KR102288646B1 - Glycan Biomarker for Diagnosing Osteoarthritis of Pets and Method for Diagnosing Osteoarthritis of Pets Using the Same - Google Patents
Glycan Biomarker for Diagnosing Osteoarthritis of Pets and Method for Diagnosing Osteoarthritis of Pets Using the Same Download PDFInfo
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Abstract
본 발명은 반려동물의 미량의 혈액 시료에서 면역 정보를 내포하고 있는 당사슬의 조성과 발현 정도를 측정하여 퇴행성 질환인 골관절염을 진단하는 방법 및 그 바이오마커에 관한 것이다. 본 발명은 최초로 반려동물 골관절염 진단을 위한 바이오마커 및 이를 이용한 반려동물 골관절염 진단방법을 제공한다.The present invention relates to a method for diagnosing osteoarthritis, a degenerative disease, and a biomarker thereof by measuring the composition and expression level of sugar chains containing immune information in a trace blood sample of a companion animal. The present invention provides a biomarker for diagnosing osteoarthritis in companion animals and a method for diagnosing osteoarthritis in companion animals for the first time.
Description
본 발명은 반려동물의 미량의 혈액 시료에서 면역 정보를 내포하고 있는 당사슬의 조성과 발현 정도를 측정하여 퇴행성 질환인 골관절염을 진단하는 방법 및 그 바이오마커에 관한 것이다. 본 발명은 최초로 반려동물 골관절염 진단을 위한 바이오마커 및 이를 이용한 반려동물 골관절염 진단방법을 제공한다.The present invention relates to a method for diagnosing osteoarthritis, a degenerative disease, and a biomarker thereof by measuring the composition and expression level of sugar chains containing immune information in a trace blood sample of a companion animal. The present invention provides a biomarker for diagnosing osteoarthritis in companion animals and a method for diagnosing osteoarthritis in companion animals for the first time.
고도의 산업 사회화, 핵가족화, 만혼, 저출산, 독신가구, 노인가구 등의 증가와 같은 사회적 환경 변화에 따라 반려동물과 함께 사는 가구수가 증가하고, 반려동물에 대한 인식이 변화하고 있다. 특히, 노령 반려동물의 증가에 따라 반려동물 수의진료/진단 매출 규모는 2010년 기준으로 약 2,178억원 수준으로 연간 급속도로 성장하는 추세이다. 그러나 수의 임상 분야는 사료첨가제, 생물학적 약제의 개발에 의존하거나 사람의 의료기술을 답습하는 수준에 머물러 있으며, 질병 진단에 대한 수요는 폭발적이나 활용될 수 있는 기술은 미미한 실정이다.The number of households living with companion animals is increasing, and the perception of companion animals is changing due to changes in the social environment such as high-level industrial socialization, nuclear familyization, late marriage, low fertility, single households, and elderly households. In particular, as the number of elderly companion animals increases, the sales volume of companion animal veterinary treatment/diagnosis is approximately KRW 217.8 billion as of 2010, which is a rapidly growing trend annually. However, the veterinary clinical field depends on the development of feed additives and biologics, or remains at the level of imitating human medical technology.
골관절염 (osteoarthritis, OA)은 반려견 5마리 중 1마리 꼴로 발병되는 흔한 쇠약 및 퇴행성 대표 질환이나, 질병의 임상 진행을 예방할 수 있는 조기 진단 방법은 전무하며, 질환과 관련된 증상이 발견되면 방사선 이미지를 이용하여 확인하는 방법과 행동요법에 의한 치료법만 존재하고 있다.Osteoarthritis (OA) is a common debilitating and degenerative disease that occurs in 1 in 5 dogs, but there is no early diagnosis method that can prevent the clinical progression of the disease. There is only a method to confirm it and a treatment method by behavioral therapy.
혈액은 시료 채취에 부담이 없는 다양한 기초 진단 및 비침습적 체외 진단에 널리 사용되는 시료이다. 혈액에 존재하는 대부분의 단백질은 이뮤노글로불린 계열의 면역 당단백질 (glycoprotein)로 혈액에 존재하는 당사슬 (glycan)은 다양한 단백질의 생물학적 기능을 수행할 뿐만 아니라 면역정보를 상당량 내포하여 신체의 상태를 반영한다. 특히, 질병과 같은 생물학적 변화 상태에 따라 촉매나 전이물질로 인해 특정 당쇄가 비정상적으로 과다 또는 과서 발현되거나 또는 비정상적인 당쇄화가 일어나 세포 변이 및/또는 기능의 소실을 일으키는 사례가 관찰되었고, 최근 이를 이용하여 당단백질 및/또는 당사슬을 이용한 질병 바이오마커 개발 연구가 새로운 패러다임이 되고 있다.Blood is a widely used sample for various basic diagnostics and non-invasive in vitro diagnostics that do not burden the sample collection. Most of the proteins in the blood are immunoglobulin-type immune glycoproteins. The sugar chains in the blood not only perform the biological functions of various proteins, but also reflect the state of the body by containing a significant amount of immune information. do. In particular, cases have been observed that cause cell mutation and/or loss of function due to abnormal overexpression or overexpression of specific sugar chains or abnormal glycosylation due to catalysts or transfer substances depending on biological change conditions such as diseases. Research on the development of disease biomarkers using glycoproteins and/or sugar chains is becoming a new paradigm.
류마티스성 관절염의 경우 자가면역질환으로 염증 지표 (혈액 CRP 수치)를 확인하는 진단법과 염증 수치를 낮춰주는 약물 요법이 다양하게 존재한다. 반면, 쇠퇴와 노화로 인해 발병되는 퇴행성 질환인 골관절염에 관하여는 병태 생리학적 연구가 현재 미비한 상태이므로 조기 진단방법은 전무하며, 생활치료를 통한 완화 요법 외에는 뚜렷한 치료법도 제시되지 않은 상황으로서 반려동물의 관절염 조기진단과 통증치료가 매우 필요하다.In the case of rheumatoid arthritis, as an autoimmune disease, there are various diagnostic methods to check the inflammatory marker (blood CRP level) and drug therapy to lower the inflammation level. On the other hand, with regard to osteoarthritis, a degenerative disease caused by decline and aging, pathophysiological studies are currently lacking, so there is no early diagnosis method, and no clear treatment has been proposed other than palliative care through lifestyle therapy. Early diagnosis of arthritis and pain treatment are very important.
당쇄화는 가장 널리 알려진 번역 후 변형 (post-translational modification; PTM) 과정으로 단백질의 기능에 크게 관여하고 있으며, 체내 단백질의 약 50% 이상은 당쇄화된 단백질인 당단백질이다. 혈액, 침, 눈물 등의 체액은 물론 대부분의 생체 조직에도 당단백질이 광범위하게 존재하며, 체외진단에 많이 사용되는 혈액에 존재하는 당단백질의 대부분은 면역계열의 단백질이다. 당쇄화의 결과물인 당사슬은 체내외의 환경 변화에 매우 민감하여 암을 포함한 여러 종류의 질병에 대한 바이오마커로 활용되고 있다. 또한, 바이오 의약품에서 당사슬은 의약품의 체내 지속시간 및 약효에 중요한 역할을 하는 것으로 알려져 있다.Glycosylation is the most widely known post-translational modification (PTM) process and is largely involved in the function of proteins, and about 50% or more of proteins in the body are glycoproteins, which are glycosylated proteins. Glycoproteins are widely present in body fluids such as blood, saliva, and tears, as well as in most living tissues. Sugar chains, the result of glycosylation, are very sensitive to changes in the environment inside and outside the body, and are being used as biomarkers for various types of diseases including cancer. In addition, in biopharmaceuticals, sugar chains are known to play an important role in the duration and efficacy of drugs in the body.
당사슬 분석을 위해서는 효소처리로 단백질에서 당사슬만을 잘라내어 분리하는 당사슬 분리 단계와 분리된 당사슬을 추출 및 농축하는 당사슬 정제의 2단계 전처리 과정을 거치게 된다. 전처리 단계 후 얻어진 당사슬은 고민감도 고분해능의 액체크로마토그래피 질량분석을 통해 포괄적 스크리닝을 하여 조성 및 분포를 확인할 수 있다.For the analysis of sugar chains, two pre-treatment steps are performed: a sugar chain separation step in which only sugar chains are cut and separated from proteins by enzymatic treatment, and a sugar chain purification step of extracting and concentrating the separated sugar chains. The sugar chains obtained after the pretreatment step can be comprehensively screened through high-sensitivity and high-resolution liquid chromatography mass spectrometry to confirm the composition and distribution.
본 발명의 목적은 상대적으로 쉽게 채취할 수 있는 반려동물의 혈액을 미량 사용하여 신체의 면역 상태를 대표할 수 있는 당사슬을 모니터링하고, 혈액에 포함되는 수백 종의 많은 당사슬 중 질병상태일 때 특이적 변화가 관찰되는 당사슬 발현량을 비교하여 반려동물의 대표 질환인 골관절염을 정확하게 진단하는 것이다.An object of the present invention is to monitor sugar chains that can represent the immune state of the body using a small amount of companion animal blood, which can be collected relatively easily, and to be specific when a disease state among hundreds of sugar chains contained in blood. It is to accurately diagnose osteoarthritis, a representative disease of companion animals, by comparing the expression level of sugar chains, where changes are observed.
본 발명자들은 상대적으로 채취가 용이한 반려동물 혈액을 시료로 이용하였으며, 신체 상태를 민감하게 반영하는 당사슬을 프로파일링하여 퇴행성 골관절염 특이적 당사슬 바이오마커를 발굴해내었다.The present inventors used companion animal blood, which is relatively easy to collect, as a sample, and discovered degenerative osteoarthritis-specific sugar chain biomarkers by profiling the sugar chain sensitively reflecting the physical condition.
본 발명에서 반려동물은 개, 고양이 등을 비롯한 반려동물을 의미한다. 또한, 본 발명에서 사용하는 용어는 특별한 언급이 없는 경우 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 알려진 의미로 사용하는 것임을 밝힌다.In the present invention, companion animals refer to companion animals including dogs and cats. In addition, it is revealed that the terms used in the present invention are used in the meaning known to those of ordinary skill in the art to which the present invention pertains unless otherwise specified.
본 발명은the present invention
(1) (Hex)3(HexNAc)2, 이론적 단일동위원소 질량값 910.3,(1) (Hex)3(HexNAc)2, theoretical monoisotope mass value 910.3,
(2) (Hex)3(HexNAc)2(Fuc)1, 이론적 단일동위원소 질량값 1056.4,(2) (Hex)3(HexNAc)2(Fuc)1, theoretical monoisotope mass value 1056.4,
(3) (Hex)4(HexNAc)4(Fuc)2(NeuAc)1, 이론적 단일동위원소 질량값 2061.8,(3) (Hex)4(HexNAc)4(Fuc)2(NeuAc)1, theoretical monoisotope mass value 2061.8,
(4) (Hex)6(HexNAc)5(NeuAc)1, 이론적 단일동위원소 질량값 2296.8,(4) (Hex)6(HexNAc)5(NeuAc)1, theoretical monoisotope mass value 2296.8,
(5) (Hex)5(HexNAc)4(Fuc)1(NeuAc)2, 이론적 단일동위원소 질량값 2368.8,(5) (Hex)5(HexNAc)4(Fuc)1(NeuAc)2, theoretical monoisotope mass value 2368.8,
(6) (Hex)6(HexNAc)4(Fuc)1(NeuAc)1, 이론적 단일동위원소 질량값 2239.8,(6) (Hex)6(HexNAc)4(Fuc)1(NeuAc)1, theoretical monoisotope mass value 2239.8,
(7) (Hex)4(HexNAc)3(NeuAc)1, 이론적 단일동위원소 질량값 1566.6,(7) (Hex)4(HexNAc)3(NeuAc)1, theoretical monoisotope mass value 1566.6,
(8) (Hex)4(HexNAc)3, 이론적 단일동위원소 질량값 1275.5,(8) (Hex)4(HexNAc)3, theoretical monoisotope mass value 1275.5,
(9) (Hex)3(HexNAc)3(Fuc)1, 이론적 단일동위원소 질량값 1259.5,(9) (Hex)3(HexNAc)3(Fuc)1, theoretical monoisotope mass value 1259.5,
(10) (Hex)5(HexNAc)3, 이론적 단일동위원소 질량값 1437.5,(10) (Hex)5(HexNAc)3, theoretical monoisotope mass value 1437.5,
(11) (Hex)6(HexNAc)4(NeuAc)1, 이론적 단일동위원소 질량값 2093.7,(11) (Hex)6(HexNAc)4(NeuAc)1, theoretical monoisotope mass value 2093.7,
(12) (Hex)5(HexNAc)4(Fuc)1(NeuAc)1, 이론적 단일동위원소 질량값 2277.7,(12) (Hex)5(HexNAc)4(Fuc)1(NeuAc)1, theoretical monoisotope mass value 2277.7,
(13) (Hex)3(HexNAc)4(Fuc)1, 이론적 단일동위원소 질량값 1462.5,(13) (Hex)3(HexNAc)4(Fuc)1, theoretical monoisotope mass value 1462.5,
(14) (Hex)3(HexNAc)4(Fuc)3, 이론적 단일동위원소 질량값 1754.7,(14) (Hex)3(HexNAc)4(Fuc)3, theoretical monoisotope mass value 1754.7,
(15) (Hex)4(HexNAc)4(Fuc)3, 이론적 단일동위원소 질량값 1916.7,(15) (Hex)4(HexNAc)4(Fuc)3, theoretical monoisotope mass value 1916.7,
(16) (Hex)7(HexNAc)7(Fuc)5(NeuAc)2(NeuGc)2, 이론적 단일동위원소 질량값 4500.6,(16) (Hex)7(HexNAc)7(Fuc)5(NeuAc)2(NeuGc)2, theoretical monoisotope mass value 4500.6,
(17) (Hex)5(HexNAc)4(Fuc)3, 이론적 단일동위원소 질량값 2078.8,(17) (Hex)5(HexNAc)4(Fuc)3, theoretical monoisotope mass value 2078.8,
(18) (Hex)4(HexNAc)5(Fuc)2, 이론적 단일동위원소 질량값 1973.7,(18) (Hex)4(HexNAc)5(Fuc)2, theoretical monoisotope mass value 1973.7,
(19) (Hex)7(HexNAc)7(Fuc)3(NeuAc)2(NeuGc)1, 이론적 단일동위원소 질량값 3901.4,(19) (Hex)7(HexNAc)7(Fuc)3(NeuAc)2(NeuGc)1, theoretical monoisotope mass value 3901.4,
(20) (Hex)6(HexNAc)7(Fuc)4, 이론적 단일동위원소 질량값 2996.1,(20) (Hex)6(HexNAc)7(Fuc)4, theoretical monoisotope mass value 2996.1,
(21) (Hex)7(HexNAc)4, 이론적 단일동위원소 질량값 1964.7,(21) (Hex)7(HexNAc)4, theoretical monoisotope mass value 1964.7,
(22) (Hex)7(HexNAc)6(Fuc)3(NeuAc)2(NeuGc)1, 이론적 단일동위원소 질량값 3698.3,(22) (Hex)7(HexNAc)6(Fuc)3(NeuAc)2(NeuGc)1, theoretical monoisotope mass value 3698.3,
(23) (Hex)6(HexNAc)3, 이론적 단일동위원소 질량값 1599.6,(23) (Hex)6(HexNAc)3, theoretical monoisotope mass value 1599.6,
(24) (Hex)7(HexNAc)6(Fuc)1(NeuGc)1, 이론적 단일동위원소 질량값 2824.0 및(24) (Hex)7(HexNAc)6(Fuc)1(NeuGc)1, theoretical monoisotopic mass value of 2824.0 and
(25) (Hex)6(HexNAc)3(Fuc)1(NeuAc)1, 이론적 단일동위원소 질량값 2036.7 중 선택된 반려동물 골관절염 진단용 당사슬 바이오마커 {단, HexNAc, N-아세틸헥소사민 (N-acetylhexosamine); Hex, 헥소스 (Hexose); Fuc, 퓨코스 (Fucose); NeuAc, N-아세틸뉴라민산 (N-acetylneuraminic acid); NeuGc, N-글리콜릴뉴라민산 (N-glycolylneuraminic Acid)}에 관한 것이다.(25) (Hex)6(HexNAc)3(Fuc)1(NeuAc)1, a sugar chain biomarker for diagnosing osteoarthritis in companion animals selected from theoretical monoisotope mass values of 2036.7 {however, HexNAc, N-acetylhexosamine (N-) acetylhexosamine); Hex, Hexose; Fuc, Fucose; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid (N-glycolylneuraminic Acid)} relates to.
또한, 본 발명은 상기 바이오마커 AUC 값이 0.9 이상인, 반려동물 골관절염 진단용 당사슬 바이오마커에 관한 것이다.In addition, the present invention relates to a sugar chain biomarker for diagnosing osteoarthritis in companion animals, wherein the biomarker AUC value is 0.9 or more.
또한, 본 발명은 상기 (1), (2), (4), (5), (7), (8), (9), (13) 및 (24)의 바이오마커가 정상군에 비하여 환자군에서 유의하게 적게 발현되는 것을 특징으로 한다.In addition, the present invention provides that the biomarkers of (1), (2), (4), (5), (7), (8), (9), (13) and (24) are higher in the patient group than in the normal group. It is characterized in that it is expressed significantly less in
또한, 본 발명은 상기 (3), (6), (10), (11), (12), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23) 및 (25)의 바이오마커가 정상군에 비하여 환자군에서 유의하게 많이 발현되는 것을 특징으로 한다.In addition, the present invention provides the above (3), (6), (10), (11), (12), (14), (15), (16), (17), (18), (19), It is characterized in that the biomarkers of (20), (21), (22), (23) and (25) are expressed significantly more in the patient group than in the normal group.
또한, 본 발명은 AUC 값 1인 (1)의 당사슬을 포함하는 반려동물 골관절염 진단용 당사슬 바이오마커에 관한 것이다.In addition, the present invention relates to a sugar chain biomarker for diagnosing osteoarthritis in companion animals, including a sugar chain having an AUC value of 1 (1).
또한, 본 발명은 정상군에서는 발견되지 않는 (14) 또는 (22) 중 하나 이상의 당사슬을 포함하는 반려동물 골관절염 진단용 당사슬 바이오마커에 관한 것이다.In addition, the present invention relates to a sugar chain biomarker for diagnosing osteoarthritis in companion animals, which contains one or more sugar chains of (14) or (22) that are not found in the normal group.
또한, 본 발명은 Also, the present invention
(가) 골관절염 의심 반려동물로부터 혈청을 얻는 단계;(A) obtaining serum from a companion animal suspected of osteoarthritis;
(나) 상기 혈청 단백질을 변성하는 단계;(B) denaturing the serum protein;
(다) 변성된 혈청 단백질에 효소 처리하여 N-당사슬을 분리하는 단계;(C) separating N-saccharide chains by enzymatically treating the denatured serum protein;
(라) 분리한 N-당사슬을 질량분석하는 단계; 및(D) mass spectrometry of the separated N-saccharide chain; and
(마) 질량분석 결과, 상기 (1) 내지 (25) 중 선택된 1종 이상의 당사슬 바이오마커가 정상 반려동물군의 해당 당사슬 질량분석 결과와 비교하여 유의한 발현량 차이를 나타내는 경우 골관절염으로 진단하는 단계;를 포함하는 반려동물 골관절염 진단방법에 관한 것이다.(E) Diagnosing osteoarthritis when, as a result of mass spectrometry, one or more sugar chain biomarkers selected from (1) to (25) above shows a significant difference in expression level compared to the corresponding sugar chain mass spectrometry result of the normal companion animal group It relates to a method for diagnosing osteoarthritis in companion animals, including.
또한, 본 발명은 상기 (마) 단계가 질량분석으로 얻은 질량 값으로 N-당사슬 조성을 확인하고, 확인된 N-당사슬을 총 이온량 (total abundance)으로 표준화하여 정상 반려동물군과 환자 간의 각 N-당사슬 증감도를 비교하는 단계를 포함하는 것을 특징으로 하는 반려동물 골관절염 진단방법에 관한 것이다.In addition, the present invention confirms the N-saccharide composition with the mass value obtained in step (E) by mass spectrometry, and normalizes the identified N-saccharide chains to the total abundance of each N- sugar chain between the normal companion animal group and the patient. It relates to a method for diagnosing osteoarthritis in companion animals, comprising the step of comparing the sensitivity of sugar chains.
또한, 본 발명은 상기 (다) 단계의 효소 처리가 변성된 혈청 단백질에 PNGase F 효소를 16~24시간 처리함을 특징으로 한다.In addition, the present invention is characterized in that the enzyme treatment of step (c) is treated with the enzyme PNGase F enzyme to the denatured serum protein for 16 to 24 hours.
또한, 본 발명은 상기 (마) 단계에서 상기 (1)의 당사슬 바이오마커 발현량이 정상 대조군에 비해 유의하게 낮을 경우 골관절염으로 진단하는, 반려동물 골관절염 진단방법에 관한 것이다.In addition, the present invention relates to a method for diagnosing osteoarthritis in companion animals, wherein in the step (E), when the expression level of the sugar chain biomarker of (1) is significantly lower than that of a normal control group, osteoarthritis is diagnosed.
또한, 본 발명은 상기 (마) 단계에서 상기 (14) 또는 (22) 중 하나 이상의 당사슬 바이오마커가 발현되는 경우 골관절염으로 진단하는, 반려동물 골관절염 진단방법에 관한 것이다.In addition, the present invention relates to a method for diagnosing osteoarthritis in companion animals, wherein in the step (E), when one or more sugar chain biomarkers of (14) or (22) are expressed, osteoarthritis is diagnosed.
또한, 본 발명은Also, the present invention
a) 피검 물질을 반려동물 유래 연골세포에 처리하는 단계; 및a) treating the test material to companion animal-derived chondrocytes; and
b) a)의 처리 결과, 처리 전 정상 대조군과 비교하여 청구항 1의 (1) 내지 (25)의 당사슬 바이오마커 중 1종 이상의 발현량에서 유의한 차이가 있었다가 처리 후 정상 대조군과 유의한 차이가 없어지는 변화를 나타내는 피검 물질을 선별하는 단계;를 포함하는, 반려동물 골관절염 치료제를 스크리닝하는 방법에 관한 것이다.b) As a result of the treatment in a), there was a significant difference in the expression level of one or more of the sugar chain biomarkers of claim 1 (1) to (25) compared to the normal control group before treatment, and there was a significant difference with the normal control group after treatment It relates to a screening method for treating companion animal osteoarthritis, including;
또한, 본 발명은Also, the present invention
가) 반려동물의 혈액시료 즉, 혈장 25 ㎕에 PNGase F 효소를 처리하여 시료 내의 N-당사슬을 분리하는 단계;A) treating a blood sample of companion animal, that is, 25 μl of plasma, with PNGase F enzyme to separate N-saccharide chains in the sample;
나) 다공성 흑연화 탄소 고체상 추출로 상기 가) 단계에서 얻은 N-당사슬을 탈염하고 N-당사슬을 분리, 정제 및/또는 농축하는 단계;b) desalting the N-sugar chains obtained in step a) by extraction of the porous graphitized carbon solid phase and separating, purifying and/or concentrating the N-saccharide chains;
다) 분리, 정제 및/또는 농축된 N-당사슬을 다공성 흑연화 탄소 기반 나노 액체크로마토그래피 - 고민감도 고분해능 질량분석 (PGC-nanoLC/Q-TOF-MS)으로 정성·정량 분석하는 단계;C) Qualitative and quantitative analysis of the separated, purified and/or concentrated N-saccharide chains by porous graphitized carbon-based nano liquid chromatography-high-sensitivity high-resolution mass spectrometry (PGC-nanoLC/Q-TOF-MS);
라) 질량분석으로 얻어진 Mass 값으로 N-당사슬의 조성을 확인하고, 확인된 N-당사슬을 총 이온량 (total abundance)으로 표준화하여 정상군과 환자군 간의 각 N-당사슬 증감도를 비교하는 단계를 포함한다.d) Checking the composition of N-saccharide chains with the mass value obtained by mass spectrometry, standardizing the identified N-saccharide chains with the total abundance, and comparing the sensitivity of each N-saccharide chain between the normal group and the patient group. .
또한, 본 발명은 상기 가) 단계의 효소 처리방법이 In addition, the present invention provides an enzyme treatment method of step a)
a) 25 ㎕의 반려동물 혈장 시료를 취하는 단계;a) taking a 25 μl companion animal plasma sample;
b) 시료에 단백질 변성 시약을 가하고 90~110℃로 1~3분간 처리하여 단백질을 변성하는 단계;b) denaturing the protein by adding a protein denaturing reagent to the sample and treating it at 90 to 110° C. for 1 to 3 minutes;
c) 시료에 PNGase F 효소 2 ㎕를 16~24시간 처리하여 N-당사슬을 분리하는 단계;c) separating N-saccharide chains by treating the sample with 2 μl of PNGase F enzyme for 16 to 24 hours;
d) 시료 내의 단백질을 침전하는 단계; 및d) precipitating the protein in the sample; and
e) 단백질을 침전시킨 시료를 원심분리하고 상층액을 얻는 단계;를 순차적으로 포함하는 것을 특징으로 한다.e) centrifuging the protein-precipitated sample and obtaining a supernatant; characterized in that it comprises sequentially.
또한, 본 발명은 상기 나) 단계의 다공성 흑연화 탄소 고체상 추출이 In addition, the present invention is the extraction of the porous graphitized carbon solid phase in step b)
a) 다공성 흑연화 탄소 컬럼을 정제수 2.5~3.5㎖로 2회 이상 세척하는 단계;a) washing the porous graphitized carbon column twice or more with 2.5 to 3.5 ml of purified water;
b) 상기 a)의 세척한 컬럼을 0.1% 트리플루오로아세트산이 든 80% 아세토나이트릴 용액 3㎖로 2회 이상 세척하는 단계;b) washing the washed column of a) twice or more with 3 ml of 80% acetonitrile solution containing 0.1% trifluoroacetic acid;
c) 상기 b) 단계를 거친 컬럼을 정제수 2.5~3.5㎖로 2회 이상 세척하는 단계;c) washing the column subjected to step b) twice or more with 2.5 to 3.5 ml of purified water;
d) 상기 c) 단계 이후 컬럼에 시료를 로딩하는 단계;d) loading a sample into the column after step c);
e) 정제수 2.5~3.5㎖로 2회 이상 컬럼을 세척하는 단계;e) washing the column twice or more with 2.5-3.5 ml of purified water;
f) 10% 아세토나이트릴 용액 2.5~3.5㎖로 2회 이상 시료를 용출하는 단계;f) eluting the sample twice or more with 2.5-3.5 ml of a 10% acetonitrile solution;
g) 20% 아세토나이트릴 용액 2.5~3.5㎖로 2회 이상 시료를 용출하는 단계; 및g) eluting the sample twice or more with 2.5-3.5 ml of a 20% acetonitrile solution; and
h) 0.05% 트리플루오로아세트산이 든 40% 아세토나이트릴 용액 2.5~3.5㎖로 2회 이상 시료를 용출하는 단계;를 순차적으로 적용하는 것을 특징한다.h) eluting the sample twice or more with 2.5 to 3.5 ml of a 40% acetonitrile solution containing 0.05% trifluoroacetic acid;
또한, 본 발명은 상기 다) 다공성 흑연화 탄소 기반 나노액체크로마토그래피 - 고민감도 고분해능 질량분석 (PGC-nanoLC/Q-TOF-MS) 정성·정량 분석하는 단계가In addition, the present invention includes the steps of c) porous graphitized carbon-based nano liquid chromatography-high-sensitivity high-resolution mass spectrometry (PGC-nanoLC/Q-TOF-MS) qualitative and quantitative analysis
a) 용매 A (10mM 폼산암모늄) 와 용매 B (100% 아세토나이트릴)의 기울기 용리(Gradient Elution)를 이용, 흑연화 탄소 충진 나노컬럼으로 N-당사슬 이성질체를 액체크로마토그래피로 분리·분석하는 단계;a) Separation and analysis of N-saccharide chain isomers by liquid chromatography in a graphitized carbon-filled nanocolumn using gradient elution of solvent A (10 mM ammonium formate) and solvent B (100% acetonitrile) ;
b) 고민감도 고분해능 Q-TOF MS 탐지기를 이용하여 m/z 500부터 2500까지 해당되는 범위의 이온을 분석하는 단계: 및b) analyzing ions in the range from m/z 500 to 2500 using a high-sensitivity, high-resolution Q-TOF MS detector: and
c) 질량분석 후, LC/MS의 데이터를 Mass Hunter Qualitative Analysis software (version B.07.00 SP2, Agilent Technologies)에 포함된 분자 특성 추출 알고리즘 (Molecular Feature Extractor algorithm)을 이용하여 Mass 값을 확인하는 단계;를 포함하는 것을 특징으로 한다.c) After mass spectrometry, the LC/MS data are analyzed using a Molecular Feature Extractor algorithm included in Mass Hunter Qualitative Analysis software (version B.07.00 SP2, Agilent Technologies) to confirm the mass value; It is characterized in that it includes.
또한, 본 발명은 상기 라) 질량분석으로 얻어진 Mass 값으로 N-당사슬의 조성을 확인하고, 확인된 N-당사슬을 총 이온량 (total abundance)으로 표준화하여 정상과 환자 간의 증감 정도를 비교하는 단계가In addition, the present invention provides a step of comparing the degree of increase or decrease between normal and patient by confirming the composition of N-saccharide chains with the mass value obtained by the above d) mass spectrometry, and standardizing the identified N-saccharide chains with the total abundance.
a) 추출된 Mass 값을 이론적으로 가능한 당사슬 조성과 20ppm (±0.001Da) 이내로 비교하여 N-당사슬의 조성을 부여하는 단계;a) comparing the extracted mass value with the theoretically possible composition of sugar chains within 20ppm (±0.001Da) to give the composition of N-saccharides;
b) 확인된 당사슬을 전체 이온량 (Total abundance)을 기준으로 표준화하는 단계; 및b) normalizing the identified sugar chains based on total abundance; and
c) 정상과 환자의 N-당사슬 발현량을 비교하는 단계;를 포함하는 것을 특징으로 한다.c) comparing the normal and patient N-saccharide expression levels; characterized in that it comprises a.
본 발명은 최초로 반려동물 퇴행성 골관절염 바이오마커 및 이를 이용한 반려동물 골관절염 진단방법을 제공한다.The present invention provides, for the first time, a companion animal degenerative osteoarthritis biomarker and a companion animal osteoarthritis diagnosis method using the same.
본 발명의 반려동물 퇴행성 골관절염 바이오마커는 AUC 0.9 이상의 고민감성 및 고선택성 특징을 나타내어 골관절염을 높은 정확도로 진단할 수 있다.The companion animal degenerative osteoarthritis biomarker of the present invention can diagnose osteoarthritis with high accuracy by exhibiting high sensitivity and high selectivity with an AUC of 0.9 or more.
또한, 본 발명의 바이오마커를 이용한 반려동물 퇴행성 골관절염 진단방법은 ROC (receiver operating characteristic) 커브 외에 발현량 비율을 계산함으로써 정상과 환자의 차이를 이중으로 구별해낼 수 있다.In addition, the companion animal degenerative osteoarthritis diagnosis method using the biomarker of the present invention can double-distinguish the difference between the normal and the patient by calculating the expression rate in addition to the ROC (receiver operating characteristic) curve.
또한, 본 발명의 반려동물 퇴행성 골관절염 특이적 N-당사슬 바이오마커는 추후 진단키트에 활용할 수 있을 뿐만 아니라 질병 활성도 측정 및 특이 치료제 개발에 널리 적용될 가능성이 있다.In addition, the companion animal degenerative osteoarthritis-specific N-saccharide biomarker of the present invention can be used for diagnostic kits in the future, and has the potential to be widely applied to disease activity measurement and development of specific therapeutic agents.
도 1은 본 발명의 바이오마커를 찾기 위한 실험방법을 나타내는 흐름도이다.
도 2a 내지 도 2e는 본 발명의 질병 진단이 가능한 혈액 당쇄체의 당사슬 조성 및 정보와 정상과 환자의 발현 (box plot)의 그리고 ROC 곡선을 표현하였다.
도 2a 내지 도 2c는 본 발명의 반려동물 골관절염 마커로서 주요 당사슬의 예상되는 구조와, 정상 (왼쪽의 검은 원), 환자 (오른쪽의 하얀 원)의 분포차이 및 바이오마커로의 활용을 위한 ROC 곡선이다.
도 2d와 도 2e는 본 발명의 반려동물 골관절염 마커로서 미량 당사슬의 예상되는 구조와, 정상 (왼쪽의 검은 원), 환자 (오른쪽의 하얀 원)의 분포차이 및 바이오마커로의 활용을 위한 ROC 곡선이다.
도 2a 내지 도 2e에 나타난 도형 중 녹색 원은 만노스 (Man), 노란색 원은 갈락토오즈 (Gal), "OAc"는 O-아세틸화를 의미하며, 청색 네모는 N-아세틸글루코사민 (GlcNAc), 빨간색 삼각형은 퓨코스 (Fuc), 보라색 마름모는 N-아세틸뉴라민산 (NeuAc)(또는 시알산이라고도 함), 회색 마름모는 N-글리콜릴뉴라민산 (NeuGc)을 나타낸다. 1 is a flowchart showing an experimental method for finding a biomarker of the present invention.
2A to 2E show the composition and information of sugar chains of blood sugar chains capable of diagnosing the disease of the present invention, and the expression (box plot) and ROC curves of normal patients.
2a to 2c show the expected structure of major sugar chains as a companion animal osteoarthritis marker of the present invention, distribution differences between normal (black circle on the left), patient (white circle on the right), and ROC curves for use as biomarkers am.
2D and 2E show the expected structure of trace sugar chains as a companion animal osteoarthritis marker of the present invention, differences in distribution between normal (black circle on the left), and patients (white circle on the right), and ROC curves for use as biomarkers am.
In the figures shown in FIGS. 2A to 2E , the green circle indicates mannose (Man), the yellow circle indicates galactose (Gal), and “OAc” indicates O-acetylation, and the blue square indicates N-acetylglucosamine (GlcNAc), The red triangle represents fucose, the purple diamond represents N-acetylneuraminic acid (NeuAc) (also known as sialic acid), and the gray diamond represents N-glycolylneuraminic acid (NeuGc).
아래에서는 구체적인 실시예를 들어 본 발명의 구성을 좀 더 자세히 설명한다. 그러나, 본 발명의 범위가 실시예의 기재에만 한정되는 것이 아님은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 자명하다.Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited only to the description of the examples.
(1) 시료 (1) sample
정상 반려견 41마리, 골관절염 반려견 92마리를 포함하는 총 133마리 반려견의 혈청 시료를 본 실험에 사용하였다. 정상 시료와 환자 시료는 동일하게 25㎕씩 취하였다.Serum samples from a total of 133 dogs, including 41 normal dogs and 92 osteoarthritis dogs, were used in this experiment. 25 μl of the normal sample and the patient sample were the same.
(2) 단백질 변성(2) protein denaturation
원활한 N-당사슬 분리를 위하여 혈청 시료는 먼저 단백질 변성 과정을 통하여 3차 구조를 제거하였다. 혈청시료 25㎕에 200mM NH4HCO3 (10mM dithiothreitol 포함) 25㎕를 넣고 교반한 다음 90℃ 이상의 뜨거운 물과 4℃의 차가운 물에 10초씩 번갈아가며 2분 동안 처리하여 단백질 변성을 유도함과 동시에 과도한 열로 인한 단백질 집적을 방지하였다.For smooth separation of N-saccharide chains, serum samples were first removed from the tertiary structure through protein denaturation. 200mM NH 4 HCO 3 in 25 μl of serum sample 25 μl of (including 10mM dithiothreitol) was added, stirred, and then treated in hot water at 90°C or higher and cold water at 4°C for 10 seconds each for 2 minutes alternately to induce protein denaturation and to prevent protein accumulation due to excessive heat.
(3) N-당사슬 분리를 위한 효소처리(3) Enzyme treatment for separation of N-saccharide chains
시료에서 N-당사슬을 추출하기 위하여 New England BioLabs (Ipswich, MA)에서 구입한 PNGase F (peptide N-glycosidase F; 500,000 unit/㎖)를 시료에 처리하였다. 상기 단백질 변성 과정이 끝난 시료에 1㎕ (500 units)의 PNGase F를 가하고 항온수조를 이용하여 37℃로 16시간 동안 배양하여 N-당사슬을 분리하였다.PNGase F (peptide N-glycosidase F; 500,000 unit/ml) purchased from New England BioLabs (Ipswich, MA) was treated in the sample to extract N-saccharide chains from the sample. After the protein denaturation process, 1 μl (500 units) of PNGase F was added to the sample and incubated at 37° C. for 16 hours using a constant temperature water bath to separate N-saccharide chains.
(4) 단백질 제거를 위한 단백질 침전 처리(4) Protein precipitation treatment for protein removal
N-당사슬 분리를 위한 효소 반응이 완료된 시료에 차가운 상태의 에탄올 200㎕를 가한 후 -45℃에서 1시간 동안 방치하여 N-당사슬이 분리되고 남은 단백질을 침전시켰다. 단백질 침전 후 4℃에서 분당 14,400rpm으로 20분간 원심분리한 다음 각 시료마다 200㎕의 상층액을 취하여 완전히 건조하였다.After adding 200 μl of cold ethanol to the sample on which the enzymatic reaction for separation of N-sugar chains was completed, it was allowed to stand at -45° C. for 1 hour to precipitate the protein remaining after the separation of N-saccharide chains. After protein precipitation, centrifugation was performed at 4° C. at 14,400 rpm per minute for 20 minutes, and then 200 μl of the supernatant was taken from each sample and completely dried.
(5) N-당사슬 분리, 정제 및 농축(5) N-saccharide chain separation, purification and concentration
시료에서 N-당사슬만을 분리 및 정제하기 위하여 흑연화 탄소 카트리지 (Graphitized carbon cartridge)를 이용한 고체상 추출법 (solid phase extraction, SPE)을 적용하였다. 흑연화 탄소 카트리지는 Agilent에서 구입하였다. 먼저 카트리지를 초순수 6㎖, 80% (v/v) 아세토나이트릴 (0.1% 트리플루오로초산 포함) 6㎖, 초순수 6㎖의 순서로 세척하였다. 이후 초순수 1㎖로 용해한 당사슬 시료를 흑연화 탄소 카트리지에 주입한 후, 6㎖의 초순수를 흘려주어 염을 제거하는 과정을 수행하였다. 카트리지에 남은 당사슬은 각각 6㎖의 10% (v/v) 아세토나이트릴, 6㎖의 20% (v/v) 아세토나이트릴, 40% (v/v) 아세토나이트릴 (0.05% 트리플루오로초산 포함)의 순서로 용출하였다. 각 구간 분획을 수집하여 진공 원심 증발기로 건조하였으며 질량분석에 앞서 15㎕의 초순수에 용해하였다.In order to separate and purify only N-saccharide chains from the sample, a solid phase extraction (SPE) method using a graphitized carbon cartridge was applied. Graphitized carbon cartridges were purchased from Agilent. First, the cartridge was washed with 6 ml of ultrapure water, 6 ml of 80% (v/v) acetonitrile (containing 0.1% trifluoroacetic acid), and 6 ml of ultrapure water in this order. Thereafter, a sugar chain sample dissolved in 1 ml of ultrapure water was injected into the graphitized carbon cartridge, and then 6 ml of ultrapure water was flowed to remove salts. The sugar chains remaining in the cartridge are each 6 ml of 10% (v/v) acetonitrile, 6 ml of 20% (v/v) acetonitrile, and 40% (v/v) acetonitrile (0.05% trifluoro acetic acid) was eluted in the following order. Each fraction was collected, dried by a vacuum centrifugal evaporator, and dissolved in 15 μl of ultrapure water prior to mass spectrometry.
(6) N-당사슬 분석(6) N-sugar chain analysis
얻어진 당사슬은 nanoLC chip/Q-TOF MS로 분석을 진행하였다. 더 자세히는 희석된 당사슬을 오토샘플러 (4℃로 유지됨), 세관 펌프, 나노펌프, HPLC-Chip/MS 인터페이스 및 6540 Q-TOF MS 탐지기를 갖춘 HPLC Chip/Q-TOF (Chip Quadrupole Time-of-Flight) MS system (Agilent Technologies, Santa Clara, CA)을 이용하여 분석하였다. 4㎜ 농축 컬럼 및 43×0.075㎜ 분석 컬럼의 정지상 (stationary phase)으로 통합된 나노-ESI 스프레이 팁과 함께 PGC가 패킹된 칩을 사용하였다. 크로마토그래피 방법에 의한 분리는 최적화된 분리 조건에 따라 수행되었다. 간단히 설명하면, 시료를 로딩한 후, 온전한 당사슬의 용출 농도구배 용액을 분당 0.3 ㎕씩 흘려주었다. 농도구배 용액은 (A) 0.1% 포름산이 함유된 3% 아세토나이트릴 (v/v) 용액, 및 (B) 0.1% 포름산이 함유된 90% 아세토나이트릴 (v/v) 용액이고, 각각 아래 시각에 아래 농도로 처리하였다: 3% B, 0~2.5min; 3~16% B, 2.5~20min; 16~44% B, 20~40min; 44~100% B, 40~45min; 100% B, 45~55min; 100~3% B, 55~57min. 최종적으로 분석 컬럼은 3% B로 18분간 재평형화하였다. 질량분석한 후, LC/MS의 원래 데이터는 Mass Hunter Qualitative Analysis software (version B.06.00 SP2, Agilent Technologies) & Bioconfirm software (version B.06.00, Agilent Technologies)에 포함된 Deconvolution algorithm (Maximum Entropy)을 이용하여 처리되었다.The obtained sugar chains were analyzed by nanoLC chip/Q-TOF MS. In more detail, the diluted sugar chains were subjected to an autosampler (maintained at 4°C), a tubular pump, a nanopump, an HPLC-Chip/MS interface and an HPLC Chip/Q-TOF (Chip Quadrupole Time-of-toF) with a 6540 Q-TOF MS detector. Flight) was analyzed using the MS system (Agilent Technologies, Santa Clara, CA). A PGC packed chip was used with a nano-ESI spray tip integrated into the stationary phase of a 4 mm concentration column and a 43×0.075 mm analytical column. Separation by chromatography was performed under optimized separation conditions. Briefly, after loading the sample, 0.3 μl of an elution gradient solution of intact sugar chains was flowed per minute. The gradient solutions are (A) a 3% acetonitrile (v/v) solution with 0.1% formic acid, and (B) a 90% acetonitrile (v/v) solution with 0.1% formic acid, each of the following Time was treated with the following concentrations: 3% B, 0-2.5 min; 3-16% B, 2.5-20 min; 16-44% B, 20-40 min; 44-100% B, 40-45 min; 100% B, 45-55 min; 100-3% B, 55-57 min. Finally, the analytical column was re-equilibrated with 3% B for 18 min. After mass spectrometry, the original data of LC/MS were analyzed using the Deconvolution algorithm (Maximum Entropy) included in Mass Hunter Qualitative Analysis software (version B.06.00 SP2, Agilent Technologies) & Bioconfirm software (version B.06.00, Agilent Technologies). was processed by
(7) N-당사슬 조성 확인(7) Confirmation of N-sugar chain composition
질량분석으로 얻어진 질량 값과 이론적으로 가능한 당사슬 조성이 가지는 질량 값을 20ppm (±0.005Da) 이내로 비교하여 N-당사슬 조성을 부여하고, 밝혀진 당사슬이 가지는 강도 (intensity) 들의 총합 (Total abundance)으로 당사슬 각각의 강도를 표준화하였다. 후에 정상과 환자가 가지는 N-당사슬 함량의 비교를 통해 차이를 확인하였고, 정상과 환자 간의 차이가 p value 10-10보다 작고, 발현양의 차이가 1.5배 이상 나는 당사슬들을 마커로 선정하였다.The N-sugar chain composition is given by comparing the mass value obtained by mass spectrometry and the theoretically possible mass value of the sugar chain composition within 20ppm (±0.005Da), and the total abundance of the revealed sugar chains is calculated for each sugar chain. was standardized. Later, the difference was confirmed by comparing the N-saccharide content of the normal and the patient, and the sugar chains with a difference between the normal and the patient less than p value 10 -10 and the difference in expression amount more than 1.5 times were selected as markers.
5. 결과5. Results
정상 반려견 41마리, 골관절염 반려견 92마리를 포함하는 총 133개의 혈액 샘플을 대상으로 혈액 25㎕에 존재하는 N-당사슬을 분리·분석하여 얻어진 수백 종의 당사슬 중 정상과 환자간의 차이를 보이는 특이적인 당사슬을 선정하여 발현량을 관찰하였다.Specific sugar chains showing differences between normal and patients among hundreds of sugar chains obtained by isolating and analyzing N-sugar chains present in 25 μl of blood from a total of 133 blood samples, including 41 normal dogs and 92 dogs with osteoarthritis. was selected and the expression level was observed.
표 1은 반려동물의 혈청 당사슬 추출 과정에 따른 당사슬 갯수를 나타낸다.Table 1 shows the number of sugar chains according to the serum sugar chain extraction process of companion animals.
(가) 정상 41개, 환자 92개 혈청 시료를 질량 분석하여 얻어진 질량 값을 이용하여 당사슬 생합성 이론에 근거하여 조합 가능한 당사슬의 이론값과 비교 (±0.001Da)하여 당사슬을 1차로 확인하였다 (정상군 316종, 환자군 597종).(A) Using the mass values obtained by mass analysis of serum samples from 41 normal and 92 patients, sugar chains were first confirmed by comparing (±0.001 Da) with the theoretical values of sugar chains that can be combined based on the theory of sugar chain biosynthesis (normal). 316 in the group and 597 in the patient group).
(나) 출현 빈도 10% 이상 (정상군에서 4회 이상, 환자군에서 9회 이상)인 당사슬을 필터링하였다 (정상군 163종, 환자군 463종, 총 466종).(B) Sugar chains with an appearance frequency of 10% or more (4 or more in the normal group, 9 or more in the patient group) were filtered (163 types in the normal group, 463 types in the patient group, 466 types in total).
(다) 상기 (나) 단계를 거친 각 당사슬의 신호강도 (peak abundance)를 총 이온량 (total abundance)으로 표준화하였다. (C) The signal intensity (peak abundance) of each sugar chain that went through step (B) above was normalized to the total abundance.
NAPI (%)= (해당 당사슬의 abundance/전체 당사슬의 abundance 총합)X100 NAPI (%) = (abundance of the corresponding sugar chain/sum of the abundance of all sugar chains)X100
(라) 표준화된 abundance의 분포 별로 정렬한 뒤, (D) After sorting by distribution of standardized abundance,
1) 상위 95%에 해당하는 주요 (major) 당사슬을 그룹화하였다 (정상군 36종, 환자군 50종, 총 51종).1) Major sugar chains corresponding to the top 95% were grouped (36 in normal group, 50 in patient group, total of 51).
2) 95-99%에 해당하는 미량 (minor) 당사슬을 그룹화하였다 (정상군 30종, 환자군 102종, 총 101종).2) Minor sugar chains corresponding to 95-99% were grouped (30 types in the normal group, 102 types in the patient group, 101 types in total).
3) 하위 1%에 해당하는 극미량 (trace) 당사슬을 그룹화하였다 (정상군 97종, 311종, 총 314종).3) Trace sugar chains corresponding to the lower 1% were grouped (97 species in the normal group, 311 species, a total of 314 species).
of N-of N-
glycansglycans
표 2는 정상에서 발견되는 당사슬 316종과 환자에서 발견된 당사슬 597종을 비교하여 유의미한 차이가 있는 34종 당사슬 조성 정보 및 발현량에 대한 것이다. 정상 대조군과 환자군 당사슬 발현의 직접적인 비교를 위해 10% 이상 빈도를 보이는 당사슬 466종을 대상으로 표준화한 abundance (NAPI)로 통합한 뒤 두 군의 전체적 경향을 대변할 수 있는 평균값으로 비교하였다. 마커를 발견하기 위해 t-검정을 수행하였고, 466종 당사슬 중 정상 대조군와 환자군 사이의 유의미한 차이 (P<10-10)를 나타내는 34종을 진단 마커로 1차 선별하였다.Table 2 shows the composition information and expression levels of 34 types of sugar chains, which are significantly different by comparing 316 types of sugar chains found in normal and 597 types of sugar chains found in patients. For direct comparison of sugar chain expression between normal control and patient groups, the normalized abundance (NAPI) of 466 sugar chains with a frequency of 10% or more was integrated and compared as an average value that can represent the overall trend of the two groups. To find the marker, a t-test was performed, and among the 466 types of sugar chains, 34 types showing a significant difference (P<10 -10 ) between the normal control group and the patient group were primarily selected as diagnostic markers.
1차 선별된 34종의 마커 후보군 중 ROC 곡선의 AUC가 0.90보다 높은 경우에만 최종 진단 마커로 선택하였다. 본 발명자들이 선택한 25개의 진단 마커 중 13종은 상위 95%에 속하는 주요 당사슬이며, 12종은 95~99%에 속하는 미량 당사슬이다. 우리가 확인한 당사슬 마커 중 극미량 그룹에 속하는 당사슬은 포함되지 않았다. 반려동물 골관절염 진단을 위한 최상의 N-당사슬 마커는 주요 당사슬 그룹에 속하고, 두 표본 간에 통계적으로 유의미한 차이를 보이며, AUC 1을 나타내는 (Hex)3(HexNAc)2이다.Only when the AUC of the ROC curve was higher than 0.90 among the 34 primary selected marker candidates, it was selected as the final diagnostic marker. Among the 25 diagnostic markers selected by the present inventors, 13 are major sugar chains belonging to the top 95%, and 12 are trace sugar chains belonging to 95-99%. Among the sugar chain markers we identified, sugar chains belonging to the trace trace group were not included. The best N-saccharide marker for diagnosing osteoarthritis in companion animals is (Hex) 3 (HexNAc) 2 , which belongs to the major sugar chain group, shows a statistically significant difference between the two samples, and represents
우리는 추가로 정상 대조군 대비 환자군에서 마커 당사슬의 발현 정도를 비율 즉, 환자군의 당사슬 발현량/정상군의 당사슬 발현량으로 환산하였고, 우리가 선택한 25종 당사슬이 최소 1.3배에서 최대 603.7배의 차이를 보이는 것을 확인하였다. 마커 2종 ((Hex)3(HexNAc)4(Fuc)3 및 (Hex)7(HexNAc)6(Fuc)3(NeuAc)2(NeuGc)1) 의 경우 정상에서는 발현되지 않은 특이적 당사슬이다.We additionally converted the expression level of the marker sugar chains in the patient group compared to the normal control group into the ratio, that is, the sugar chain expression level of the patient group/the sugar chain expression level of the normal group. was confirmed to be visible. 2 markers ((Hex) 3 (HexNAc) 4 (Fuc) 3 and (Hex) 7 (HexNAc) 6 (Fuc) 3 (NeuAc) 2 (NeuGc) 1 ) In the case of normal, it is a specific sugar chain that is not expressed.
상기 표 2에서 사용한 약자는 다음과 같은 의미이다. m/z, 분자량 (molecular mass); NOTE, Hex_HexNAc_Fuc_NeuAc_NeuGc; AVE, 평균 (average); SD, 표준편차 (standard deviation); CV, 변동계수 (coefficient of variation); Acc, 축적강도 (accumulation intensity).The abbreviations used in Table 2 have the following meanings. m/z, molecular mass; NOTE, Hex_HexNAc_Fuc_NeuAc_NeuGc; AVE, average; SD, standard deviation; CV, coefficient of variation; Acc, accumulation intensity.
상기 표 3에서 사용된 약자는 다음과 같은 의미이다. m/z, 분자량 (molecular mass); NOTE, Hex_HexNAc_Fuc_NeuAc_NeuGc; CLASS, 당사슬 타입 (C: complex, HB: hybrid, C/HB: complex or hybrid, F: fucosylated, S: sialylated); CATEGORY, Major: NAPI≤95%, Minor: NAPI 95~99%; P/C ratio: 환자/정상 강도 비 (patient/control intensity ratio); A/P, absent/present; AUC, area under the curve.The abbreviations used in Table 3 have the following meanings. m/z, molecular mass; NOTE, Hex_HexNAc_Fuc_NeuAc_NeuGc; CLASS, sugar chain type (C: complex, HB: hybrid, C/HB: complex or hybrid, F: fucosylated, S: sialylated); CATEGORY, Major: NAPI≤95%, Minor: NAPI 95~99%; P/C ratio: patient/control intensity ratio; A/P, absent/present; AUC, area under the curve.
Claims (12)
(나) 상기 혈청 내에 함유된 혈청 단백질을 변성하는 단계;
(다) 변성된 혈청 단백질에 효소 처리하여 N-당사슬을 분리하는 단계;
(라) 분리한 N-당사슬을 질량분석하는 단계; 및
(마) 질량분석 결과, 아래 (1) 내지 (25) 중 선택된 1종 이상의 당사슬 바이오마커가 정상 반려견군의 해당 당사슬 질량분석 결과와 비교하여 발현량 차이를 나타내는 경우 골관절염으로 진단하는 단계;를 포함하는 반려견 골관절염 진단방법;
(1) (Hex)3(HexNAc)2, 이론적 단일동위원소 질량값 910.3,
(2) (Hex)3(HexNAc)2(Fuc)1, 이론적 단일동위원소 질량값 1056.4,
(3) (Hex)4(HexNAc)4(Fuc)2(NeuAc)1, 이론적 단일동위원소 질량값 2061.8,
(4) (Hex)6(HexNAc)5(NeuAc)1, 이론적 단일동위원소 질량값 2296.8,
(5) (Hex)5(HexNAc)4(Fuc)1(NeuAc)2, 이론적 단일동위원소 질량값 2368.8,
(6) (Hex)6(HexNAc)4(Fuc)1(NeuAc)1, 이론적 단일동위원소 질량값 2239.8,
(7) (Hex)4(HexNAc)3(NeuAc)1, 이론적 단일동위원소 질량값 1566.6,
(8) (Hex)4(HexNAc)3, 이론적 단일동위원소 질량값 1275.5,
(9) (Hex)3(HexNAc)3(Fuc)1, 이론적 단일동위원소 질량값 1259.5,
(10) (Hex)5(HexNAc)3, 이론적 단일동위원소 질량값 1437.5,
(11) (Hex)6(HexNAc)4(NeuAc)1, 이론적 단일동위원소 질량값 2093.7,
(12) (Hex)5(HexNAc)4(Fuc)1(NeuAc)1, 이론적 단일동위원소 질량값 2277.7,
(13) (Hex)3(HexNAc)4(Fuc)1, 이론적 단일동위원소 질량값 1462.5,
(14) (Hex)3(HexNAc)4(Fuc)3, 이론적 단일동위원소 질량값 1754.7,
(15) (Hex)4(HexNAc)4(Fuc)3, 이론적 단일동위원소 질량값 1916.7,
(16) (Hex)7(HexNAc)7(Fuc)5(NeuAc)2(NeuGc)2, 이론적 단일동위원소 질량값 4500.6,
(17) (Hex)5(HexNAc)4(Fuc)3, 이론적 단일동위원소 질량값 2078.8,
(18) (Hex)4(HexNAc)5(Fuc)2, 이론적 단일동위원소 질량값 1973.7,
(19) (Hex)7(HexNAc)7(Fuc)3(NeuAc)2(NeuGc)1, 이론적 단일동위원소 질량값 3901.4,
(20) (Hex)6(HexNAc)7(Fuc)4, 이론적 단일동위원소 질량값 2996.1,
(21) (Hex)7(HexNAc)4, 이론적 단일동위원소 질량값 1964.7,
(22) (Hex)7(HexNAc)6(Fuc)3(NeuAc)2(NeuGc)1, 이론적 단일동위원소 질량값 3698.3,
(23) (Hex)6(HexNAc)3, 이론적 단일동위원소 질량값 1599.6,
(24) (Hex)7(HexNAc)6(Fuc)1(NeuGc)1, 이론적 단일동위원소 질량값 2824.0 및
(25) (Hex)6(HexNAc)3(Fuc)1(NeuAc)1, 이론적 단일동위원소 질량값 2036.7. {단, HexNAc, N-아세틸헥소사민 (N-acetylhexosamine); Hex, 헥소스 (Hexose); Fuc, 퓨코스 (Fucose); NeuAc, N-아세틸뉴라민산 (N-acetylneuraminic acid); NeuGc, N-글리콜릴뉴라민산 (N-glycolylneuraminic Acid)}.
(A) obtaining serum from a dog suspected of osteoarthritis;
(B) denaturing the serum protein contained in the serum;
(C) separating N-saccharide chains by enzymatically treating the denatured serum protein;
(D) mass spectrometry of the separated N-saccharide chain; and
(E) Diagnosis of osteoarthritis when the mass spectrometry result shows a difference in expression level of one or more sugar chain biomarkers selected from the following (1) to (25) compared with the corresponding sugar chain mass spectrometry result of the normal dog group; including; canine osteoarthritis diagnosis method;
(1) (Hex)3(HexNAc)2, theoretical monoisotope mass value 910.3,
(2) (Hex)3(HexNAc)2(Fuc)1, theoretical monoisotope mass value 1056.4,
(3) (Hex)4(HexNAc)4(Fuc)2(NeuAc)1, theoretical monoisotope mass value 2061.8,
(4) (Hex)6(HexNAc)5(NeuAc)1, theoretical monoisotope mass value 2296.8,
(5) (Hex)5(HexNAc)4(Fuc)1(NeuAc)2, theoretical monoisotope mass value 2368.8,
(6) (Hex)6(HexNAc)4(Fuc)1(NeuAc)1, theoretical monoisotope mass value 2239.8,
(7) (Hex)4(HexNAc)3(NeuAc)1, theoretical monoisotope mass value 1566.6,
(8) (Hex)4(HexNAc)3, theoretical monoisotope mass value 1275.5,
(9) (Hex)3(HexNAc)3(Fuc)1, theoretical monoisotope mass value 1259.5,
(10) (Hex)5(HexNAc)3, theoretical monoisotope mass value 1437.5,
(11) (Hex)6(HexNAc)4(NeuAc)1, theoretical monoisotope mass value 2093.7,
(12) (Hex)5(HexNAc)4(Fuc)1(NeuAc)1, theoretical monoisotope mass value 2277.7,
(13) (Hex)3(HexNAc)4(Fuc)1, theoretical monoisotope mass value 1462.5,
(14) (Hex)3(HexNAc)4(Fuc)3, theoretical monoisotope mass value 1754.7,
(15) (Hex)4(HexNAc)4(Fuc)3, theoretical monoisotope mass value 1916.7,
(16) (Hex)7(HexNAc)7(Fuc)5(NeuAc)2(NeuGc)2, theoretical monoisotope mass value 4500.6;
(17) (Hex)5(HexNAc)4(Fuc)3, theoretical monoisotope mass value 2078.8,
(18) (Hex)4(HexNAc)5(Fuc)2, theoretical monoisotope mass value 1973.7,
(19) (Hex)7(HexNAc)7(Fuc)3(NeuAc)2(NeuGc)1, theoretical monoisotope mass value 3901.4,
(20) (Hex)6(HexNAc)7(Fuc)4, theoretical monoisotope mass value 2996.1,
(21) (Hex)7(HexNAc)4, theoretical monoisotope mass value 1964.7,
(22) (Hex)7(HexNAc)6(Fuc)3(NeuAc)2(NeuGc)1, theoretical monoisotope mass value 3698.3,
(23) (Hex)6(HexNAc)3, theoretical monoisotope mass value 1599.6,
(24) (Hex)7(HexNAc)6(Fuc)1(NeuGc)1, theoretical monoisotopic mass value of 2824.0 and
(25) (Hex)6(HexNAc)3(Fuc)1(NeuAc)1, theoretical monoisotopic mass value 2036.7. {However, HexNAc, N-acetylhexosamine (N-acetylhexosamine); Hex, Hexose; Fuc, Fucose; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic Acid}.
상기 당사슬 바이오마커는 AUC 값이 0.9 이상인, 반려견 골관절염 진단방법.
The method according to claim 1,
The sugar chain biomarker has an AUC value of 0.9 or more, a method for diagnosing osteoarthritis in dogs.
상기 (1), (2), (4), (5), (7), (8), (9), (13) 및 (24) 중 1종 이상의 당사슬 바이오마커는 정상 반려견군에 비하여 골관절염 의심 반려견에서 적게 발현되는, 반려견 골관절염 진단방법.
The method according to claim 1,
One or more sugar chain biomarkers of (1), (2), (4), (5), (7), (8), (9), (13) and (24) above showed osteoarthritis compared to the normal dog group. A method for diagnosing canine osteoarthritis, which is less common in suspected dogs.
상기 (3), (6), (10), (11), (12), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23) 및 (25) 중 1종 이상의 당사슬 바이오마커는 정상 반려견군에 비하여 골관절염 의심 반려견에서 많이 발현되는, 반려견 골관절염 진단방법.
The method according to claim 1,
(3), (6), (10), (11), (12), (14), (15), (16), (17), (18), (19), (20), ( 21), (22), (23) and (25), one or more sugar chain biomarkers are more expressed in dogs with osteoarthritis than in the normal dog group, a method for diagnosing canine osteoarthritis.
AUC 값 1인 (1)의 당사슬 바이오마커를 포함하는 반려견 골관절염 진단방법.
The method according to claim 1,
A method for diagnosing canine osteoarthritis including a sugar chain biomarker of (1) with an AUC value of 1.
정상 반려견군에서는 발견되지 않는 (14) 또는 (22) 중 하나 이상의 당사슬 바이오마커를 포함하는 반려견 골관절염 진단방법.
The method according to claim 1,
A method for diagnosing canine osteoarthritis comprising one or more sugar chain biomarkers in (14) or (22) that are not found in the normal canine group.
상기 (마) 단계는 질량분석으로 얻은 질량 값으로 N-당사슬 조성을 확인하고, 확인된 N-당사슬을 총 이온량 (total abundance)으로 표준화하여 정상 반려견군과 골관절염 의심 반려견 간의 각 N-당사슬 증감도를 비교하는 단계를 포함하는 반려견 골관절염 진단방법.
The method according to claim 1,
In step (E), the N-saccharide chain composition was confirmed with the mass value obtained by mass spectrometry, and the N-saccharide chain sensitization was determined between the normal dog group and the osteoarthritis-suspected dog group by normalizing the identified N-saccharide chains to the total abundance. A method for diagnosing canine osteoarthritis comprising the step of comparing.
상기 (다) 단계의 효소 처리는 변성된 혈청 단백질에 PNGase F 효소를 처리함을 특징으로 하는 반려견 골관절염 진단방법.
The method according to claim 1,
The enzyme treatment in step (c) is a dog osteoarthritis diagnosis method, characterized in that the denatured serum protein is treated with PNGase F enzyme.
상기 (마) 단계는 청구항 1의 (1) 당사슬 바이오마커 발현량이 정상 반려견군에 비해 낮을 경우 골관절염 의심 반려견이 골관절염에 걸린 것으로 진단하는, 반려견 골관절염 진단방법.
The method according to claim 1,
In the step (E), (1) of claim 1, when the expression level of the sugar chain biomarker is lower than that of the normal dog group, the dog suspected of osteoarthritis is diagnosed as having osteoarthritis, the dog osteoarthritis diagnosis method.
상기 (마) 단계는 청구항 1의 (14) 또는 (22) 중 하나 이상의 당사슬 바이오마커가 발현되는 경우 골관절염 의심 반려견이 골관절염에 걸린 것으로 진단하는, 반려견 골관절염 진단방법.
The method according to claim 1,
In the step (E), when one or more sugar chain biomarkers of claim 1 (14) or (22) are expressed, the canine osteoarthritis diagnosis method for a dog suspected of osteoarthritis is diagnosed as having osteoarthritis.
b) a)의 피검 물질 처리 결과, 피검 물질 처리 전 정상 대조군과 비교하여 청구항 1의 (1) 내지 (25)의 당사슬 바이오마커 중 1종 이상의 발현량에서 유의한 차이가 있었다가 피검 물질 처리 후 정상 대조군과 유의한 차이가 없어지는 변화를 나타내는 피검 물질을 선별하는 단계;를 포함하는, 반려견 골관절염 치료제를 스크리닝하는 방법.a) treating the test material to dog-derived chondrocytes; and
b) As a result of treatment with the test substance in a), there was a significant difference in the expression level of one or more of the sugar chain biomarkers of claim 1 (1) to (25) compared to the normal control before the test substance treatment, but after the test substance treatment A method of screening for a treatment for canine osteoarthritis, comprising a; selecting a test material that exhibits a change that is not significantly different from the normal control.
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JP2010526299A (en) | 2007-05-01 | 2010-07-29 | ヒルズ・ペット・ニュートリシャン・インコーポレーテッド | Methods and compositions for diagnosing osteoarthritis in cats |
KR101484969B1 (en) | 2014-03-26 | 2015-01-22 | 충남대학교산학협력단 | Method for cancer diagnosis using N-glycopeptide |
JP2019517007A (en) | 2016-04-15 | 2019-06-20 | イーエスエム テクノロジーズ エルエルシーESM Technologies, LLC | Method for evaluating joint treatment |
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