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Received October 17, 2016
Accepted November 7, 2016
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질량분석기를 활용한 효과적 이황화결합 분석법 개발
Mass Spectrometry-Based Strategy for Effective Disulfide Bond Identification
오송첨단의료산업진흥재단 신약개발지원센터, 28160 충청북도 청주시 흥덕구 오송읍 오송생명로 123 1한국과학기술연구원 도핑콘트롤센터, 02792 서울특별시 성북구 화랑로14길 5 2고려대학교 기계공학과, 02841 서울특별시 성북구 안암로 145 3광운대학교 화학공학과, 01897 서울특별시 노원구 광운로 20
New Drug Development Center, Osong Medical Innovation Foundation, 123, Osong Saengmyung-ro, Osong-eup, Heungdeok-gu, Cheongju, Chungbuk, 28160, Korea 1Doping Control Center, Korea Institute of Science and Technology (KIST), 5, Hwarang-ro 14-gil, Seongbuk-gu, Seoul, 02792, Korea 2School of Mechanical Engineering, Korea University, 145, Anam-ro, Seongbuk-gu, Seoul, 02841, Korea 3Department of Chemical Engineering, Kwangwoon University, 20, Kwangwoon-ro, Nowon-gu, Seoul, 01897, Korea
jonwkim@kbiohealth.kr
Korean Chemical Engineering Research, February 2017, 55(1), 27-33(7), 10.9713/kcer.2017.55.1.27 Epub 1 February 2017
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Abstract
이황화결합(Disulfide Bond)은 다양한 생리학적 혹은 병리학적 과정 중 단백질번역 후 변형(Post-Translational Modifications) 과정 중에 형성된다. 그러므로 이황화결합에 대한 정보는 단백질의 화학적 구조를 보다 종합적으로 이해하는데 매우 중요한 일이다. 질량분석기를 이용한 이황화결합 분석은 매우 효과적이며, 현재까지 질량 분석기를 활용한 다양한 이황화결합 분석법들이 개발되었다. 그러나, 대부분의 이황화결합 분석법의 경우, 이황화결합 분석 시 자유-시스테인잔기(Free Thiol Residues) 분석을 고려하지 않았다. 본 연구에서는 이황화결합에 관여하는 시스테인/자유-시스테인에 초점을 두고 총 4단계(1단계: 아미노산 서열을 통한 이황화결합 가능 부위를 예측, 2단계: 자유시스테인의 존재 유무의 확인, 3단계: 질량 분석기를 활용한 이황화결합 분석, 4단계: 이황화결합 분석법의 종합적인 검증)의 분석법을 개발하였다. 나아가, 본 연구에서 개발된 분석 기법을 실제 휴먼 유래 재조합 단백질(HRPE1)에 적용함으로써 개발된 이황화결합 분석법의 효용성을 확인하였다. HRPE1의 경우, 총 6개의 이황화결합(Inter-chain 형태: 1, Intra-chain 형태: 5)으로 구성된 것을 최종 확인하였다.
The determination of disulfide bonds is important for comprehensive understanding of the chemical structure of protein. So far, many strategies for the disulfide bond analysis have been suggested in terms of speed and sensitivity. However, most of these strategies have not considered free thiol residues in the target protein in the process of determining the disulfide bond. We suggested the strategy which was composed of four steps for the identification of disulfide bonds; the first step was the prediction of possible disulfide bonds, the second step was the determination of free cysteine residues, the third step was the analysis of disulfide bond using a high-resolution mass spectrometry, and the final step was the determination of disulfide bonds based on the comprehensive verification. In this study, we performed the characterization of disulfide bonds for the recombinant protein (HRPE1), where 1 and 5 inter- and intra-chain disulfide bonds were identified, respectively.
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