leu Tyr Ser Ala Ser Pro Ala Leu             180 185 190 Ala Pro His Glu Gly Leu Tyr Ala Ser Asn Ala Leu Ala Ala Ser Asn         195 200 205 Ser Glu Arg Ala Leu Ala Ala Arg Gly Gly Leu Tyr Pro His Glu Pro     210 215 220 Arg Val Ala Leu Ile Leu Glu Ala Ser Pro Ala Arg Gly Met Glu Thr 225 230 235 240 Leu Tyr Ser Ala Leu Ala Leu Ala Val Ala Leu Gly Leu Ser Glu                 245 250 255 Arg Ala Leu Ala Cys Tyr Ser Pro Arg Ala Arg Gly Thr His Arg Val             260 265 270 Ala Leu Ser Glu Arg Cys Tyr Ser Ala Leu Ala Ala Ser Pro Leu Glu         275 280 285 Leu Glu Thr His Arg Ile Leu Glu Ala Leu Ala Ala Leu Ala Gly Leu     290 295 300 Asn Gly Leu Asn Ser Glu Arg Val Ala Leu Thr His Arg Leu Glu Ala 305 310 315 320 Leu Ala Gly Leu Tyr Gly Leu Tyr Pro Arg Ser Glu Arg Thr Arg Pro                 325 330 335 Ala Arg Gly Val Ala Leu Pro Arg Leu Glu Gly Leu Tyr Ala Arg Gly             340 345 350 Ala Arg Gly Ala Ser Ser Glu Arg Leu Glu Gly Leu Asn Ala Leu         355 360 365 Ala Pro His Glu Leu Glu Ala Ser Pro Leu Glu Ala Leu Ala Ala Ser     370 375 380 Asn Ala Leu Ala Ala Ser Asn Leu Glu Pro Arg Ala Leu Ala Pro Arg 385 390 395 400 Pro His Glu Pro His Glu Thr His Arg Leu Glu Pro Arg Gly Leu Asn                 405 410 415 Leu Glu Leu Tyr Ser Ala Ser Pro Ser Glu Arg Pro His Glu Ala Arg             420 425 430 Gly Ala Ser Asn Val Ala Leu Gly Leu Tyr Leu Glu Ala Ser Asn Ala         435 440 445 Arg Gly Ser Glu Arg Ser Glu Arg Ala Ser Pro Leu Glu Val Ala Leu     450 455 460 Ala Leu Ala Leu Glu Ser Glu Arg Gly Leu Tyr Gly Leu Tyr His Ile 465 470 475 480 Ser Thr His Arg Pro His Glu Gly Leu Tyr Leu Tyr Ser Ala Ser Asn                 485 490 495 Gly Leu Asn Cys Tyr Ser Ala Arg Gly Pro His Glu Ile Leu Glu Met             500 505 510 Glu Thr Ala Ser Pro Ala Arg Gly Leu Glu Thr Tyr Arg Ala Ser Asn         515 520 525 Pro His Glu Ser Glu Arg Ala Ser Asn Thr His Arg Gly Leu Tyr Leu     530 535 540 Glu Pro Arg Ala Ser Pro Pro Arg Thr His Arg Leu Glu Ala Ser Asn 545 550 555 560 Thr His Arg Thr His Arg Thr Tyr Arg Leu Glu Gly Leu Asn Thr His                 565 570 575 Arg Leu Glu Ala Arg Gly Gly Leu Tyr Leu Glu Cys Tyr Ser Pro Arg             580 585 590 Leu Glu Ala Ser Asn Gly Leu Tyr Ala Ser Asn Leu Glu Ser Glu Arg         595 600 605 Ala Leu Ala Leu Glu Val Ala Leu Ala Ser Pro Pro His Glu Ala Ser     610 615 620 Pro Leu Glu Ala Arg Gly Thr His Arg Pro Thr His Arg Ile Leu 625 630 635 640 Glu Pro His Glu Ala Ser Pro Ala Ser Asn Leu Tyr Ser Thr Tyr Arg                 645 650 655 Thr Tyr Arg Val Ala Leu Ala Ser Asn Leu Glu Gly Leu Gly Leu Gly             660 665 670 Leu Asn Leu Tyr Ser Gly Leu Tyr Leu Glu Ile Leu Glu Gly Leu Asn         675 680 685 Ser Glu Arg Ala Ser Pro Gly Leu Asn Gly Leu Leu Glu Pro His Glu     690 695 700 Ser Glu Arg Ser Glu Arg Pro Ala Ser Ser Asn Ala Leu Ala Thr His 705 710 715 720 Arg Ala Ser Pro Thr His Arg Ile Leu Glu Pro Arg Leu Glu Val Ala                 725 730 735 Leu Ala Arg Gly Ser Glu Arg Pro His Glu Ala Leu Ala Ala Ser Asn             740 745 750 Ser Glu Arg Thr His Arg Gly Leu Asn Thr His Arg Pro His Glu Pro         755 760 765 His Glu Ala Ser Asn Ala Leu Ala Pro His Glu Val Ala Leu Gly Leu     770 775 780 Ala Leu Ala Met Glu Thr Ala Ser Pro Ala Arg Gly Met Glu Thr Gly 785 790 795 800 Leu Tyr Ala Ser Asn Ile Leu Glu Thr His Arg Pro Arg Leu Glu Ala                 805 810 815 Thr His Arg Gly Leu Tyr Thr His Arg Gly Leu Asn Gly Leu Tyr Gly             820 825 830 Leu Asn Ile Leu Glu Ala Arg Gly Leu Glu Ala Ala Ser Asn Cys Tyr         835 840 845 Ser Ala Arg Gly Val Ala Leu Val Ala Leu Ala Ser Asn Ser Glu Arg     850 855 860 Ala Ser Asn Ser Glu Arg 865 870 <210> 6 <211> 927 <212> DNA <213> Artificial Sequence <220> <223> gene of Horseradish peroxidase <400> 6 atgcagttaa cccctacatt ctacgacaat agctgtccca acgtgtccaa catcgttcgc 60 gacacaatcg tcaacgagct cagatccgat cccaggatcg ctgcttcaat attacgtctg 120 cacttccatg actgcttcgt gaatggttgc gacgctagca tattactgga caacaccacc 180 agtttccgca ctgaaaagga tgcattcggg aacgctaaca gcgccagggg ctttccagtg 240 atcgatcgca tgaaggctgc cgttgagtca gcatgcccac gaacagtcag ttgtgcagac 300 ctgctgacta tagctgcgca acagagcgtg actcttgcag gcggaccgtc ctggagagtg 360 ccgctcggtc gacgtgactc cctacaggca ttcctagatc tggccaacgc caacttgcct 420 gctccattct tcaccctgcc ccagctgaag gatagcttta gaaacgtggg tctgaatcgc 480 tcgagtgacc ttgtggctct gtccggagga cacacatttg gaaagaacca gtgtaggttc 540 atcatggata ggctctacaa tttcagcaac actgggttac ctgaccccac gctgaacact 600 acgtatctcc agacactgag aggcttgtgc ccactgaatg gcaacctcag tgcactagtg 660 gactttgatc tgcggacccc aaccatcttc gataacaagt actatgtgaa tctagaggag 720 cagaaaggcc tgatacagag tgatcaagaa ctgtttagca gtccaaacgc cactgacacc 780 atcccactgg tgagaagttt tgctaactct actcaaacct tctttaacgc cttcgtggaa 840 gccatggacc gtatgggtaa cattacccct ctgacgggta cccaaggcca gattcgtctg 900 aactgcagag tggtcaacag caactct 927

Claims (16)

Introducing into the cell a polynucleotide comprising a gene encoding a targeting sequence and a gene encoding a peroxidase into a cell organelle in which an intracellular target protein exists for detection;
Treating the cell with a phenol compound, a stereoisomer, a tautomer or a salt thereof having hydrogen peroxide and a structure represented by the following formula (1) to label the intracellular protein with the phenol compound;
Adding a Streptavidin (SA) bead to the cell lysate to obtain a labeled protein bound to the streptavidin bead; And
Identifying a protein of interest in said product;
[Chemical Formula 1]

The method according to claim 1, wherein the step of identifying the target protein in the product comprises: treating the labeled protein bound to the streptavidin bead with an enzyme to cleave the protein; separating the cleaved fragment bound to the streptavidin bead And analyzing the separated cleaved fragments. &Lt; Desc / Clms Page number 19 &gt; 3. The method of claim 2, wherein separating the cleaved fragments is performed by desalting. 3. The method according to claim 2, wherein the enzyme is trypsin. 2. The method of detecting a protein according to claim 1, wherein the step of analyzing the intracellular position of the target protein is performed prior to the step of introducing the polynucleotide into the cell. The method of claim 1, wherein the phenolic compound produces a radical by peroxidase. 7. The method of claim 6, wherein the peroxidase is selected from the group consisting of Soybean ascorbate peroxidase, Arabidopsis ascorbate peroxidase, Spinach ascorbate peroxidase, Saccharomyces cerevisiae cytochrome c peroxidase, Phanerochaete chrysosporium PCL1, Phanerochaete chrysosporium PCM (PCM), Phanerochaete chrysosporium PCM (PCM) An enzyme comprising the amino acid sequence of SEQ ID NO: 1, an enzyme consisting of the amino acid sequence of SEQ ID NO: 3, an enzyme consisting of the amino acid sequence of SEQ ID NO: 3, an enzyme consisting of the amino acid sequence of SEQ ID NO: And an enzyme consisting of the amino acid sequence of SEQ ID NO: 5 The one or more selected enzymes, detection methods of protein. The method according to claim 6, wherein the generated radical forms a covalent bond with a tyrosine group present on the protein or peptide surface and binds to the protein or peptide. 5. The method of claim 4, wherein the distance that the radical binds to the protein or peptide is 5 to 50 nm. The method of claim 1, wherein the organelle is selected from the group consisting of mitochondria, endoplasmic reticulum, nucleus, cytoplasm and Golgi. The method for detecting a protein according to claim 1, wherein the phenol compound is treated with a compound having a structure of the following formula (1) at a concentration of 0.1 uM to 1 mM for 10 to 60 minutes:
[Chemical Formula 1]

2. The method of claim 1, wherein the treatment with hydrogen peroxide comprises treating the hydrogen peroxide with a concentration of 0.1 to 2 mM for 1 to 10 minutes. The method according to claim 1, wherein the gene coding for the targeting sequence to the organelle is selected from the group consisting of mitochondrial substrate expression gene, cytoplasmic expression gene, mitochondrial bilayer membrane spacer gene, nuclear expression gene, extracellular membrane expression gene, endoplasmic reticulum expression gene, An expression gene, or an intracellular membrane expression gene. The method of claim 1, wherein the cells are selected from the group consisting of kidney cells, cancer cells, skin cells, ovarian cells, synovial cells, peripheral blood mononuclear cells, fibroblasts, fibroblasts, neurons, epithelial cells, keratinocytes, hematopoietic cells, Chondrocytes, macrophages, muscle cells, blood cells, bone marrow cells, lymphocyte cells, mononuclear cells, lung cells, pancreatic cells, liver cells, gastric cells, intestinal cells, cardiac cells, bladder cells, urethral cells, A method of detecting a protein that is a cell. The method of claim 1, wherein the streptavidin bead is a streptavidin-coated magnetic bead. The method of claim 1, wherein identifying the target protein is mass spectrometry, Western blot, fluorescence microscopy, dot blot, or ELISA.

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