多功能纳米材料的制备及其在磷酸化多肽高灵敏度质谱检测中的应用

发布时间：2018-02-04 10:32

本文关键词： 复合纳米纤维超顺磁性复合纳米材料有序介孔结构蛋白质磷酸化 MALDI-TOF MS　出处：《苏州大学》2016年硕士论文　论文类型：学位论文

【摘要】：随着生命科学与临床研究水平的不断发展,对翻译后修饰的蛋白质的富集技术引起了研究者们越来越多的兴趣。蛋白质磷酸化的异常,往往会引起众多疾病,这使得对蛋白质磷酸化的研究成为许多疾病早期诊断的突破口。近些年随着纳米技术的快速发展,功能纳米复合材料被赋予良好的生物相容性、稳定的理化性质及方便的多功能化,并很快被用于生物分离及生物医药等众多领域。本论文基于以上磷酸化蛋白及纳米技术展开了相关研究,具体工作主要有以下两方面:第一,在物化性质稳定的细菌纤维素的基础上,合成了一种多功能复合纳米纤维(细菌纤维素-二氧化钛)以用于磷酸化多肽的特异性富集及辅助质谱检测。由细菌纤维素产生菌生物合成的细菌纤维素(BC)表面富含羟基,为接下来的表面溶胶-凝胶反应提供反应位点,从而形成二氧化钛层。然后在氨水的催化下,通过水热反应,促进有序介孔结构的形成,同时提升了二氧化钛纳米颗粒的结晶度。我们利用BC@mTiO_2复合纳米纤维,实现了对磷酸化多肽的特异性富集,且灵敏度可以达到1.7pmol,这与商业化磷酸化多肽富集Tip相比灵敏度高出两个数量级。第二,在具有超顺磁性质的四氧化三铁的基础上,经过溶胶凝胶过程成功包裹上二氧化锆层。利用水热过程获得的有序介孔结构比表面积可达到338.3m~2/g。该磁性纳米复合材料(Fe_3O_4@mZrO_2)具有路易斯酸性更强的二氧化锆亲和层,提高对单磷酸化多肽的作用。另外利用材料直接检测和原位洗脱方法来避免多磷酸化多肽的损失,最终达对单磷酸化多肽和多磷酸化多肽同时灵敏检测的目的。在标准蛋白评价中,Fe_3O_4@mZrO_2的检测灵敏度达到了飞摩尔级别,同时我们利用这种很好的富集方法对癌细胞(A549)裂解液进行了检测。综上所述,本论文对复合纳米材料的合成及其在磷酸化蛋白及多肽方面进行了较为系统的研究和论述,我们的研究结果为这一类复合材料在蛋白质磷酸化的富集检测提供方法及优化条件,同时也为其以后可能应用于相关疾病早期诊断打下一个良好的基础。
[Abstract]:With the development of life science and clinical research, the enrichment of post-translational modified proteins has attracted more and more interest of researchers. Abnormal phosphorylation of proteins often leads to many diseases. In recent years, with the rapid development of nanotechnology, functional nanocomposites have been given good biocompatibility. Stable physical and chemical properties and convenient multifunctional, and soon used in many fields such as biological separation and biomedicine. Based on the above phosphorylated protein and nanotechnology, this thesis has carried out related research. The specific work mainly includes the following two aspects: first, on the basis of stable physical and chemical properties of bacterial cellulose. A multifunctional composite nanofiber (bacterial cellulose-titanium dioxide) was synthesized for specific enrichment of phosphorylated polypeptides and assisted mass spectrometry. The surface of BC is rich in hydroxyl groups. The formation of ordered mesoporous structure was promoted by hydrothermal reaction under the catalysis of ammonia. At the same time, the crystallinity of TIO _ 2 nanoparticles was improved. We realized the specific enrichment of phosphorylated polypeptides by using BC@mTiO_2 composite nanofibers, and the sensitivity was 1.7 pmol. This is two orders of magnitude higher than commercial phosphorylated polypeptides in the enrichment of Tip. Second, it is based on the superparamagnetic ferric oxide. Zirconia layer was successfully coated by sol-gel process. The specific surface area of ordered mesoporous structure obtained by hydrothermal process could reach 338.3 mg / g. Fe3O _ 4 / Fe3O _ 4 / mZrO _ 2) has a stronger Lewis acidic zirconia affinity layer. In addition, direct material detection and in situ elution were used to avoid the loss of polyphosphorylated polypeptides. In the evaluation of standard protein, the detection sensitivity of Fe3O _ (3) O _ (4) M _ (ZrOO) _ (2) reached the flying molar level. At the same time, we used this good enrichment method to detect the cancer cell A549) lytic solution. In this paper, the synthesis of composite nanomaterials and their phosphorylation proteins and peptides were systematically studied and discussed. Our results provide a method and optimization condition for the detection of protein phosphorylation in this kind of composite materials, and also lay a good foundation for its application in the early diagnosis of related diseases in the future.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TB383.1;Q503

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