KR101424217B1 - Composition for the diagnosis, therapy and prophylaxis of age-realted macular deteneration and method for obtaining information on age-realted macular deteneration - Google Patents

Abstract

The present invention relates to a protein biomarker for diagnosis and treatment of age-related macular degeneration.

Description

TECHNICAL FIELD The present invention relates to a composition for diagnosis, treatment and prevention of age-related macular degeneration, and a method for obtaining information on age-related macular degeneration (hereinafter referred to as " age-real macular deterioration &

The present invention relates to a protein biomarker for diagnosis and treatment of age-related macular degeneration.

The average life expectancy of Koreans as well as the rest of the world is living in an age of over 80. In Korea, aging is on the fastest pace in the world due to rapid increase in average life expectancy and decrease in fertility rate. In order to maintain a healthy life as the life expectancy increases, the treatment of diseases caused by aging and the external environment is very important. The most common and representative disease caused by aging is eye related disease. The age of the onset of ophthalmologic disease, which was considered to be a disease caused by aging, has been lowered due to the changed environmental factors and the incidence is also increasing. Elderly macular degeneration, the most common cause of blindness in adults aged 50 years and older, is rapidly increasing in Korea, and its prevalence is high.

Age-related macular degeneration (AMD) is a disease in which retinas and retinal pigment epithelium, choriocapillaris are progressive, degenerated, and atrophy in adults over 50 years. AMD is already an important disease in the western world to overtake diabetic retinopathy and glaucoma and become the leading cause of blindness among adults aged 50 years and older. In the United States, more than 8 million people are now reported to have the more common form of this disease, non-vascular or atrophic AMD, and more than 3 million people die of this disease each year. In Korea, the rate of incidence is rapidly increasing with westernization of food, environmental factors, and life expectancy. It is estimated that about 250,000 people, aged 65 and over, 5% of the 5 million people, are suffering from age-related macular degeneration. Such geriatric diseases are likely to become not only economic problems such as medical burden but also a great social problem such as a decrease in "quality of life". Therefore, there is an urgent need for early diagnosis and efficient treatment of age - related macular degeneration.

However, the pathogenesis of AMD is multifactorial and only partly revealed. In addition to the genetic predisposition, there is a link between environmental pollution, (excessive) light exposure, and the accumulation of harmful environments such as oxidative stress, but the specific mechanism is not yet fully understood.

Currently, the treatment status of patients with AMD is as follows.

The treatment of choroidal neovascularization (CNV), a mature macular degeneration in the elderly, is largely divided into destructive therapy (laser photocoagulation, metastatic surgery, surgical surgery) and pathway-based therapy [ Anti-vascular endothelial growth factor, anti-vascular endothelial growth factor].

However, to date, none of the above methods have resulted in serious visual loss or slow visual acuity improvement. In particular, these therapies are limited to the treatment of angiogenic AMD [wet AMD, or neovascular AMD] and include non-angiogenic formation, which accounts for 70-80% of AMD patients Old age macular degeneration Adult atrophic degeneration [dry AMD, geographic atrophy] has rarely been treated.

Currently, there is only one repetitive intravitreal injection of anti-VEGF that has been proven to have a limited visual acuity improvement in the treatment of mood-old age-related macular degeneration. It is a treatment targeting vascular endothelial growth factor (VEGF), an angiogenic cytokine of choroidal neovascularization (CNV) of mature senile macular degeneration. However, only one-third of the patients who received anti-VEGF treatment had limited visual improvement and one sixth of the treated patients progressed to legal blindness. In addition, safety issues such as retinal toxicity, which can be achieved by continuously suppressing VEGF present in the normal adult retina, have been raised. This treatment is repeatedly injected repeatedly due to short half-life of intravitreal drug, and repeated injections increase the likelihood of complications such as vitreous hemorrhage, retinal detachment, and endophthalmitis. VEGF acts as a potent vasodilator, which helps maintain coronary artery relaxation and blood circulation in the heart. Since AMD patients are older, they are at higher risk for cardiovascular disease and there is also a risk of serious side effects. Therefore, in addition to VEGF, it is necessary to develop materials targeting and targeting AMD specific molecules or protein markers.

The present invention is a system for discovering biomarkers by introducing a proteomics technique for the early diagnosis and effective treatment of the above-mentioned age-related macular degeneration.

Changes in waterproofing proteins and cytokines in some guiding diseases, especially in the anterior segment inflammatory disease, have been reported, but changes in the total waterproofing protein of the elderly macular degeneration patients have not been previously investigated. Patients with mild age-related macular degeneration who have no previous history of treatment, and patients with cataract surgery whose age and gender are in agreement with each other, will be analyzed by proteomics.

In particular, we have used proteomics to identify materials that may have developed into retina and retinal pigment epithelium as a kind of 'adaptation' and endogenous neuroprotection under stress conditions including oxidative stress, and have been used to treat dry AMD It is a core technology to be utilized in the present invention to be usefully used in target treatment and monitoring of therapeutic response.

The present invention solves the above problems and aims at providing a protein biomarker for diagnosis and treatment of age-related macular degeneration.

In order to achieve the above object, the present invention provides a lysozyme precursor of GI No. 4557894, autophagy-related protein 7 isoform c of 222144229, car of 4503143, Cathepsin D preproprotein, 14-3-3 protein zeta / delta of 208973244, keratin of 4504919, type II cytoskeletal 8,55956899 Keratin, Type I Saitoskeletal 9,24430190 Keratin Type I Saitoskeletal 15,88853069 Bittern Nectin Precursor 24234699 Keratin Type I Saitoskeletal 19,58530842 Desmoplakin Isoform II 119392081 Complement (IX) chain precursor of the complement factor I pre-protein, the complement factor H-related protein 1 precursor of 118442839, the proteasome 26S non-ATPase subunit 1,11386161 of 25777600, , SPARC of 190341024 -Like protein 1 precursor, 4507149 superoxide dismutase, 4504517 heat shock protein beta -1,124256496 heat shock 70 kDa protein 1-analog, 109255234 290 kDa centrosomal protein (centrosomal protein and one or more proteins selected from the group consisting of glyceraldehyde-3-phosphate dehydrogenase (7669492).

The present invention also relates to a kit comprising a lysozyme precursor of GI No. 4557894, an autophagy-related protein 7 isoform c of 222144229, a cathepsin D-free of 4503143, A preprotein, a 14-3-3 protein zeta / delta of 208973244, a keratin of 4504919, a keratin of type II cytoskeletal 8,55956899, Keratin type I of Saitosuketaltal 15,88853069 of Retal 9,24430190, desmoplakin isform II of keratin type I cytoskeletal 19,58530842 of 24234699, complement factor I of 119392081 (IX) chain precursor of the proteasome 26S non-ATPase subunit 1,11386161 of 25777600, SPARC-like (SEQ ID NO: 1) of 190341024, the precursor of the complement factor H- like protein 1 precursor, 4507149 of the heat shock protein beta-1, 124256496, heat shock 70 kDa protein 1-analogue, 109255234 290 kDa centrosomal protein, and 7669492 glyceraldehyde 3-phosphate dehydrogenase, and an antibody against at least one protein selected from the group consisting of glyceraldehyde-3-phosphate dehydrogenase.

The present invention also provides a method for obtaining information on old age macular degeneration in a subject,

(a) measuring one or more biomarkers in a biological sample from a subject, wherein the one or more biomarkers are selected from the compositions according to claim 1 of the present invention; And

(b) comparing the measurements or measurements with normal subjects and correlating them with age-old macular degeneration when the measurements are higher than normal.

In the present invention, the term subject is a patient with an age-old macular degeneration and the immunogenic fragment means a fragment of a biomarker protein having at least one epitope that can be recognized by an antibody to the biomarker protein of the present invention .

In order to accomplish still another object of the present invention, the present invention provides a diagnostic agent for old age-old macular degeneration diseases comprising an antibody which specifically binds to the biomarker protein of the present invention or an immunogenic fragment thereof as an active ingredient.

The antibody of the present invention may be a polyclonal antibody, but is preferably a monoclonal antibody.

The polyclonal antibody can be prepared by injecting an immunogen-causing biomarker protein or a fragment thereof into an external host according to conventional methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, and rabbits. The immunogen is injected intraperitoneally, intraperitoneally or subcutaneously, and is generally administered with an adjuvant to increase the antigenicity. Blood is routinely collected from an external host to collect the sera showing improved titer and specificity for the antigen or isolating and purifying the antibody therefrom.

Monoclonal antibodies can be prepared by immortalized cell line generation techniques by fusion as disclosed in the art [Koeher and Milstein 1975, Nature, 256: 495). The manufacturing method will be briefly described as follows. First, 20 μg of pure protein is obtained to immunize Balb / C mice or to synthesize peptides and bind to bovine serum albumin and immunize mice. Then hybridizing the antigen-producing lymphocytes isolated from the mice with the myeloma of a human or mouse to produce an immortalized hybridoma, and using hybridoma (ELISA) method to generate the desired monoclonal antibody After selective proliferation, the monoclonal antibody is separated and purified from the culture.

In order to achieve another object of the present invention, the present invention provides a method for obtaining information on the age-old macular degeneration of an individual, comprising the step of detecting the presence of the biomarker protein or an immunogenic fragment thereof of the present invention in the body fluid of an individual do.

In the method of the present invention, the detection step is a step of detecting directly the presence of the biomarker protein or its immunogenic fragment by two-dimensional (2-D) electrophoresis from the liver blood solution of the individual, Is contacted with the antibody of the present invention to indirectly confirm the presence of the biomarker protein or its immunogenic fragment through the antigen-antibody reaction

.

Immunoassay, which is widely known as an antigen-antibody reaction, is an enzyme-linked immunosorbent assay (ELISA, Coated Tube), an antibody-bound magnetic particle conjugated to a tube, A magnetic particle method in which an enzyme reaction is caused to occur by competitively reacting a degradable contaminant with each other, and a latex particle method using antibody-bonded latex particles.

In order to accomplish another object of the present invention, the present invention provides a diagnostic kit for old age-old macular degeneration comprising an antibody specifically binding to the biomarker protein of the present invention or an immunogenic fragment thereof as an active ingredient.

The diagnostic kits of the present invention are prepared by conventional methods known to those skilled in the art and typically include lyophilized forms of antibodies and buffers, stabilizers, inert proteins, and the like. The antibody may be labeled with a radionuclide, a fluorescent source, an enzyme, or the like.

The monoclonal antibodies of the present invention can be used in a wide variety of immunoassay kits (ELISA, antibody coated tube test, lateral-flow test, potable biosensor) Can be used for the development of protein chips having various age-old macular degeneration detection spectra through the development of antibodies exhibiting higher specificity and sensitivity.

In order to accomplish another object of the present invention, the present invention provides a composition for treating and preventing old age-related macular degeneration diseases, which comprises as an active ingredient a protein selected from the group of increase or decrease in the aqueous humor of an old age-related macular degeneration patient.

The pharmaceutical composition of the present invention may be formulated into injectable preparations by mixing with a pharmaceutically acceptable carrier, excipient, diluent or the like by a method known in the pharmaceutical field and administered by intravenous injection or the like. The dosage of the pharmaceutical composition according to the present invention can be suitably selected according to the patient's age, sex, condition, and symptoms of the disease, and preferably 0.001 to 100 mg of protein per day on an adult basis.

Hereinafter, the present invention will be described.

The present invention utilizes the proteomics technique to exclude biomarkers from aqueous humor samples of elderly patients with macular degeneration.

Therefore, we analyzed the changes of protein levels in two patients before treatment, two patients after treatment, and two patients in normal group. For the reproducibility of the experiment, three experiments were repeated with the same sample, and a list of proteins detected more than twice was selected. As a result, 131 and 161 proteins were detected in patient 1 and 2 before treatment, respectively, and 264 and 183 proteins were detected in patients 1 and 2 after treatment for the same patient. 161 and 144 proteins were detected in normal samples 1 and 2 (FIG. 2).

In order to confirm the mass change of the protein between the patient group and the normal group, the label-free quantification method was used to select 92 and 75 proteins more than 1.5 times in the pre-treatment group compared to the normal group (Table 1-2), and 67 and 64 proteins that decreased after treatment were selected (Table 3-4). The western blot was performed on representative proteins of cathepsin D and desmoplakin expected to be significant candidates for biomarkers in elderly macular degeneration patients (Fig. 3) And the change of mass was observed.

Table 1-2 shows the list of proteins increased more than 1.5 times in the patient group compared with the normal group.

A. Protein list increased more than 1.5 times in patient 1 No. GI No . MW Reference One 4502101 38690 annexin A1 2 109255249 63871.45 keratin, type II cytoskeletal 4 3 213688375 41981.82 actin, aortic smooth muscle 4 4504517 22768.49 heat shock protein beta-1 5 45439327 204552.8 periplakin 6 44890059 68437.3 involucrin 7 4503475 50438.36 eukaryotic translation elongation factor 1 alpha 2 8 31543169 52080.16 histone-lysine N-methyltransferase SUV420H2 9 5454052 27756.7 14-3-3 protein sigma 10 4506773 13233.5 protein S100-A9 11 4501887 41765.8 actin, cytoplasmic 2 12 77539758 11360.38 histone H4 13 291045225 3710441 titin isoform N2-A 14 27436948 70618.16 lamin-A / C isoform 3 15 4504183 23341.02 glutathione S-transferase P 16 214010185 59114.2 amyloid-like protein 2 isoform 4 17 94536793 102933.8 tubulin polyglutamylase TTLL7 18 167466177 92553.75 cell division cycle protein 27 isoform 1 19 186659512 177860.6 calmodulin-regulated spectrin-associated protein 1 20 61743954 628705.2 neuroblast differentiation-associated protein AHNAK isoform 1 21 4504253 15135.42 histone H2A.x 22 157653329 47942.04 procollagen C-endopeptidase enhancer 1 23 109255234 290205.6 The centrosomal protein of 290 kDa 24 57864582 282225.7 hornerin 25 41327708 154080.4 crumbs homolog 1 precursor 26 124028519 160892.4 hypothetical protein LOC158401 isoform 2 27 226246665 51091.87 keratin, type II cytoskeletal 72 isoform 2 28 73858564 45095.96 corticosteroid-binding globulin precursor 29 212549782 222518.8 caspase 8 associated protein 2 30 115511024 64831.51 lipase maturation factor 1 31 53729346 82807.91 plakophilin-1 isoform 1b 32 208973244 27727.73 14-3-3 protein zeta / delta 33 47607492 518156.3 plectin isoform 1c 34 32455266 22096.28 peroxiredoxin-1 35 28173554 13899.54 histone H2B type 3-B 36 4505821 16561.8 전사인 인 인피티비 전구체 37 195947389 101902.1 ectonucleotide pyrophosphatase /
phosphodiesterase family member 2 isoform 3 preproprotein 38 178557739 192629.6 complement C4-B preproprotein 39 5031863 65289.4 galectin-3-binding protein 40 94536788 103344.8 THAP domain-containing protein 9 41 5031579 76061.17 A-kinase anchor protein 8 42 222144229 68573.8 autophagy-related protein 7 isoform c 43 262359929 253301.3 protein ELYS 44 163659918 520794.2 sacsin 45 154146262 571640.9 ィGFGF-binding protein precursor 46 206597522 106915.3 arf-GAP with SH3 domain,
ANK repeat and PH domain-containing protein 2 isoform b 47 124256496 70331.45 heat shock 70 kDa protein 1-like 48 169211906 22688.56 PREDICTED: hypothetical protein 49 166795236 35686.62 tumor-associated calcium signal transducer 2 precursor 50 12667788 226390.6 myosin-9 51 57013276 50119.64 tubulin alpha-1B chain 52 29788768 49920.98 tubulin beta-2B chain 53 5902072 44536.6 serpin B3 54 60097902 434921.4 filaggrin 55 194248072 70009.18 heat shock 70 kDa protein 1A / 1B 56 16507237 72288.52 78 kDa glucose-regulated protein 57 10190712 11654.76 S100 calcium binding protein A14 58 21614499 69369.84 ezrin 59 5803227 27746.77 14-3-3 protein theta 60 27894339 48101.24 keratin 23 61 5032161 12465 transcription elongation factor B polypeptide 1 62 109148552 64377.6 keratin, type II cytoskeletal 3 63 34734068 61538.3 fibulin-1 isoformed precursor 64 21071030 54219.66 alpha 1B-glycoprotein precursor 65 5031839 60008.32 keratin, type II cytoskeletal 6A 66 189083782 81890.13 gelsolin isoform c 67 58530842 259954.2 desmoplakin isoform II 68 239753016 13156.05 PREDICTED: similar to hCG2042913 69 239752583 15863.81 PREDICTED: hypothetical protein XP_002348171 70 209915573 80782.38 amyloid beta A4 protein isoform f precursor 71 239753442 194818.7 PREDICTED: HEAT repeat containing 7B1 72 4557373 61093.34 비오티 니티스 전구체 73 154277116 1010419 nesprin-1 isoform 1 74 169207532 24015.14 PREDICTED: similar to hCG2042717 75 37588869 148420 E3 ubiquitin-protein ligase RNF123 76 24430190 49167.11 keratin, type I cytoskeletal 15 77 50592994 11729.74 thioredoxin 78 116295258 129214.8 integrin alpha-2 precursor 79 5902010 45749.07 transcription termination factor, mitochondrial precursor 80 155969697 59988.35 keratin, type II cytoskeletal 6C 81 24430192 51236.25 keratin, type I cytoskeletal 16 82 12056468 81692.87 junction plagoglobin 83 33620759 39754.96 LRP2-binding protein 84 119703753 60030.33 keratin, type II cytoskeletal 6B 85 7669492 36030.4 glyceraldehyde-3-phosphate dehydrogenase 86 260436922 60504.64 suprabasin isoform 1 precursor 87 4503143 44523.66 cathepsin D preproprotein 88 192807292 55053.49 keratin 24 89 4504489 59540.94 histidine-rich glycoprotein precursor 90 4507725 15877.05 트렌치 하이레틴 전구체 91 116734860 174651.4 amylo-1,6-glucosidase, 4-alpha-glucanotransferase isoform 1 92 25777600 105768.7 proteasome 26S non-ATPase subunit 1

B. Protein list increased more than 1.5 times in patient 2 No. GI No . MW Reference One 206597441 50362.9 aldehyde dehydrogenase, dimeric NADP-preferring 2 4504935 55766.54 keratin, type II cuticular Hb5 3 4557287 53120.61 angiotensinogen preproprotein 4 4503571 47139.37 alpha-enolase 5 5902010 45749.07 transcription termination factor, mitochondrial precursor 6 25777600 105768.7 proteasome 26S non-ATPase subunit 1 7 118442839 37625.96 complement factor H-related protein 1 precursor 8 55743122 22995.26 retinol-binding protein 4 precursor 9 213688375 41981.82 actin, aortic smooth muscle 10 261278368 76506.45 lipid phosphate phosphatase-related protein type 4 isoform 2 11 115527097 194191.4 serine / threonine-protein kinase MRCK beta 12 4505529 23587.64 alpha-1-acid glycoprotein 2 precursor 13 190341024 75161.32 SPARC-like protein 1 precursor 14 222144229 68573.8 autophagy-related protein 7 isoform c 15 58530840 331567.8 desmoplakin isoform I 16 11386161 65090.96 collagen alpha-2 (IX) chain precursor 17 33620759 39754.96 LRP2-binding protein 18 109255234 290205.6 The centrosomal protein of 290 kDa 19 75677365 507374.4 dynein heavy chain 2, axonemal 20 134031945 547136.1 SCO-spondin precursor 21 4507509 23155.55 metalloproteinase inhibitor 1 precursor 22 34787409 166691.8 ral GTPase-activating protein beta subunit 23 74271828 44385.62 mTERF domain-containing protein 3, mitochondrial precursor 24 4501887 41765.8 actin, cytoplasmic 2 25 4557373 61093.34 비오티 니티스 전구체 26 4506145 26541.09 trypsin-1 preproprotein 27 156627579 22522.28 테트라네틴 기체 28 4507149 15925.91 슈퍼 산화물 29 4505047 38404.8 lumican precursor 30 33413418 42232.73 signal peptide peptidase-like 3 31 4504489 59540.94 histidine-rich glycoprotein precursor 32 162809334 163759.1 전국화 기체 전구체 33 4826762 45176.59 haptoglobin isoform 1 preproprotein 34 144226251 42598.43 chitinase 3-like 1 precursor 35 119392081 65676.66 complement factor I preproprotein 36 4502149 11167.9 apolipoprotein A-II preproprotein 37 4503143 44523.66 cathepsin D preproprotein 38 58530842 259954.2 desmoplakin isoform II 39 89357932 56830.53 keratin, type II cytoskeletal 78 40 62122917 247926.3 filaggrin family member 2 41 209862791 69012.77 zinc finger protein 90 42 77539758 11360.38 histone H4 43 156104891 58205.11 general transcription factor IIF subunit 1 44 4502067 38973.99 protein AMBP preproprotein 45 42734397 43628.38 ubiquitin-conjugating enzyme E2C binding protein 46 117320537 147775.9 1-phosphatidylinositol-4,5-bisphosphate
phosphodiesterase gamma-2 47 61743957 28173.1 C-> U-editing enzyme APOBEC-1 48 239755764 128617.5 PREDICTED: hypothetical protein 49 22538442 33846.23 cathepsin Z preproprotein 50 4505821 16561.8 전사인 인 인피티비 전구체 51 239757666 14577.9 PREDICTED: similar to hCG2040021 52 148276990 134171.8 zinc finger and BTB domain-containing protein 38 53 57529737 119521 PDZ domain containing ring finger 3 54 62526026 19104.11 protein CutA isoform 3 55 5729770 61209.71 tripeptidyl-peptidase 1 preproprotein 56 111125011 41499.8 WNT inhibitory factor 1 precursor 57 239758153 23120.24 PREDICTED: similar to Ig heavy chain 58 153791670 65800.08 keratin, type II cytoskeletal 2 oral 59 190684677 163715.5 mitogen-activated protein kinase-binding protein 1 isoform b 60 57863250 184587.1 terminal uridylyltransferase 4 isoform c 61 4507467 74634.1 transforming growth factor-beta-induced protein ig-h3 precursor 62 4503009 53117.17 carboxypeptidase E preproprotein 63 222446620 124963 hypothetical protein LOC342346 64 115298678 187029.3 complement C3 precursor 65 50659080 47620.63 alpha-1-antichymotrypsin precursor 66 4504619 29111.45 insulin-like growth factor binding protein 7 67 4502261 52568.98 antithrombin-III precursor 68 21071030 54219.66 alpha 1B-glycoprotein precursor 69 4557894 16526.29 lysozyme precursor 70 7669492 36030.4 glyceraldehyde-3-phosphate dehydrogenase 71 239752583 15863.81 PREDICTED: hypothetical protein XP_002348171 72 169207532 24015.14 PREDICTED: similar to hCG2042717 73 239756207 13240.43 PREDICTED: similar to pre-B lymphocyte gene 2 74 110347423 90913.51 spondin-1 precursor 75 205277441 46294.67 serine (or cysteine) proteinase inhibitor,
cladee, member 7 precursor

The following Table 3-4 shows a list of proteins that increased more than 1.5 times in the patients group compared with the normal group and decreased after 1 month of treatment.

A. List of proteins that decrease after treatment in Patient 1 No. GI No . MW Protein name One 192807292 55053.49 keratin 24 2 31543169 52080.16 histone-lysine N-methyltransferase SUV420H2 3 41327708 154080.4 crumbs homolog 1 precursor 4 109255249 63871.45 keratin, type II cytoskeletal 4 5 94536793 102933.8 tubulin polyglutamylase TTLL7 6 167466177 92553.75 cell division cycle protein 27 isoform 1 7 186659512 177860.6 calmodulin-regulated spectrin-associated protein 1 8 178557739 192629.6 complement C4-B preproprotein 9 5031863 65289.4 galectin-3-binding protein 10 109148552 64377.6 keratin, type II cytoskeletal 3 11 4502101 38690 annexin A1 12 239753016 13156.05 PREDICTED: similar to hCG2042913 13 212549782 222518.8 caspase 8 associated protein 2 14 115511024 64831.51 lipase maturation factor 1 15 4557373 61093.34 비오티 니티스 전구체 16 37588869 148420 E3 ubiquitin-protein ligase RNF123 17 291045225 3710441 titin isoform N2-A 18 214010185 59114.2 amyloid-like protein 2 isoform 4 19 94536788 103344.8 THAP domain-containing protein 9 20 33620759 39754.96 LRP2-binding protein 21 222144229 68573.8 autophagy-related protein 7 isoform c 22 262359929 253301.3 protein ELYS 23 163659918 520794.2 sacsin 24 154146262 571640.9 ィGFGF-binding protein precursor 25 206597522 106915.3 arf-GAP with SH3 domain,
ANK repeat and PH domain-containing protein 2 isoform b 26 169211906 22688.56 PREDICTED: hypothetical protein 27 166795236 35686.62 tumor-associated calcium signal transducer 2 precursor 28 116734860 174651.4 amylo-1,6-glucosidase, 4-alpha-glucanotransferase isoform 1 29 124028519 160892.4 hypothetical protein LOC158401 isoform 2 30 226246665 51091.87 keratin, type II cytoskeletal 72 isoform 2 31 73858564 45095.96 corticosteroid-binding globulin precursor 32 239752583 15863.81 PREDICTED: hypothetical protein XP_002348171 33 209915573 80782.38 amyloid beta A4 protein isoform f precursor 34 239753442 194818.7 PREDICTED: HEAT repeat containing 7B1 35 154277116 1010419 nesprin-1 isoform 1 36 109255234 290205.6 The centrosomal protein of 290 kDa 37 34734068 61538.3 fibulin-1 isoformed precursor 38 189083782 81890.13 gelsolin isoform c 39 195947389 101902.1 ectonucleotide pyrophosphatase /
phosphodiesterase family member 2 isoform 3 preproprotein 40 4504183 23341.02 glutathione S-transferase P 41 169207532 24015.14 PREDICTED: similar to hCG2042717 42 116295258 129214.8 integrin alpha-2 precursor 43 5031579 76061.17 A-kinase anchor protein 8 44 25777600 105768.7 proteasome 26S non-ATPase subunit 1 45 157653329 47942.04 procollagen C-endopeptidase enhancer 1 46 44890059 68437.3 involucrin 47 4503143 44523.66 cathepsin D preproprotein 48 4504517 22768.49 heat shock protein beta-1 49 4504489 59540.94 histidine-rich glycoprotein precursor 50 4505821 16561.8 전사인 인 인피티비 전구체 51 4503475 50438.36 eukaryotic translation elongation factor 1 alpha 2 52 4501887 41765.8 actin, cytoplasmic 2 53 5902010 45749.07 transcription termination factor, mitochondrial precursor 54 45439327 204552.8 periplakin 55 4507725 15877.05 트렌치 하이레틴 전구체 56 21071030 54219.66 alpha 1B-glycoprotein precursor 57 61743954 628705.2 neuroblast differentiation-associated protein AHNAK isoform 1 58 4504253 15135.42 histone H2A.x 59 32455266 22096.28 peroxiredoxin-1 60 50592994 11729.74 thioredoxin 61 10190712 11654.76 S100 calcium binding protein A14 62 21614499 69369.84 ezrin 63 213688375 41981.82 actin, aortic smooth muscle 64 47607492 518156.3 plectin isoform 1c 65 24430190 49167.11 keratin, type I cytoskeletal 15 66 4506773 13233.5 protein S100-A9 67 208973244 27727.73 14-3-3 protein zeta / delta

B. List of Proteins Decreasing After Treatment in Patient 2 No. GI No . MW Reference One 4504935 55766.54 keratin, type II cuticular Hb5 2 25777600 105768.7 proteasome 26S non-ATPase subunit 1 3 115527097 194191.4 serine / threonine-protein kinase MRCK beta 4 5902010 45749.07 transcription termination factor, mitochondrial precursor 5 55743122 22995.26 retinol-binding protein 4 precursor 6 261278368 76506.45 lipid phosphate phosphatase-related protein type 4 isoform 2 7 190341024 75161.32 SPARC-like protein 1 precursor 8 222144229 68573.8 autophagy-related protein 7 isoform c 9 134031945 547136.1 SCO-spondin precursor 10 34787409 166691.8 ral GTPase-activating protein beta subunit 11 74271828 44385.62 mTERF domain-containing protein 3, mitochondrial precursor 12 4501887 41765.8 actin, cytoplasmic 2 13 4507149 15925.91 슈퍼 산화물 14 205277441 46294.67 serine (or cysteine) proteinase inhibitor,
cladee, member 7 precursor 15 4502149 11167.9 apolipoprotein A-II preproprotein 16 209862791 69012.77 zinc finger protein 90 17 77539758 11360.38 histone H4 18 156104891 58205.11 general transcription factor IIF subunit 1 19 4502067 38973.99 protein AMBP preproprotein 20 42734397 43628.38 ubiquitin-conjugating enzyme E2C binding protein 21 61743957 28173.1 C-> U-editing enzyme APOBEC-1 22 239755764 128617.5 PREDICTED: hypothetical protein 23 22538442 33846.23 cathepsin Z preproprotein 24 4505821 16561.8 전사인 인 인피티비 전구체 25 109255234 290205.6 The centrosomal protein of 290 kDa 26 57863250 184587.1 terminal uridylyltransferase 4 isoform c 27 4503571 47139.37 alpha-enolase 28 206597441 50362.9 aldehyde dehydrogenase, dimeric NADP-preferring 29 4507467 74634.1 transforming growth factor-beta-induced protein ig-h3 precursor 30 222446620 124963 hypothetical protein LOC342346 31 239752583 15863.81 PREDICTED: hypothetical protein XP_002348171 32 162809334 163759.1 전국화 기체 전구체 33 4505529 23587.64 alpha-1-acid glycoprotein 2 precursor 34 58530840 331567.8 desmoplakin isoform I 35 4503009 53117.17 carboxypeptidase E preproprotein 36 118442839 37625.96 complement factor H-related protein 1 precursor 37 239758153 23120.24 PREDICTED: similar to Ig heavy chain 38 4503143 44523.66 cathepsin D preproprotein 39 213688375 41981.82 actin, aortic smooth muscle 40 89357932 56830.53 keratin, type II cytoskeletal 78 41 117320537 147775.9 1-phosphatidylinositol-4,5-bisphosphate
phosphodiesterase gamma-2 42 148276990 134171.8 zinc finger and BTB domain-containing protein 38 43 4557894 16526.29 lysozyme precursor 44 4504619 29111.45 insulin-like growth factor binding protein 7 45 4502261 52568.98 antithrombin-III precursor 46 75677365 507374.4 dynein heavy chain 2, axonemal 47 4507509 23155.55 metalloproteinase inhibitor 1 precursor 48 4506145 26541.09 trypsin-1 preproprotein 49 156627579 22522.28 테트라네틴 기체 50 4505047 38404.8 lumican precursor 51 239756207 13240.43 PREDICTED: similar to pre-B lymphocyte gene 2 52 11386161 65090.96 collagen alpha-2 (IX) chain precursor 53 4557287 53120.61 angiotensinogen preproprotein 54 50659080 47620.63 alpha-1-antichymotrypsin precursor 55 4826762 45176.59 haptoglobin isoform 1 preproprotein 56 119392081 65676.66 complement factor I preproprotein 57 190684677 163715.5 mitogen-activated protein kinase-binding protein 1 isoform b 58 4557373 61093.34 비오티 니티스 전구체 59 33413418 42232.73 signal peptide peptidase-like 3 60 62122917 247926.3 filaggrin family member 2 61 239757666 14577.9 PREDICTED: similar to hCG2040021 62 57529737 119521 PDZ domain containing ring finger 3 63 62526026 19104.11 protein CutA isoform 3 64 5729770 61209.71 tripeptidyl-peptidase 1 preproprotein

As can be seen from the present invention, the protein identified in the present invention can be used for diagnosis of senile macular degeneration.

FIG. 1 is a view showing a waterproof protein sample of a patient with age-related macular degeneration obtained through one-dimensional electrophoresis,
FIG. 2 is a histogram showing the analysis of the identified proteins in groups,
Figure 3 is a western blot result of Cathepsin D and Desmoplakin.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Example 1: One-dimensional electrophoresis and coomassie blue staining

50ug of the aqueous humor of old age-related macular degeneration patients was subjected to one-dimensional electrophoresis (120V, 1 hr 30min) and the gel was stained by the method described below.

Proteins were separated by 1-D electrophoresis, washed three times for 15 minutes with 3 rd distilled water, and stained with gel code blue staining solution for 1 hour (Fig. 2).

Example  2: Protein Peptideization  Step (perform the steps below sequentially)

The dyed gel was divided into 6 sections according to the compartments and completely dyed using a de-staining reagent (50 mM ammonium bicarbonate in 50% acetonitrile) and 100% acetonitrile And dehydrated it.

The dehydrated sample was completely dried for 10 minutes using speed-vacuum.

Since the protein in the dried gel was connected to the disulfide bonds, dithiothreitol (DTT) was added to give a final concentration of 10 mM, and the reaction was carried out at 56 ° C for 45 minutes.

55 mM iodoacetamide was reacted in the dark for 30 minutes in order to prevent the -SH group, which had been broken by the preceding reaction, from being attached again, and the alkyl residue was then alkylated with the -SH residue.

To hydrolyze the protein, trypsin was added to the protein at a ratio of 1:20 and reacted overnight at 37 ° C.

After Overnight reaction, the sample was transferred to a 500-μL tube, and the tube was washed with 50 μl of an extraction solution [5% formic acid, 50% acetonitrile] And the mixture was reacted at room temperature for 20 minutes. (Repeat the above procedure twice)

The sample obtained in the above procedure was speed-dried by vacuum until the solution was completely blown.

Example  3: LC - ESI - MS / MS  And data analysis

The samples obtained in the previous procedure were analyzed using LTQ ion trap mass spectrometer.

A C18 microcapillary column (12 cm x 75 um) was connected to the mass spectrometer and the sample was injected into the column and analyzed on a mass spectrometer using RP-HPLC.

The mobile phase was diluted with solvent A [tertiary distilled water / 0.1% formic acid] and solvent B [acetonitrile / 0.1% formic acid] for 5-45 min gradient [3-40% solvent B]. (Table 3)

The flow rate of the column is 250 nL / min, allowing MS / MS to have 5 peaks with the highest intensity in the MS. MS / MS uses data-dependent scan mode, electrospray voltage is 1.95 kV, CE (collision energy) is 35%

The MS / MS data obtained were based on a human database and identified proteins through SEQUEST.

Time Flow (uL / min) Solventa Solvent B 0 0.2 97 3 5 0.2 97 3 45 0.2 60 40 50 0.2 10 90 60 0.2 10 90 65 0.2 97 3 85 0.2 97 3

Table 3 shows the LC gradient conditions used in the waterproof sample analysis.

Claims (4)

Lysozyme precursors of GI No. 4557894,
222144229 autophagy-related protein 7 isoform c,
4503143 < / RTI > of cathepsin D preproprotein,
0.0 > 14-3-3 < / RTI > protein zeta / delta,
4504919, keratin, type II cytoskeletal 8,
55956899 keratin, type I Saitoskeletal 9,
24430190 keratin type I Saitoskeletal 15,
88853069, Keratin Type I Saitoscapeal 19, 24234699,
Desmoplakin isoform II of < RTI ID = 0.0 > 58530842, <
The complement factor I < RTI ID = 0.0 > I <
The complement factor H-related protein 1 precursor of 118442839,
25777600 of the proteasome 26S non-ATPase subunit 1,
Collagen alpha-2 (IX) chain precursor of 11386161,
SPARC-like protein 1 precursor of 190341024,
4507149 < / RTI > superoxide dismutase,
4504517 < / RTI > heat shock protein beta-1,
124256496 < / RTI > of the heat shock 70 kDa protein 1-analog,
The 290 kDa centrosomal protein of 109255234, and
A composition for the diagnosis of age-related macular degeneration comprising at least one protein selected from the group consisting of glyceraldehyde-3-phosphate dehydrogenase of Korean Patent No. 7669492 Lysozyme precursors of GI No. 4557894,
222144229 autophagy-related protein 7 isoform c,
4503143 < / RTI > of cathepsin D preproprotein,
0.0 > 14-3-3 < / RTI > protein zeta / delta,
4504919, keratin, type II cytoskeletal 8,
55956899 keratin, type I Saitoskeletal 9,
24430190 keratin type I Saitoskeletal 15,
88853069, Keratin Type I Saitoscapeal 19, 24234699,
Desmoplakin isoform II of < RTI ID = 0.0 > 58530842, <
The complement factor I < RTI ID = 0.0 > I <
The complement factor H-related protein 1 precursor of 118442839,
25777600 of the proteasome 26S non-ATPase subunit 1,
Collagen alpha-2 (IX) chain precursor of 11386161,
SPARC-like protein 1 precursor of 190341024,
4507149 < / RTI > superoxide dismutase,
4504517 < / RTI > heat shock protein beta-1,
124256496 < / RTI > of the heat shock 70 kDa protein 1-analog,
The 290 kDa centrosomal protein of 109255234, and
One or more proteins selected from the group consisting of glyceraldehyde-3-phosphate dehydrogenase of < RTI ID = 0.0 > As a method for obtaining information on old age macular degeneration in a subject,
(a) measuring one or more biomarkers in a biological sample from a subject, wherein the one or more biomarkers are selected from the compositions according to claim 1; And
(b) comparing the measurements or measurements with a normal person, and correlating the measurement with the age-old macular degeneration when the measurement is higher than normal.
Lysozyme precursors of GI No. 4557894,
222144229 autophagy-related protein 7 isoform c,
4503143 < / RTI > of cathepsin D preproprotein,
0.0 > 14-3-3 < / RTI > protein zeta / delta,
4504919, keratin, type II cytoskeletal 8,
55956899 keratin, type I Saitoskeletal 9,
24430190 keratin type I Saitoskeletal 15,
88853069, Keratin Type I Saitoscapeal 19, 24234699,
Desmoplakin isoform II of < RTI ID = 0.0 > 58530842, <
The complement factor I < RTI ID = 0.0 > I <
The complement factor H-related protein 1 precursor of 118442839,
25777600 of the proteasome 26S non-ATPase subunit 1,
Collagen alpha-2 (IX) chain precursor of 11386161,
SPARC-like protein 1 precursor of 190341024,
4507149 < / RTI > superoxide dismutase,
4504517 < / RTI > heat shock protein beta-1,
124256496 < / RTI > of the heat shock 70 kDa protein 1-analog,
The 290 kDa centrosomal protein of 109255234, and
Wherein the composition comprises at least one protein selected from the group consisting of glyceraldehyde-3-phosphate dehydrogenase as an active ingredient.

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