KR20130117260A - Biomarker composition for diagnosis of androgenetic alopecia and the method of diagnosis using the same - Google Patents

Abstract

PURPOSE: A biomarker composition for diagnosing male pattern alopecia is provided to easily and early diagnose male pattern alopecia and to effectively treat male pattern alopecia. CONSTITUTION: A biomarker composition for diagnosing male pattern alopecia contains proteins which are increased in hair papilla cells of alopecia. The proteins are selected among argininosuccinate synthase (ASS1, Accession No. IPI00020632); phosphoribosyl aminoimidazole carboxylase (PAICS[ADE2], Accession No. IPI00217223); isoform 1 of cytoskeleton-associated protein 4 (CKAP4, Accession No. IPI00141318); isoform 1 of gelsolin (GSN, Accession No. IPI00026314); Ras GTPase-activating-like protein 1 (IQGAP1, Accession No. IPI00009342); isoform 1 of S-phase kinase-associated protein 1 (SKP1, Accession No. IPI00301364); isoform 3 of spectrin alpha chain, brain (SPTAN1, Accession No. IPI00843765); and isoform 9 of plectin-1 (PLEC1, Accession No. IPI00398775). [Reference numerals] (AA,CC) Alopecia hair papilla cells; (BB,DD) Non-Alopecia hair papilla cells

Description

Biomarker composition for diagnosing male hair loss and diagnostic method using the same {Biomarker composition for diagnosis of androgenetic alopecia and the method of diagnosis using the same}

The present invention relates to a biomarker composition for diagnosing male hair loss and a diagnostic method using the same.

Mammalian hair follicles include dermal papilla cells and dermal sheath cells derived from the mesenchyme. It also includes epithelial cells, matrix, and hair shaft of outer and inner root sheaths (Non-Patent Document 1). The epithelial-mesenchymal interaction plays an important role in the growth of postnatal hair as well as the formation of hair follicles in the embryonic state (Non-Patent Documents 2 and 3), but the results of this research are not known yet.

On the other hand, postnatal hair follicles are grown and maintained through the cycles of anagen (the active growth phase), catagen (the apoptotic regression phase), and telogen (the resting phase). Symptoms of androgenetic alopecia include androgen-dependent chronic alopecia and hair loss caused by seborrheic dermatitis, and the effect of male hormone shortens the hair follicle growth period (Non-Patent Document 4) and degenerates in the growth phase of the hair follicle growth cycle. Hair loss begins with the early migration phenomenon (Non-Patent Literature 5), or testosterone, a male hormone, is another male hormone type, dihydrotestosterone (5α-reductase). DHT) is caused by abnormal overproduction of the dihydrotestosterone and the resulting sebum production.

Male baldness occurs mainly in people with a family history of male baldness, and hair loss progresses from the beginning of the 20's or 30's to thinning hair. At this time, the boundary line of the forehead and the hair is pushed backwards, and the forehead is widened in the shape of M on both sides of the head and hair loss progresses gradually in the center of the head. The rate of hair loss varies from person to person, and the back of the scalp (back of the head) and side hair are often maintained without hair loss progressing. It is generally known that the earlier the hair loss begins, the worse it progresses. Koreans have a lower incidence, a weaker degree, and tend to start later than white people.

The most well known treatments for androgenetic alopecia are the three most well known treatments: application of minoxidil solution, taking finasteride drug, and autologous hair transplantation. Except in the case of surgery, the treatment of hair loss is known that the earlier the treatment, the more hair loss does not progress and the therapeutic effect is increased. On the other hand, the diagnosis of androgenetic alopecia can be diagnosed as a typical pattern of hair loss, such as thinning hair, but the deviation is severe for each individual to observe it with the naked eye. Therefore, early and accurate diagnosis of androgenetic alopecia is the most important factor of treatment.

Recently, bald dermal papilla cells (balding dermal papilla cells) taken from the forehead of a man with alopecia areata are undergoing a bald dermal papilla cells without any baldness. DNA macroarray analysis of gene expression differences in non-balding dermal papilla cells has been reported, but studies using proteomics have been reported. (Hereinafter, 'hair loss all milk cells' refers to' all hair cells collected from the forehead of a man who is undergoing male hair loss', and 'hair loss hair milk cells' refers to the' no hair loss at the back of the head. Collected milk cells'), and the present inventors determined that both hair loss cells and non-hair loss cells were treated by proteomic analysis. It was identified proteins expressing different from each other in, as a result of studying the diagnostic agent and diagnostic method that can be pre-diagnosis and prevention of progression of male pattern baldness through the method development had to complete the present invention.

Hardy, M. H., The secret life of the hair follicle. Trends Genet, 1992, 8, 55-61. Botchkarev, V. A., and Kishimoto, J., Molecular control of epithelial-mesenchymal interactions during hair follicle cycling. J. Investig. Dermatol. Symp. Proc., 2003, 8, 46-55. Millar, S. E., Molecular mechanisms regulating hair follicle development. J. Invest. Dermatol., 2002, 118, 216-225. Rushton, D. H., Ramsay, I. D., Norris, M. J., and Gilkes, J. J., Natural progression of male pattern baldness in young men. Clin. Exp. Dermatol., 1991, 16, 188-192. Jahoda, C. A. Cellular and developmental aspects of androgenetic alopecia. Exp Dermatol., 1998, 7, 235-248.  Han, C. L., et al., A multiplexed quantitative strategy for membrane proteomics: opportunities for mining therapeutic targets for autosomal dominant polycystic kidney disease. Mol. Cell Proteomics, 2008, 7, 1983-1997.

An object of the present invention is to provide a biomarker composition for diagnosing male hair loss and a diagnostic method using the same.

The present invention relates to a biomarker composition for diagnosing male hair loss and a diagnostic method using the same.

The biomarker composition for diagnosing male hair loss of the present invention may include at least three proteins selected from the following protein groups or immunogenic fragments thereof.

Protein increases in all hair loss

Argininosuccinate synthase (ASS1, Accession No. IPI00020632);

Phosphoribosyl aminoimidazole carboxylase (PAICS [ADE2], Accession No. IPI00217223);

Isoform 1 of Cytoskeleton-associated protein 4 (CKAP4, Accession No. IPI00141318);

Isoform 1 of Gelsolin (GSN, Accession No. IPI00026314);

Ras GTPase-activating-like protein 1 (IQGAP1, Accession No. IPI00009342);

Isoform 1 of S-phase kinase-associated protein 1 (SKP1, Accession No. IPI00301364);

Isoform 3 of Spectrin alpha chain, brain (SPTAN1, Accession No. IPI00843765);

Isoform 9 of Plectin-1 (PLEC1, Accession No. IPI00398775).

The immunogenic fragments refer to protein fragments having one or more epitopes that can be recognized by antibodies to increasing proteins in all hair loss hair cells of the present invention.

The biomarker composition compares the expression of three or more proteins, thereby allowing a more significant and accurate comparative analysis of the masculine hair loss phenomenon than comparing the expression of one or two proteins.

In addition, the present invention provides a diagnostic agent containing an antibody that specifically recognizes three or more proteins selected from the group of proteins increasing in all hair loss. In addition, the diagnostic agent may include a labeled secondary antibody, a chromophore, an enzyme conjugated with the antibody, and a substrate or other substance capable of binding to the antibody. The diagnostic agent contains three or more antibodies, thereby enabling a more significant and accurate comparative analysis of the phenomenon of masculine hair loss than using a diagnostic agent containing one or two antibodies.

By antibody is meant a specific protein molecule directed against the antigenic site. Preferably, the antibody refers to an antibody that specifically binds to an increasing protein in all hair loss hair cells of the present invention, and may include all polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. Producing such antibodies can be readily prepared using techniques well known in the art. The polyclonal antibody can be obtained from serum collected after injecting an animal with a protein (antigen) which is increased in all hair loss cells. The animal may be any animal host such as goat, rabbit, or pig. The monoclonal antibody, as is well known in the art, uses a hybridoma method (Kohler G. and Milstein C.), or phage antibody library (Clackson et al .; Marks et al.) Technology Can be prepared. In order to perform the hybridoma method, cells of an immunologically suitable host animal such as a mouse and cancer or myeloma cell line may be used. Thereafter, by using a method such as polyethylene glycol, that is, a method well known in the art to which the present invention belongs, after fusion of these two types of cells, the antibody-producing cells can be propagated by a standard tissue culture method. have. Subsequently, after obtaining a uniform cell population by subcloning by the limited dilution technique, hybridomas capable of producing antibodies specific for the proteins of the present invention are in vitro or in vivo according to standard techniques. Can be cultured in large quantities. The phage antibody library method, by obtaining an antibody gene for an increasing protein in all the hair loss cells of the present invention, expressing it in the form of a fusion protein on the surface of the phage (phage) to produce an antibody library in vitro, From the library, monoclonal antibodies that bind to proteins of increasing genes in all hair loss cells of the present invention can be isolated and produced. Antibodies prepared by the above methods can be separated by electrophoresis, dialysis, ion exchange chromatography, affinity chromatography and the like.

The antibody may include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. The functional fragment of an antibody molecule means the fragment which has at least antigen binding function, and includes Fab, F (ab '), F (ab') 2, F (ab) 2, Fv.

On the other hand, in addition to the diagnostic agent, the present invention provides a diagnostic kit containing an antibody that specifically recognizes three or more proteins selected from the group of proteins increasing in all the hair loss hair cells, the hair loss is increased in all Provided is a protein chip containing an antibody that recognizes at least three proteins selected from the protein group or at least three proteins selected from the protein group increasing in both hair loss cells. The diagnostic kit is prepared by conventional manufacturing methods known to those skilled in the art, and typically include lyophilized antibodies, buffers, stabilizers, inactive proteins, and the like. The antibody may be labeled by radioactive species, fluorescent sources, enzymes, and the like. The antibody can be used in a variety of immunoassay kits (ELISA, antibody coated tube test, lateral-flow test, potable biosensor), as well as various male types through the development of antibodies showing higher specificity and sensitivity. It can also be used to develop protein chips with a hair loss detection spectrum.

The present invention also provides a diagnostic method using an antibody that specifically recognizes three or more proteins selected from the group of proteins increasing in all hair loss.

The diagnostic method,

(Step 1) separating the protein from the sample to be analyzed;

(Step 2) contacting the protein of step 1 with an antibody that specifically binds to the three or more proteins to form an antigen-antibody complex; And

(Step 3) quantitatively detecting and analyzing the antigen-antibody complex generated in step 2; may include.

In all the above procedures, the gene expression levels of all the alopecia all hair cells (all hair cells suspected of developing male alopecia) and the control sample (the alopecia all hair cells) are compared and analyzed. That is, the sample of step 1 can be extracted from a patient suspected of having a male hair loss progressing.

The process of separating the protein in the first step can be carried out using a known process, the amount of protein can be measured by various methods known to those skilled in the art.

The antigen-antibody complex of step 2 refers to a combination of a protein and an antibody specific thereto that are increased in all hair loss cells of the present invention. That is, the antigen refers to a protein that increases in all hair loss hair cells.

In other words, the amount of formation of the antigen-antibody complex of the control group and the amount of formation of the antigen-antibody complex in all-milk cells or in all-hair loss hair cells suspected that male-type hair loss is progressing can be compared through the above analysis method. All of the hair loss can be diagnosed whether the actual male hair loss is progressing by determining the protein expression amount of the gene that is increased in the milk cells.

The amount of formation of the antigen-antibody complex can be quantitatively determined through the signal magnitude of the detection label. The detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, and radioisotopes, but is not limited thereto. When the enzyme is used as the detection label, available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase. , Acetylcholinesterase, glucose oxidase, hexokinase, and the like, but are not limited to the ranges described above. The fluorescent material may be fluorescein, phycocyanin, fluorescarmine, etc., but is not limited to the above-described materials. Such ligands include, but are not limited to, biotin derivatives. Examples of the light emitting material include luciferin, but are not limited thereto. The microparticles include, but are not limited to, colloidal and gold. The redox molecule includes, but is not limited to, quinone, 1,4-benzoquinone, hydroquinone, and the like. The radioisotope includes, but is not limited to, ³ H, ¹⁴ C, and the like.

The diagnostic method includes Western blot, ELISA (enzyme linked immunosorbent assay), RIA (radioimmunoassay), radioimmunodiffusion, oaklononiy immunodiffusion, rocket immunoelectrophoresis , Tissue immunostaining (immunohistochemistry), immunoprecipitation (immunoprecipitation), complement fixation assay (complement fixation assay), fluorescense activated cell sorter (FACS), protein chip (protein chip), etc., but is not limited to the described method no.

Therefore, preferably, the present invention provides a diagnostic method using a diagnostic kit containing an antibody that specifically recognizes three or more proteins selected from the group of proteins increasing in all the hair loss cells, and in all the hair loss cells Provided is a diagnostic method using a protein chip containing an antibody that recognizes at least three proteins selected from an increasing protein group or at least three proteins selected from an increasing protein group in both hair loss cells.

The present invention relates to a biomarker composition for diagnosing male hair loss and a diagnostic method using the same, through the biomarker composition and the diagnostic method, it is possible to easily diagnose the progress of male hair loss. In addition, the above method can provide a method for early diagnosis of masculine hair loss and effective treatment associated with it.

Figure 1 shows the forehead and laryngeal region of male hair loss patients from which both hair loss and non-hair loss cells are collected.
FIG. 2 shows an androgen receptor (AR) and type 2 5α-reductase (type 2 5α-) between both hair loss and non-hair loss cells obtained from two male hair loss patients (Patient 1 and Patient 2). The difference in RNA expression of reductase, 5αR2) was confirmed using RT-PCR.
FIG. 3 shows ASS1 (argininosuccinate synthase 1), ADE2 (phosphoribosylaminoimidazole carboxylase (PAICS), and CKAP4 between alopecia areata and alopecia areata cells of alopecia areata in two male alopecia patients (Patient 1 and Patient 2). The protein expression of cytoskeleton-associated protein 4), GSN (gelsolin), IQGAP1 (Ras GTPaseactivating-like protein 1) and SKP1 (S-phase kinase-associated protein 1) was confirmed by Western blot.
Figure 4 shows IQGAP1 (Ras GTPaseactivating-like protein 1), PLEC1 (Isoform 9 of Plectin-1), GSN (Gelsolin) and SPTAN1 (Isoform 3 of Spectrin alpha) chain, brain) protein expression was confirmed by immunofluorescence therapy.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

Example 1 Isolation and Culture of Breast Papillary Cells

Hair loss all milk cells and non-hair loss both milk cells used in the experiment of the present invention were obtained from hair follicles separated from the scalp tissue of two male patients (Patient 1 and Patient 2) undergoing hair transplantation (see FIG. 1). The cells were harvested from the injured hair follicles, and then DMEM (Dulbecco's modified Eagle's) containing penicillin (100 U / ml), streptomycin (100 µg / ml), and 20% heat inactivated fetal calf serum (FCS). medium; Gibco BRL, Gaithersburg, MD) in a 37 ° C., 5% CO ₂ wet incubator. The cells were then subcultured with trypsin to maintain and culture the cells in DMEM containing 10% FCS.

<Example 2. Confirmation of all hair loss milk cells>

In order to confirm the well-separated hair loss and non-hair loss both milk cells in Example 1, androgen receptor (AR) and type 2 5α-reductase (5αR2) The expression of RNA was confirmed.

In general, hair loss-producing cells are known to have increased expression of androgen receptor (AR) and type 2 5-reductase (5αR2) than cells without hair loss. have.

To this end, total RNA was extracted using Trizol solution (Invitrogen, Carlsbad, Calif.) In both alopecia and non-alopecia milk cells, and cDNA was synthesized from a total of 3 μg of RNA (superscript of Promega's cDNA synthesis kit. Ⅱ reverse transcriptase and oligo-dT primers). Thereafter, RT-PCR was performed using 1 μl of cDNA and the primers described below. PCR products were separated by electrophoresis on 1% agarose gel and confirmed by UV, and the amount of RNA was compared with β-actin.

AR PCR conditions :

1 cycle: 94 ℃-2min

30 cycles: 94 ° C-1 minute, 60 ° C-45 seconds, 72 ° C-45 seconds

1 cycle: 72 ℃-5min

forward primer: 5’-GGTAAGGGAAGTAGGTGGAA-3 ’

reverse primer: 5'-CCTTCTAGCCCTTTGGTGTA-3 '.

5αR2 PCR conditions :

1 cycle: 94 ℃-2min

30 cycles: 94 ° C-1 minute, 55 ° C-45 seconds, 72 ° C-45 seconds

1 cycle: 72 ℃-5min

forward primer: 5’-TGAGGTTACATGCTGCTTGC-3 ’

reverse primer: 5'-TCCAATTACAAGCGTTCGG-3 '.

β-actin PCR conditions : 1 cycle: 94 ℃-2 minutes

23 cycles: 94 ° C-1 minute, 58 ° C-45 seconds, 72 ° C-45 seconds

1 cycle: 72 ℃-5min

forward primer: 5’-GGGAAATCGTGCGTGACATT-3 ’

reverse primer: 5'-GGAGTTGAAGGTAGT TTCGTG-3 '.

Referring to the results of FIG. 2, the expression of the androgen receptor and type 2α-reductase in the cells of all hair loss hair cells compared to the non-hair loss both milk cells in the cells collected from two male hair loss patients (Patient 1 and Patient 2) It was confirmed that this is significantly increased, and it was found that this resulted in good separation of both hair loss and non-hair loss milk cells.

On the other hand, in order to confirm whether the analysis conditions of the present invention is correct, before performing the experiments of Examples 3 to 6 using both hair loss and non-hair loss milk cells, the four standard proteins glycogen kinase ( glycogen phosphorylase, serum albumin precursor, enolase 1, and alcohol dehydrogenase 1 were mixed with E. coli extraction standard protein to produce 1-D LC-MS and 2-D LC IDEAL-Q analysis on MS. Glycogen phosphorylase, serum albumin precursor, enolase 1, alcohol dehydrogenase 1, and E. coli extracted standard proteins in the same pattern repeatedly After confirming that the analysis conditions of the present invention are correct, the following experiments were performed.

< Example  3. Protein Degradation Using Trypsin>

In order to perform proteolysis using trypsin, both hair loss and non-hair loss milk cells collected from male hair loss patients (Patient 1 and Patient 2) were collected, and then the protein pellet contained a protease inhibitor. The prepared RIPA buffer was reacted (RIPA buffer: 20 mM Tris-HCl, pH 7.5, 1 mM Na ₂ EDTA, 1 mM Na ₂ EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate) , 1 mM Na ₃ VO ₄ , 1 / ml leupeptin, 1 mM Na ₃ VO ₄ ). Thereafter, the reaction was placed on ice for 15 minutes and centrifuged at 4 ° C. and 12,000 rpm for 5 minutes to collect only supernatants. The harvested cell extracts were quantified for protein using a BCA assay kit (Pierce).

Tryptic digestion was performed using the method of Han, C. L. et al. (Non-Patent Document 6). To this end, the cell extract was dissolved in 50 μl of lysis buffer at 37 ° C. for 30 minutes (dissolution buffer: 6 M urea, 5 mM EDTA, 2% (w / v) SDS in 0.1 M TEABC [Triethylammonium bicarbonate]) Once dissolved, the proteins were transferred to vials containing gel (Eppendorf vial), the gel was cut into small pieces, and washed several times with 1 ml of TEABC containing 50% (v / v) ACN. When the protein was dissolved, 3 μl of 10 mM dithiothreitol (DTT) was added, and 3 μl of 50 mM IAA (iodoactamide) was added for 30 minutes to react at room temperature. Then 15 μl of acrylamide / bisacrylamide solution (30%, v / v, 29: 1), 2.5 μl of 10% (w / v) APS (ammonium persulfate) and 1 μl of 100% TEMED (tetramethylethylene diamine) was added to polymerize in a gel state. The polymerized gel was cut into small pieces, washed three times with 1 ml of TEABC solution containing 50% (v / v) ACN (acetonitrile), and then dehydrated. Then, the gel sample was further dehydrated with 100% ACN. After that, it was dried completely. Thereafter, proteolysis was performed by using trypsin (protein: trypsin = 50: 1, g / g) dissolved in 25 mM TEABC for 18 hours at 37 ° C. After proteolysis, peptides were extracted from the gel sample by sequential dissolution (extraction solvent: 200 μl of 25 mM TEABC, 200 μl of 0.1% (v / v) TFA in water, 200 μl of 0.1% (v / v)). ) TFA in ACN, 200 μl of 100% ACN). The extracted peptide sample was concentrated using SpeedVac.

< Example  4. LC Analysis of Proteins Through

In Example 3, liquid chromatography (LC) was performed for the trypsin digested protein, but both 1-D one-dimensional liquid chromatography (LC) and 2-D LC (two-dimensional liquid chromatography) were used for protein expression. The difference was confirmed.

Example  4-1. 1-D LC

Nanoscale LC separations of trypsin digested protein (0.8 μg) were performed using a nanoAcquity system (nanoAcquity system, Waters Corporation, Milford, Mass.) Equipped with the following columns.

* Column: Symmetry C18 5㎛, 5mm × 300㎛ precolumn / BEH C18 1.7㎛, 25cm × 75㎛ analytical reversed phase column (Waters Corporation)

To this end, first, the trypsin digested protein was transferred to a precolumn for 5 minutes at a flow rate of 10 μl / min while suspended in 0.1% formic acid solution. At this time, the mobile phase A is composed of 0.1% formic acid solution, and the mobile phase B is composed of 0.1% formic acid solution dissolved in acetonitrile. Subsequently, the peptide was separated in a concentration gradient of 3 to 55% of the solvent at a flow rate of 300 nl / min for 120 minutes in the B mobile phase of the reversed phase column and washed for 5 minutes at 90% concentration.

Example  4-2. 2-D LC

Nanoscale LC separations of trypsin digested protein (0.8 μg) were performed using a nanoAcquity system (nanoAcquity system, Waters Corporation, Milford, Mass.) Equipped with the following columns.

* Column: Symmetry C18 precolumn (5㎛, 5㎜ × 300㎛) / hybrid silica XTerra MS C18 first dimensional column (100㎜ × 300㎛, 5㎜, 130A ° Column) / BEH C18 analytical reversed phase column (1.7㎛, 25cm × 75㎛) (Waters Corporation)

To this end, first, the trypsin digested protein was transferred to a first dimensional column for 5 minutes at a flow rate of 0.5 μl / min while suspended in 0.1% formic acid aqueous solution. At this time, the peptide of the primary column is separated into four concentration gradients of 11, 21, 35, and 62% in the B mobile phase and attached to a precolumn, followed by a second dimensional column, analytical reversed phase. column), separated by a gradient of 3 ~ 55% of solvent at a flow rate of 300nl / min for 60 min in mobile B phase, and washed for 5 min at 90% concentration. In this case, the mobile phase A of the primary column is composed of 20 mM ammonium formate and the B mobile phase B is composed of 20 mM ammonium formate / ACN (pH 10.0). In addition, the mobile phase A of the secondary column (mobile phase A) is composed of 0.1% formic acid solution and B mobile phase (mobile phase B) is composed of 0.1% formic acid solution dissolved in ACN.

< Example  5. MS Of MS Collection of Data>

Results analyzed using the 1-D LC and 2-D LC carried out in Example 4 is The Q-Tof mass spectrometer (Q-Tof Premier mass spectrometer, Waters Corporation, Manchester, UK) was used.

To this end, LC-MS data was collected in a data dependent mode of acquisition. The mass spectrometer system was performed in ESI positive V mode with a resolving power of 10,000. Nano Lock Spray source was used for accurate mass measurement, and lock mass channel was used as a sample every 30 seconds. Mass spectrometers were calibrated with synthetic human [Glu1] -fibrinopeptide B solution, 100 fmol / μl; Sigma-Aldrich, delivered from a nano-lock spray source. This method includes MS / MS scans under [m / z 4001600, 0.6s] and [m / z 1001990, 1.2s per scan]. In addition, MS / MS data was obtained from the difference in the amount of charge of the ions, and the MS / MS collision energy was selected between 15 eV and 60 eV. At this time, the dynamic exclusion window was set to 300 seconds, and when the base peak intensity (BPI) exceeds the threshold of 150 count / s, the acquisition is converted from MS mode to MS / MS mode, and TIC (total ion) is obtained. When current) exceeds the threshold of 1000 count / s in the MS / MS channel, it is converted back to MS mode.

< Example  6. LC - MS Of MS  Relative Analysis of Results>

The resulting files analyzed in Examples 4 and 5 were converted to a peak list using Mascot Distiller (Matrix Science; version 2.3.2), and analysis of all LC-MS / MS samples was performed using Mascot (Matrix Science; version 2.2.1). To this end, Mascot found and compared the results of the IPI_Rat_3.70 database (version 3.70; 68,161 entries) with the analytical results. The database against Mascot was found to have a fragment ion mass tolerance of 0.05 Da and a parent ion tolerance of 0.1 Da. It was performed using. For protein quantification, IDEAL-Q software (version 1.0.1.1) capable of analyzing LC-MS / MS data was used.

Final analysis results for this are shown in Table 1 by selecting only the 1.5-fold increase in each protein expression or 0.66-fold decrease. As a result, expression of a total of 128 proteins was increased, and it was confirmed that expression of a total of 13 proteins was decreased.

Accession Number Protein Name 1-D LC 2-D LC Patient 1 Patient 2 Patient 1 Patient 2 Ratio SD Ratio SD Ratio SD Ratio SD IPI
00018398 PSMC3: 26S protease regulatory subunit 6A bald only bald only IPI
00168184 PPP2R1A: SERINE / THREONINE PROTEIN PHOSPHATASE 2A, 65 kDa REGULATORY SUBUNIT A, ALPHA ISOFORM 2.128 0 2.078 0.175 IPI
00798116 PPP2R1A: Putative uncharacterized protein PPP2R1A 1.794 0.489 IPI
00028006 PSMB2: Proteasome subunit beta type-2 1.746 0 1.214 0 IPI
00027933 PSMB10: Proteasome subunit beta type-10 9.632 5.973 IPI
00385042 GTPBP4: Nucleolar GTP-binding protein 1 1.28 0 1.802 0 1.28 0 2.152 0 IPI
00031820 FARSA: Phenylalanyl-tRNA synthetase alpha chain bald only bald only IPI
00953028 RPL7: RPL7P32 Similar to 60S ribosomal protein L7 bald only 1.216 0 IPI
00470528 RPL15: 60S ribosomal protein L15 7.748 0 1.559 0 IPI
00178440 EEF1B2: Elongation factor 1-beta 4.129 0 1.024 0 IPI
00000875 TUT1: EEF1G cDNA FLJ56389, highly similar to Elongation factor 1-gamma 4.9569 0 1.413 0.899 7.466 9.604 1.413 0.899 IPI
00013415 RPS7: 40S ribosomal protein S7 1.876 0.703 1.326 0.239 IPI
00396485 EEF1A1: Elongation factor 1-alpha 1 1.837 0.862 2.009 0 1.837 0.621 2.009 0 IPI
00023048 EEF1D: Isoform 1 of Elongation factor 1-delta 1.273 1.573 1.628 0.381 IPI
00014424 EEF1A2: Elongation factor 1-alpha 2 1.157 0 2.802 0 IPI
00008530 RPLP0: 60S acidic ribosomal protein P0 1.756 0.698 1.587 0 1.756 0.24 1.587 0 IPI
00215743 RRBP1: Isoform 3 of Ribosome-binding protein 1 1.711 0 1.486 0 IPI
00216587 RPS8: 40S ribosomal protein S8 1.219 0 1.657 0 IPI
00215719 RPL18: 60S ribosomal protein L18 1.2 0 3.256 0 IPI
00013485 RPS2: 40S ribosomal protein S2 1.18 0 1.657 0.179 IPI
00479058 RPS15: 40S ribosomal protein S15 1.108 0 bald only IPI
00000494 RPL5: 60S ribosomal protein L5 1.42 0 bald only 1.077 0 bald only IPI
00329633 TARS: Threonyl-tRNA synthetase, cytoplasmic 1.047 0 bald only IPI
00221093 RPS17: 40S ribosomal protein S17 1.0142 0 1.699 0 IPI
00411639 RPSA: RPSAP15 Laminin receptor-like protein LAMRL5 2.377 1.233 2.294 2.914 IPI
00645201 RPS8: Ribosomal protein S8 1.614 0.902 1.345 0.004 IPI
00797038 PCK2: Isoform 1 of Phosphoenolpyruvate carboxykinase [GTP], mitochondrial bald only 2.041 0 bald only 5.304 0 IPI
00646779 TUBB6: TUBB6 protein bald only 2.649 0 bald only 3.022 0 IPI
00013475 TUBB2A: Tubulin beta-2A chain bald only 2.472 0.4 IPI
00001639 KPNB1: Importin subunit beta-1 3.6484 0.902 2.141 0.024 7.811 3.024 2.301 0.202 IPI
00217236 TBCA: Tubulin-specific chaperone A 6.712 0 1.292 0 IPI
00027341 CAPG: Macrophage-capping protein 3.864 0 3.168 0 IPI
00007752 TUBB2C: Tubulin beta-2C chain 1.704 0 2.226 0 IPI
00026314 GSN: Isoform 1 of Gelsolin 1.53 0.36 2.992 0 1.53 0.01 3.457 1.54 IPI
00645452 TUBB: Tubulin, beta 1.457 0.447 1.814 0.249 IPI
00003482 DECR1: 2,4-dienoyl-CoA reductase, mitochondrial 1.418 0 4.407 0 IPI
00329801 ANXA5: Annexin A5 1.1195 0.242 1.958 0.484 1.402 0.242 1.958 0.484 IPI
00792677 TUBA1B: cDNA FLJ60097, highly similar to Tubulin alpha-ubiquitous chain 1.637 0.412 IPI
00221226 ANXA6: Annexin A6 1.573 0.13 1.555 0.149 1.573 0.211 1.555 0.149 IPI
00941465 COL6A3 325 kDa protein 1.852 0.497 IPI
00302944 COL12A1: Isoform 4 of Collagen alpha-1 (XII) chain 1.631 0.105 IPI
00072917 COL6A3: COL6A3 protein 2.486 3.921 1.627 1.545 IPI
00220113 COL6A3: COL6A3 protein 2.438 0.59 1.047 0 2.438 1.271 1.013 0.446 IPI
00291136 COL6A1: Collagen alpha-1 (VI) chain 1.316 0.506 1.786 0.136 IPI
00339225 FN1: Isoform 5 of Fibronectin 4.4563 0 1.027 0.213 6.706 0 1.054 0 IPI
00307162 VCL: Isoform 2 of Vinculin 1.62 0.81 1.051 0.273 IPI
00024157 FKBP3: Peptidyl-prolyl cis-trans isomerase FKBP3 bald only 2.642 0 bald only 3.02 0 IPI
00295857 COPA: Isoform 1 of Coatomer subunit alpha bald only 1.894 0 IPI
00220219 COPB2: Coatomer subunit beta ' bald only bald only IPI
00029557 GRPEL1: GrpE protein homolog 1, mitochondrial 3.1777 0 bald only 5.069 0 bald only IPI
00010720 CCT5: T-complex protein 1 subunit epsilon 2.066 0.141 1.069 0.118 IPI
00007765 HSPA9: Stress-70 protein, mitochondrial 1.573 0.029 1.467 0 1.573 0.029 1.737 0.542 IPI
00018465 CCT7: T-complex protein 1 subunit eta 1.466 0.18 1.56 0.229 1.466 0.18 1.56 0.229 IPI
00299571 PDIA6: Isoform 2 of Protein disulfide-isomerase A6 1.2509 0.089 1.432 0 1.139 0.089 1.811 0.513 IPI
00382470 HSP90AA1: Isoform 2 of Heat shock protein HSP 90-alpha 1.818 0.205 1.4881 0 1.818 1.187 1.141 0.292 IPI
00027230 HSP90B1: Endoplasmin 1.471 0.689 1.643 1.089 1.471 0.689 1.643 1.089 IPI
00939289 HSP90B1: cDNA FLJ58626, highly similar to Endoplasmin 2.83 4.911 4.117 3.685 IPI
00027378 UBXN1: Isoform 1 of UBX domain-containing protein 1 bald only 2.754 0 IPI
00156689 VAT1: Synaptic vesicle membrane protein VAT-1 homolog bald only 2.269 0 bald only 2.601 0 IPI
00797709 CORO1C: 18 kDa protein 4.4563 1.24 9.529 0.959 IPI
00106668 PLIN3: mannose 6 phosphate receptor binding protein 1 isoform 3 2.149 0.826 IPI
00027809 PPP3CB: protein phosphatase 3, catalytic subunit, beta isoform a 6.926 0 1.615 0 IPI
00029631 ERH: Enhancer of rudimentary homolog 1.791 0 1.4928 0.13 1.791 0 1.146 0 IPI
00218918 ANXA1: Annexin A1 1.154 0.201 1.715 0.343 1.154 0.201 1.715 0.343 IPI
00020632 ASS1: Argininosuccinate synthase 2.96 0 1.02 0.23 4.563 2.27 1.085 0 IPI
00024915 PRDX5: Isoform Mitochondrial of Peroxiredoxin-5, mitochondrial 4.193 0 1.123 0.077 IPI
00000874 PRDX1: Peroxiredoxin-1 1.58 0.876 1.5104 0 1.58 0.518 1.164 0.087 IPI
00301364 SKP1: Isoform 1 of S-phase kinase-associated protein 1 3.388 1.13 4.43 0 3.388 0 3.335 0 IPI
00165579 CNDP2: Isoform 2 of Cytosolic non-specific dipeptidase 3.071 0 2.504 0 3.071 0 2.809 0 IPI
00304409 CARHSP1: Calcium-regulated heat stable protein 1 3.068 0 bald only 3.068 0 bald only IPI
00028888 HNRNPD: Isoform 1 of Heterogeneous nuclear ribonucleoprotein D0 2.976 1.104 1.388 0.024 2.976 1.225 1.388 0.024 IPI
00479786 KHSRP: KH-type splicing regulatory protein 2.304 0 2.288 0 IPI
00844578 DHX9: ATP-dependent RNA helicase A 2.249 0 2.15 0 2.249 0 2.335 0 IPI
00012074 HNRNPR: Isoform 1 of Heterogeneous nuclear ribonucleoprotein R 1.058 0.167 1.395 0.273 1.058 0.167 1.931 0.336 IPI
00304596 NONO: Non-POU domain-containing octamer-binding protein 1.02 0 1.461 0 1.02 0 1.753 0 IPI
00303476 ATP5B: ATP synthase subunit beta, mitochondrial 2.917 1.135 1.331 0.138 2.917 1.118 1.331 0.138 IPI
00007682 ATP6V1A: V-type proton ATPase catalytic subunit A 2.635 0.605 2.568 0 IPI
00440493 ATP5A1: ATP synthase subunit alpha, mitochondrial 2.31 0 1.144 0 IPI
00848320 ATP6V1B2: ATPase, V1 complex, subunit B family protein One 0 1.532 0 IPI
00021290 ACLY: ATP-citrate synthase 1.2549 0.046 1.013 0.024 IPI
00012585 HEXB: Beta-hexosaminidase subunit beta 2.71 0 2.331 0 2.906 0 2.602 0 IPI
00291006 MDH2: Malate dehydrogenase, mitochondrial 1.855 0.671 1.131 0.331 IPI
00220271 AKR1A1: Alcohol dehydrogenase [NADP +] 1.434 0 1.001 0 1.434 0 1.106 0 IPI
00465248 ENO1: Isoform alpha-enolase of Alpha-enolase 1.148 0.509 1.026 0 IPI
00217223 PAICS: Phosphoribosyl aminoimidazole carboxylase (ADE2) 2.702 0 bald only IPI
00015029 PTGES3: Prostaglandin E synthase 3 1.22 0 bald only 1.013 0 bald only IPI
00003128 P4HA2: Isoform IIb of Prolyl 4-hydroxylase subunit alpha-2 2.612 0.748 1.129 0 IPI
00026182 CAPZA2: F-actin-capping protein subunit alpha-2 2.576 0 1.946 0 2.576 0 1.946 0 IPI
00456969 DYNC1H1: Cytoplasmic dynein 1 heavy chain 1 2.012 0.273 bald only IPI
00216694 PLS3: Plastin-3 1.622 0 1.023 0.327 IPI
00298994 TLN1: Talin-1 1.302 0.303 1.515 0.541 1.302 0.303 1.515 0.541 IPI
00100980 EHD2: EH domain-containing protein 2 1.092 0.118 1.645 0.448 1.092 0.118 1.645 0.448 IPI
00465084 DES: Desmin 1.669 0 1.43 0 1.069 0 13.233 0 IPI
00000861 LASP1: Isoform 1 of LIM and SH3 domain protein 1 1.559 0.413 1.061 0.106 IPI
00141318 CKAP4: Isoform 1 of Cytoskeleton-associated protein 4 bald only 1.491 0.2 bald only 1.716 0.61 IPI
00843765 SPTAN1: Isoform 3 of Spectrin alpha chain, brain 3.301 0.58 8.153 0 5.305 32.2 3.33 4.81 IPI
00022334 OAT Ornithine aminotransferase, mitochondrial 2.545 0 1.357 0 IPI
00032140 SERPINH1: Serpin H1 2.254 0.339 1.329 0.581 2.254 0.615 1.329 0.581 IPI
00220327 KRT1: Keratin, type II cytoskeletal 1 2.064 0.536 2.064 0.536 IPI
00220834 XRCC5: ATP-dependent DNA helicase 2 subunit 2 1.999 0.08 3.002 0.438 1.999 0.08 3.084 0.418 IPI
00398775 PLEC1: Isoform 9 of Plectin-1 1.994 0.85 1.447 0 1.994 0.85 1.768 5.06 IPI
00178431 RECQL: ATP-dependent DNA helicase Q1 1.128 0 bald only IPI
00011229 CTSD: Cathepsin D 1.281 0 1.052 0 IPI
00186711 PLEC1: Isoform 2 of Plectin-1 2.198 1.11 2.746 1.28 IPI
00010418 MYO1C: Isoform 2 of Myosin-Ic 1.947 0 2.658 0 IPI
00784156 AP2B1: Isoform 1 of AP-2 complex subunit beta 1.941 0 bald only 1.941 0 bald only IPI
00021766 RTN4: Isoform 1 of Reticulon-4 1.678 0.381 1.819 0.194 IPI
00030781 STAT1: Isoform Alpha of Signal transducer and activator of transcription 1-alpha / beta 1.583 0.393 1.405 0.086 IPI
00026260 NME2: NME1 Isoform 1 of Nucleoside diphosphate kinase B 1.5052 0.195 IPI
00015018 PPA1: Inorganic pyrophosphatase 1.65 0 1.039 0.112 1.562 0 1.031 0 IPI
00295386 CBR1: Carbonyl reductase [NADPH] 1 1.501 0 2.955 0 IPI
00028091 ACTR3: Actin-related protein 3 1.496 0.292 2.176 0 1.496 0.292 2.482 0.99 IPI
00916188 NCL: Putative uncharacterized protein NCL 1.476 0 2.279 0 IPI
00221224 ANPEP: Aminopeptidase N 1.0848 0.181 2.543 0 1.367 0.181 2.998 0 IPI
00246975 GSTM3: Glutathione S-transferase Mu 3 1.147 0 1.425 0 1.434 0 1.864 0 IPI
00019755 GSTO1: Glutathione S-transferase omega-1 3.5975 0.363 bald only 6.055 1.548 bald only IPI
00289758 CAPN2: Calpain-2 catalytic subunit 1.411 0 1.555 0 IPI
00000877 HYOU1: Hypoxia up-regulated protein 1 1.272 0 2.183 0 1.272 0 2.118 0 IPI
00016513 RAB10: Ras-related protein Rab-10 1.464 0 1.5582 0.497 IPI
00028635 RPN2: Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 2 1.27 0.05 1.691 0 1.27 0.05 2.206 0 IPI
00793199 ANXA4: annexin IV 1.245 0.097 1.498 0.088 1.245 0.097 1.498 0.088 IPI
00032064 AKAP2: PALM2-AKAP2 Isoform 2 of A-kinase anchor protein 2 1.217 0 6.185 0 IPI
00024664 USP5: Isoform Long of Ubiquitin carboxyl-terminal hydrolase 5 1.215 0 3.524 0 IPI
00021347 UBE2L3: Ubiquitin-conjugating enzyme E2 L3 1.023 0.145 1.745 0 1.023 0.145 2.054 0.233 IPI
00015148 RAP1B: Ras-related protein Rap-1b 1.2 0 1.483 0.151 1.144 0 1.722 0 IPI
00009342 IQGAP1: Ras GTPase-activating-like protein 1 IQGAP1 2.896 3.56 1.408 0.2 2.896 3.56 1.909 0.83 IPI
00005198 ILF2: Interleukin enhancer-binding factor 2 1.133 0 1.619 0 IPI
00029750 RPS24: Isoform 1 of 40S ribosomal protein S24 0.78 0 0.435 0 0.98 0 0.5244 0 IPI
00219153 RPL22: 60S ribosomal protein L22 0.794 0 0.3923 0 IPI
00014263 EIF4H: Isoform Long of Eukaryotic translation initiation factor 4H 0.5923 0 0.993 0.081 IPI
00023748 NACA: Nascent polypeptide-associated complex subunit alpha 0.5831 0 0.5907 0 IPI
00014581 TPM1: Isoform 1 of Tropomyosin alpha-1 chain 0.796 0.018 0.5797 0.1 IPI
00410693 SERBP1: Isoform 1 of Plasminogen activator inhibitor 1 RNA-binding protein 0.9129 0 1.69 0 IPI
00010740 SFPQ: Isoform Long of Splicing factor, prolineand glutamine-rich 0.5639 0 0.952 0 0.5513 0 0.856 0 IPI
00299573 RPL7A: 60S ribosomal protein L7a 0.193 0 0.463 0.357 0.3867 0 0.6035 0.164 IPI
00031812 YBX1: Nuclease-sensitive element-binding protein 1 0.5023 0.136 IPI
00215965 HNRNPA1: Isoform A1-B of Heterogeneous nuclear ribonucleoprotein A1 0.1645 0.017 0.926 0.333 IPI
00018278 H2AFV: Histone H2A.V 0.5308 0.037 0.869 0.117 IPI
00453473 HIST1H4L: Histone H4 0.4634 0.009 0.692 0.018 IPI
00011200 PHGDH: D-3-phosphoglycerate dehydrogenase non-bald only 0.163 0.039 non-bald only non-bald only

< Example  7. Expression Verification of Proteins>

Western blot analysis was performed to verify the expression of the protein identified in Example 6 above. To this end, both hair loss and non-hair loss milk cells are washed twice in an ice-cold phosphate-buffered saline (PBS), and the cell pellet is extracted with a proteinase inhibitor to extract the cell extract. RIPA buffer was added and reacted (RIPA buffer: 20 mM Tris-HCl, pH 7.5, 1 mM Na ₂ EDTA, 1 mM Na ₂ EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na ₃ VO ₄ , 1 / ml leupeptin, 1 mM Na ₃ VO ₄ ).

Thereafter, the reaction was placed on ice for 15 minutes, and then centrifuged at 4 ° C. and 12,000 rpm for 5 minutes to collect only supernatants. The harvested cell extracts were protein quantified using BCA assay kit (Pierce), and then 5 μg of protein was inactivated in SDS sample buffer and separated by electrophoresis on 10% SDS-polyacrylamide gel. The isolated protein was transferred to nitrocellulose membrane (60 minutes at 150 mA) and then blocked with 5% skim milk (PBS / 0.1% Tween-20). Each primary antibody was reacted at 4 ° C. for 18 hours or more, the next day the secondary antibody was reacted, and each antibody was washed with PBST. Thereafter, the intensity of each protein band was analyzed using an ECL solution, and the analysis results are shown in FIG. 3.

 Referring to the results of FIG. 3, hair loss cells collected from two male hair loss patients (Patient 1 and Patient 2) were identified as ASS1 (argininosuccinate synthase 1), ADE2 (phosphoribosylaminoimidazole carboxylase, PAICS), CKAP4 (cytoskeleton-associated protein 4), and GSN. (Gelsolin), IQGAP1 (Ras GTPaseactivating-like protein 1) and SKP1 (S-phase kinase-associated protein 1) were found to increase, and the protein increase pattern was consistent with LC-MS / MS analysis.

< Example  8. Immunofluorescence >

In order to verify the expression of the protein identified in Example 6, a hair follicle sample containing both hair loss cells and non-hair loss milk cells obtained from the scalp of a male patient who had undergone hair transplantation was embedded with a medium (OCED compound). , Tissue-Tek; Miles, Napierville, IL) and hardened at -80 ° C. Thereafter, it was cut to a thickness of 7 μm and fixed by reacting with 4% paraformaldehyde (4% paraformaldehyde containing 0.1% triton X-100) for 10 minutes. Next, the embedded section was washed with PBS for 15 minutes, blocked with 4% donkey serum for 1 hour, and then diluted with primary antibody (IQGAP1, PLEC1) at 4 ° C for 18 hours or more. , GSN, SPTAN1). Thereafter, the mixture was reacted with a secondary antibody, reacted with 1 mg / ml DAPI (4 ′, 6-diamidino-2-phenylindole) for 10 minutes for DNA staining, and then mounted (using VECTASHIELD mounting medium, [Vector Laboratories, Burlingame, CA]). Fluorescence images were measured using a confocal microscope (Carl Zeiss, Oberkochen, Germany) and the results are shown in FIG. 4.

Referring to Figure 4, IQGAP1 (Ras GTPaseactivating-like protein 1), PLEC1 (Isoform 9 of Plectin-1), GSN (Gelsolin) and SPTAN1 (Isoform 3 of Spectrin alpha chain, brain) are all hair loss in the hair follicles It was confirmed that the remarkably increased, it was able to prove that the protein is expressed in the same pattern as LC-MS / MS.

Claims (8)

Biomarker composition for diagnosing male hair loss comprising at least three proteins selected from the following protein groups as an active ingredient:
Protein increases in all hair loss
Argininosuccinate synthase (ASS1, Accession No. IPI00020632);
Phosphoribosyl aminoimidazole carboxylase (PAICS [ADE2], Accession No. IPI00217223);
Isoform 1 of Cytoskeleton-associated protein 4 (CKAP4, Accession No. IPI00141318);
Isoform 1 of Gelsolin (GSN, Accession No. IPI00026314);
Ras GTPase-activating-like protein 1 (IQGAP1, Accession No. IPI00009342);
Isoform 1 of S-phase kinase-associated protein 1 (SKP1, Accession No. IPI00301364);
Isoform 3 of Spectrin alpha chain, brain (SPTAN1, Accession No. IPI00843765);
Isoform 9 of Plectin-1 (PLEC1, Accession No. IPI00398775). A diagnostic agent containing an antibody that recognizes three or more proteins selected from the group of proteins increasing in all the hair loss cells described in claim 1. A diagnostic kit containing an antibody that recognizes three or more proteins selected from the group of proteins increasing in all of the hair loss cells described in claim 1. A protein chip containing an antibody that recognizes three or more proteins selected from the group of proteins increasing in all the hair loss cells described in claim 1 or three or more proteins selected from the group of proteins increasing in all the hair loss cells. A method for diagnosing male baldness using the biomarker composition of claim 1. A method for diagnosing male baldness using the diagnostic agent of claim 2. A method for diagnosing male baldness using the diagnostic kit of claim 3. Method for diagnosing male baldness using the protein chip of claim 4.

Images