초음파를 이용한 단백질 가수분해의 온도 효과 및 마이크로파를 이용한 사람의 모발 단백질 추출

다국어 초록 (Multilingual Abstract)

The goal of this thesis is to develop effective protein analysis technique. To achieve this, I investigated the temperature effect on ultrasound-assisted tryptic protein digestion and developed an effective protein extraction method from human hair.
First, the effects of temperature ultrasound-assisted tryptic protein digestion were investigated using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Three standard proteins, cytochrome c, myoglobin, and bovine serum albumin, were digested at 4？C (ice), room temperature (20-25？C), 37？C, and 55？C for 0 s, 30 s, 1 min, and 5 min, in a water bath under ultrasonication. It was found that the number of identified peptides generally increased with increasing temperature or digestion time. The best performance in terms of the number of identified peptides was observed at 55？C. Compared with conventional overnight digestion at 37？C without ultrasonication, digestions performed under ultrasonication generally produced more peptides in most of the above listed conditions. Ultrasound-assisted tryptic digestion at 55？C for 5 min showed better performance in terms of sequence coverage and the number of identified peptides for the three model proteins, compared with conventional overnight digestion. A more than doubled miscleavage ratio was observed with ultrasound-assisted tryptic digestions at 55？C for 5 min versus conventional overnight digestion.
Second, the effectiveness of microwave-assisted extraction of proteins from human hair samples was evaluated. Extractions were performed from 2 mg hair samples in an extraction solution consisting of 25 mM Tris？HCl (pH 8.5), 2.6 M thiourea, 5 M urea, and 5% mercaptoethanol. During extraction, samples were exposed to microwave radiation (600 W) for a specified incubation period (5？120 min). The extraction efficiency of samples that had been incubated for 60 min was similar to that of samples that had been heated at 50？C for 24 h using the conventional Shindai method.

목차 (Table of Contents)

• Ⅰ. 서론 1
• Ⅱ. 실험 8
• 1. 시약 및 기기 8
• 2. 초음파를 이용한 단백질 가수분해 8
• 2.1 표준 단백질 준비 9
• 2.2 온도의 변화에 따른 초음파 가수분해 실험 10
• 2.3 매트릭스 보조 레이저 탈착 이온화 비행시간 질량분석기 분석 10
• 3. 모발 단백질 추출 실험 13
• 3.1 모발 준비 및 단백질 추출 13
• 3.2 겔 전기 영동법 15
• 3.3 단백질 정량분석 15
• Ⅲ. 결과 및 토의 16
• 1. 초음파를 이용한 단백질 가수분해의 온도 효과 16
• 1.1 표준 단백질 확인 16
• 1.2 Cytochrome c의 초음파 가수분해 18
• 1.3 Myoglobin의 초음파 가수분해 21
• 1.4 BSA의 초음파 가수분해 24
• 1.5 초음파 가수분해의 조건과 동정된 펩타이드 수의 관계 26
• 1.6 초음파 가수분해와 일반적인 overnight 가수분해 비교 29
• 1.6 55oC에서 5분동안 초음파를 이용한 가수분해 방법과 일반적인 overnight 가수분해 방법 비교 29
• 2. 마이크로파를 이용한 사람 모발의 단백질 추출 43
• 2.1 혼합 모발 시료에서 단백질 추출 43
• 2.2 마이크로파 복사선 (radiation)으로 인한 단백질 분해 47
• 2.3 한 가닥 모발 분석 49
• Ⅳ. 결론 52
• Ⅴ. 참고문헌 53
• Ⅵ. Abstract 59