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학술연구/단체지원/교육 등 연구자 활동을 지속하도록 DBpia가 지원하고 있어요.
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논문 기본 정보
- 자료유형
- 학위논문
- 저자정보
- 지도교수
- 이용문
- 발행연도
- 2014
- 저작권
- 충북대학교 논문은 저작권에 의해 보호받습니다.
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초록· 키워드
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Herein, I describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry method. The analyte protein, recombinant human erythropoietin (rhEPO), was effectively hydrolyzed by incubation with 8 M hydrochloric acid at 130 ℃ for 48 h, in which at least 1 μmol/kg of rhEPO was treated to avoid possible degradation of released amino acids during hydrolysis. Prior to hydrolysis, sample solution was subjected to ultrafiltration to eliminate potential interfering substances. In a reversed-phase column, the analytes (phenylalanine, proline and valine) were separated within 3 min using gradient elution comprising 20 % (v/v) acetonitrile and 10 mM ammonium acetate, both containing 0.3 % (v/v) trifluoro- acetic acid. The optimized hydrolysis and analytical conditions in my study were strictly validated in terms of accuracy and precision, and were suitable for the accurate quantification of rhEPO. Certified rhEPO was analyzed using a conventional biochemical assay kit as an additional working calibrant for the quantification of EPO and improved the accuracy. The optimized protocol is suitable for the accurate quantification of rhEPO and satisfactorily serves as a reference analytical procedure for the certification of rhEPO and similar proteins.
목차
1. Introduction 12. Background 62.1. Amino acid 62.2. Hydrolysis of protein 112.3. Isotope dilution mass spectrometry 133. Experimental 143.1. Materials 143.2. Sample preparation 153.3. Sample purity assessment 163.4. LC-MS conditions 173.5. Hydrolysis method comparison 203.6. Method validation 203.7. Application of certified rhEPO as a reference material 204. Results and Discussion 224.1. Sample purity assessment 224.2. Elimination of AA impurities in sample solution 244.3. Optimization of hydrolysis conditions 284.4. Optimization of analytical conditions 314.5. Method validation 344.6. Application of certified rhEPO as a reference material 395. Conclusion 42References 43Summary in Korean 48Acknowledgement 50
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