Application of Surface Functionalized Superparamagnetic Nanoparticles to Selective Purification of Specific Proteins in Biological Sample
- 주제(키워드) Superparamagnetic Nanoparticles , Selective purification
- 발행기관 고려대학교 대학원
- 지도교수 이상원
- 발행년도 2009
- 학위수여년월 2009. 8
- 학위명 석사
- 학과 일반대학원 화학과
- 세부전공 생물화학전공
- 원문페이지 55 p
- 실제URI http://www.dcollection.net/handler/korea/000000009890
- 본문언어 영어
- 제출원본 000045552307
초록/요약
Nanometer-sized materials have been widely applied to biological system. The Magnetic nanoparticles especially have been most commonly used for in vivo and in vitro application such as magnetic resonance imaging (MRI), drug delivery, and cell mechanical studies. Because of the ease of isolation, the separation and purification of specific cells and biomolecules have become one of the most promising application of magnetic nanoparticles. The surface modification of the nanoparticles makes it possible to isolate the specific species from the complex solution, having a number of advantages instead of the use of conventional micron-sized bead techniques, because of higher surface-to-volume areas, faster binding rates, higher miscibility, and higher specificity. Well-defined isolation of a specific protein from complex protein mixture is a critical step in proteome research to increase information contents under the limited dynamic range of currently available proteomic technologies. In our recent experiments, the selective isolation of cysteine-containing peptide from the complex peptide mixture including human serum was achieved by using silica-coated magnetic nanoparticles that were surface-modified with SPDP (N-Succinimidly 3-(2-pyridydithio)-propionate) having reactivity toward free thiol of cysteine. On the basis of this technique, nanoparticles were biofunctionalized with bio-molecule, the tri-peptide containing a cysteine. The target proteins fused with tag protein that recognize bio-molecule as own substrate and bind specifically via enzyme-substrate interaction were isolated without nonspecifically bound proteins from the complex proteome sample. The purified proteins, which were later eluted from the nanoparticles, were confirmed by SDS-PAGE and tryptic peptides of those proteins were analyzed by doing LC/MS/MS experiments.
more목차
1. Abstract 1
2. Introduction 3
3. Experimental section 7
1. Chemical and materials 7
2. preparation of bio-functionalized superparamagnetic Fe3O4@SiO2 NPs 7
2.1. Synthesis of 29.6 (± 2.1)-nm Fe3O4@SiO2 NPs and removal of Igepal CO-520 from Fe3O4@SiO2 NPs 7
2.2. Surface modification with 3-(trihydroxysliyl)propylmethylphosphonate (THPMP) and N-[3- (trimethoxysilyl)propyl]-ethylenediamine(AEAPTMS) 8
2.3. Surface modification with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and succinimidyl 6-(3-[2-pyridldithio]-propionamido)hexanoate (LC-SPDP) 8
2.4. Surface modification with reduced glutathione 9
3. Purification of specific proteins 10
3.1. Purification of GST tagging protein from complex protein mixtures 10
3.2.Purification of GST tagging protein from complex protein mixtures using glutathione sepharose 4B bead having same function as glutathione NPs 11
4. Capillary RPLC/MS/MS experiments and data analysis 12
5. proteomic data analysis 13
6. SDS-PAGE Gel Electrophoresis 14
4. Results and discussion 16
1. Analysis of bio-funcnalized NPs 16
2. Purification of specific proteins 21
5. Conclusion 44
6. Reference 49
