【作者】 黄光明；

【导师】 张新荣；

【作者基本信息】 清华大学 ， 化学， 2007， 博士

【摘要】 糖基化是蛋白质转录后修饰最重要的几种形式之一其研究在生命科学中有重要意义。然而,糖组学的研究长期以来一直由于缺乏适当的分析方法,限制了其的发展,针对这一问题本文在寡聚糖的定性与定量分析方法的研究方面做了一些探索。本论文主要的研究内容如下:1.发展了基于在线全甲基化衍生-固相萃取的快速质谱检测方法。选用全甲基化衍生稳定糖链末端的唾液酸结构,并且便于检测糖链的连接顺序和糖苷键位置,并使用了固相衍生的方法加快反应速度同时减少副反应的发生。本文提出将碳纳米管作为全甲基化糖链的吸附剂,在强碱性的条件下实现了在线萃取,并且能够在不改变衍生条件的情况下与液相或者固相衍生反应联用。2.应用基于在线衍生的快速质谱检测方法对具有相同多肽序列的糖蛋白的糖链结构进行了检测。使用人免疫球蛋白G的N-糖链验证了本方法定性检测复杂糖链的可靠性,并定性检测了不同重组促血红蛋白生长素的N-糖链结构。结果表明,由于来自不同细胞系的两个促血红蛋白生长素具有不同的糖基化形式,N-糖链的结构有所不同。3.使用反应解吸附电喷雾离子化-多级质谱实现了尿样中类固醇化合物及其糖基化产物的直接快速检测。在离子化过程中,喷雾溶液中的羟胺与样品中的待分析物的酮羰基发生离子分子反应,反应产物被多级质谱检测并得以确认。类固醇的糖基化产物在聚四氟乙烯的表面可以不用任何水解处理而直接被检测出。反应解吸附电喷雾离子化大大提高了离子化效率,降低了样品的基体效应。4.研究了与毛细管电泳结合的糖类化合物的新型催化发光定量检测器。催化化学发光是由糖在多孔三氧化二铝表面的催化氧化反应产生的。对于缺乏紫外吸收的糖类化合物,比如蔗糖、α-乳糖、麦芽糖、棉仔糖、半乳糖、木糖和葡萄糖,本检测器能够很好地实现定量检测。本方法提供了一个不需要衍生而直接检测的方案,也不需要加入离子生色团,相比安培检测器能为毛细管电泳提供更灵活的分离条件。化学发光检测器具有较低的基线漂移、干扰小、线性范围宽且具有长使用寿命等特点。更多还原

【Abstract】 Glycosylation is considered to be the one of the most common types of post-translational modifications of proteins. Glycosylated proteins are ubiquitous components of extracellular matrices and cellular surfaces where their oligosaccharide moieties are implicated in a wide range of cell-cell and cell-matrix recognition events. The development of the glycomics is far hinged since the lack of proper analytical methods. In the present dissertation, investigations of both quality and quantity analysis methods for glycomics were studied. The main contents of the present dissertation are as follows:1. A new approach for mass spectrometry detection method of glycan was developed based on on-line permethylation-SPE technique. Permethylation derivatization is preferred to stabilize the sialic acid residues and facilitate the determination of branching and interglycosidic linkages. Solid phase permethylation was carried out to fasten the derivatization procedure and to minimize the side reactions. For the first time carbon nanotubes were used as the absorbent for permethylated glycans at extremely harsh condition. This on-line SPE could be easily combined to either liquid or solid phase permethylation without changing permethylation reaction condition.2. A new strategy was investigated for the glycan analysis of proteins with the same peptide sequence based on the on-line permethylation coupling to mass spectrometry detection. N-glycans released from human Immune Globin G were used to evaluate the reliability of the present method. Then structures of N-glycans form different recombinant EPOs were identified. Different N-glycans were identified for the two recombinant EPOs tested, illustrating the different glycosylations of the two recombinant EPOs since they were from the different cell lines.3. Fast screening for anabolic steroids and glycosteroid in raw urine was achieved using reactive desorption electrospray ionization (reactive DESI) and tandem mass spectrometry. Spray solutions containing hydroxylamine allow heterogeneous reactions of hydroxylamine with the carbonyl group of the steroids during the ionization process. The ion/molecule reaction product and the oxime formed by its dehydration were observed using reactive DESI. The glycosteroid was ionized intact without hydrolysis from polytetrafluoroethylene. Reactive DESI provided significant improvements in ionization efficiency of these steroids in raw undiluted urine as compared to conventional DESI; Suppression effects due to the sample matrix were minimal and the urine matrix had no deleterious effect.4. An aerosol chemiluminescent (CL) detector coupled to CE for quantity analysis of saccharides analysis has been investigated. The CL emission was generated due to the catalyzing oxidization of saccharides on the surface of porous alumina. The saccharides such as sucrose,α-lactose, maltose, reffinose, galactose, xylose and glucose with only weak UV absorbance can be successfully detected with this CE detector. This detector offers advantage of direct detection of the analytes without involving in a derivatization procedure. In comparing with indirect UV detector, the addition of ionic chromophore into the electrophoresis buffer is therefore no longer needed. Aerosol CL detector is also a robust means over a relative wide range of pH, while high pH value was requested by using amperometric detection. The low baseline shift, low interference, relative wide linear range and long lifetime also make CE-aerosol chemiluminescent detector a potential means for routine analysis of saccharides.更多还原