【作者】 唐佳；

【导师】 张祥民； 邓春晖；

【作者基本信息】 复旦大学 ， 分析化学， 2010， 博士

【摘要】 蛋白质组学以大规模分析细胞或生物体内的蛋白质为目的,主要开展表达蛋白质组学和功能蛋白质组学两类研究工作。生物体内蛋白质种类繁多,性质复杂,数量庞大,尤其是蛋白质翻译后修饰、蛋白质—蛋白质相互作用等现象的存在,对现行的蛋白质组学研究方法和技术提出了严峻挑战。因此,发展更多的蛋白质组学研究新技术与新方法,从而更好更快地进行蛋白质体系研究,对于解决生物体生理学、病理学以及药理学方面的重要科学问题有着重大的意义。翻译后修饰蛋白质组学在现今蛋白质组学中有着特殊的地位,比较常见的后修饰种类有磷酸化、糖基化、甲基化、乙酰化和泛素化修饰,其中磷酸化和糖基化蛋白质翻译后修饰的研究较多。规模化的识别、检测和鉴定生物体内这些后修饰蛋白质的表达及其变化,是翻译后修饰蛋白质组学研究的关键技术之一。然而,磷酸化和糖基化蛋白质组学研究中都面临着类似的困难,即这两类翻译后修饰蛋白质的种类虽然不少,丰度却通常较低,并且在通用的质谱鉴定中离子化效率不高,不易被质谱识别鉴定。因此,研究磷酸化和糖基化蛋白质组学,首要任务就是要将其从复杂体系中纯化出来,这也是翻译后修饰蛋白质组学发展的关键技术之一。功能化微米级和纳米级材料目前在科学发展的各个领域都有着很好的应用,相对于普通材料而言,它们具有极大的比表面积和极高的表面活性,特别适于生物医学领域的应用。以这些微米或纳米材料为基底,通过一些特殊的处理或者功能化基团的修饰,这些材料就能够与一些经过特定修饰的蛋白质发生相互作用,从而简化目标蛋白的分离纯化步骤,使得后续的分析更为可行。本论文针对蛋白质组学研究中面临的磷酸化和糖基化蛋白质高效选择性富集方面的热点难点问题,将功能化材料与蛋白质分析结合起来,开展了一系列研究工作,发展了一些基于功能化材料的磷酸化和糖基化蛋白质组学研究新技术新方法,并利用实际生物样品验证了这些新技术新方法的可行性,取得了一些创新性研究结果。第一章概述了蛋白质组学研究的意义和现状,集中讨论了磷酸化和糖基化蛋白质组学研究的概况、技术和方法,并就生物质谱在蛋白质组学研究技术以及新兴的翻译后修饰蛋白组学中的应用展开了一些讨论,概述了功能化材料及其在生物分析中的应用,最后提出了本论文选题的目的和意义。第二章利用水热法合成了具有多孔表面结构的二氧化钛微球,并将其用于鼠脑组织蛋白提取物中磷酸化肽段的富集。通过一系列的表征,确认多孔二氧化钛微球(直径1微米左右)具有锐钛矿的晶体结构,并且其比表面积达到了84.98m2/g,是同样粒径的具有光滑表面的二氧化钛微球比表面积的25倍,是商品化纳米级二氧化钛材料(直径90纳米左右)比表面积的2倍。经过条件优化,多孔二氧化钛材料和光滑二氧化钛材料都成功地在标准磷酸化蛋白p-酪蛋白(β-casein)的酶解产物中富集到了磷酸化肽,然而当将这两种材料进一步应用于较为复杂的体系,如β-casein与非磷酸化蛋白牛血清白蛋白(Bovine Serum Albumin, BSA)的酶解产物混合物中时,多孔二氧化钛材料显示了比光滑二氧化钛材料更优异的磷酸化肽选择性。在此基础上,我们还首次将三种二氧化钛材料,即多孔二氧化钛材料、光滑二氧化钛材料、商品化纳米级二氧化钛材料用于选择性富集鼠脑蛋白质提取物的酶解产物中的磷酸化肽段,分别分离鉴定出了223、47、90条磷酸化肽。这种多孔二氧化钛微球合成方法简单,成本低廉,使用灵活方便,对于磷酸化肽段的亲和效果好,经过实验验证,可以直接用于复杂体系中磷酸化肽段的分离。因其比表面积大,粒径合适,也可将其装填成亲和色谱柱进行在线的磷酸化肽段或蛋白的分离富集。第三章首次发展了简便的糖基化肽段靶上富集的方法用于糖基化肽段的快速分离和质谱鉴定。首先合成了纳米级金粒子,然后通过高温煅烧将这些纳米金颗粒烧结到MALDI-QIT-TOF-MS靶板上,再利用金和巯基之间的相互作用在这些纳米金颗粒表面修饰上巯基苯硼酸。硼酸分子在碱性条件下会与糖分子中的顺式二羟基发生可逆反应形成一个五元环,此五元环在酸性条件下能够解离,因此常被用来选择性富集糖基化的肽或者蛋白质。我们选用标准糖蛋白辣根过氧化物酶(Horseradish peroxidase, HRP)、胎球蛋白(Fetuin)和去唾液酸胎球蛋白(Asialofetuin, ASF)的酶解产物验证了靶板对于糖基化肽段的选择性富集能力。在HRP浓度低至2.5×10-9M时,仍能够鉴定到9条糖肽或者是糖肽碎片离子的信号。不仅仅是HRP,靶上富集糖肽的方法也成功地应用到了胎球蛋白和去唾液酸胎球蛋白酶解产物的糖肽富集中。为了进一步验证这种方法的实用性,我们用靶上富集的方法对HRP与非糖基化蛋白β-casein的酶解产物混合液中的糖肽进行了富集和鉴定,即使在HRP与β-casein的浓度比达到了l：10的条件下,也成功地从混合肽段中鉴定到了7条糖肽或糖肽碎片离子。最后,我们将HRP酶解产物与牛奶的酶解产物混合并利用靶上富集的方法对该肽段混合物进行了糖基化肽段的富集,共分离鉴定了7条HRP的糖肽或糖肽碎片离子信号。靶上富集方法快速,简便,需要的样品量少,易于实现高通量和自动化的蛋白质组学研究。在这里,我们首次发展了糖基化肽段靶上富集的方法,并通过一系列实验验证了该方法的可行性,为糖基化蛋白质组学研究开辟了新的道路。第四章创新性地利用“三明治”固定方法在硼酸纳米磁性微球表面固定了伴刀豆球蛋白(Concanavalin A, Con A)，并将其用于糖基化蛋白的分离富集。硼酸修饰的氨基磁球曾被成功地用于标准糖肽和标准糖蛋白的富集,但当用于实际生物样品中的糖蛋白富集时,效率并不理想。Con A是凝集素中最常用的一种,它与甘露糖和葡萄糖残基有着很强的亲和作用,常被用来预富集生物样品中的糖基化蛋白质。因此我们选择Con A作为修饰物,利用硼酸和糖的共价反应首先在氨基苯硼酸纳米磁球表面修饰上一层甲基α-D-吡喃甘露糖苷,用单糖作为介质,再利用单糖和Con A的亲和作用在磁球表面固定上ConA。与在硼酸磁球表面直接固定Con A相比,利用上述“三明治”方法固定的Con A量提高了三倍。Con A纳米磁球、硼酸磁球和商品化的Con A磁球(直径1微米左右)都被用来进行人肝癌细胞株7703细胞裂解液中糖蛋白的分离富集。利用Con A纳米磁球共鉴定了包含184个糖基化位点在内的172条糖肽,这些糖肽共对应到101个糖蛋白,占鉴定蛋白总数的约68%。利用商品化的Con A磁球共鉴定了69条糖肽,对应于51个糖蛋白,占鉴定蛋白总数的约65%。而利用硼酸纳米磁球富集后鉴定到了47个蛋白,其中只有12个是糖蛋白。Con A纳米磁球制备方法简单,成本较低,固定效率高,使用也极为方便,亲和效率和商品化的Con A磁球相当,并且由于其“三明治”法固定的第一步是利用了硼酸—单糖的可逆反应,还能方便地利用这一性质对作为基底的硼酸磁球进行回收再利用,极大地节省了成本。总之,本论文针对翻译后修饰蛋白质组学中面临的磷酸化和糖基化蛋白质高效富集方面的热点问题,以功能化材料为基础,以发展相关的磷酸化和糖基化蛋白质组学研究新技术新方法并进行实际的应用研究为目的,探索和发展了各种不同的功能化材料的实际应用,并建立了新颖的分析方法,为解决磷酸化／糖基化肽段或蛋白质的高效分离富集及鉴定提供了有效的研究手段和方法。更多还原

【Abstract】 Proteomics aim at large-scale analysis of proteins in all biological objects, and it constitutes of expressional proteomics and functional proteomics. Investigation of genomics and proteomics gives us insights to the physiological, pathological and pharmacological processes of cells and organisms. However, there are extremely large amount of proteins with complicated properties, e.g. post-translation, protein-protein interaction, which bring great challenge to existing analytical methods. Therefore, it is necessary to develop new techniques and methodologies for better solution of proteomic research.Post-translational proteomics is one of the most important subjects in proteome research. Protein post-translational modification plays key role in many biological processes. There are more than 200 reported PTMs, among which phosphorylation and glycosylation are the most studied protein modifications. However, phosphorylated and glycosylated proteins are usually of low abundance in biological samples. It is difficult to detect these proteins in mass spectrometry without sample pre-treatment. Development of purification and enrichment methods for post-translational modified proteins are in great demand.The application of functionalized micro-/nano-particles in biomedical research area is gaining increasing attention due to there ease of manipulation and recovery during the past decades. These particles possess great specific surface areas and high surface activity. Functional modification based on these paricles can be easily performed. Up till now, functionalized materials are extensively applied in various biomedical applications such as cell separation, drug delivery, enzyme immobilization, and protein purification. With specific modification, functionalized materials can be effectively applied in phosphorylated/glycosylated protein separation and enrichment.Based on the proteome research background and the development trend of functionalized materials, the research interest of this work focused on the preparation of several kinds of functionalized materials and developing a series of techniques and methods to resolve current problems in the separation and concentration of phosphorylated and glycosylated proteins. The feasibility of these techniques and methods was validated with real biological samples. This dissertation is divided into four parts. In Chapter 1, advances in proteome research, current research techniques and methods of phosphorylated/glycosylated proteomics, and applications of functionalized materials in biological analysis were summarized in brief. The intention and meaning of this dissertation were explained.In Chapter 2, mesoporous TiO2 microspheres were synthesized by simple hydrothermal reaction, and successfully developed for phosphopeptides enrichment from both standard protein digestion and real biological sample such as rat brain tissue extract. The mesoporous TiO2 microspheres (the diameter size of about 1.0μm) obtained by simple hydrothermal method were found to have a specific surface area of 84.98 m2/g, which is much lager than smooth TiO2 microspheres with same size. The surface area of mesoporous TiO2 microspheres is almost two times of commercial TiO2 nano particle (a diameter of 90 nm). Both of these two TiO2 microspheres are successfully applied to selective enrichment of phosphopeptides generated from P-casein digest. However, when they are used for phosphopeptides enrichment from a complicated peptide mixture such as P-casein and BSA digest mixture, mesoporous TiO2 microspheres exhibit strong specific selectivity compared with amorphous TiO2 microspheres with smooth surface. When they are further used for phosphopeptide enrichment from rat brain tissue extract, the mesoporous TiO2 microspheres show the highest binding capacity as well as capture efficiency for phosphopeptides.223,47, 90 phosphopeptides were identified using mesoporous TiO2 microspheres, smooth TiO2 microspheres and commercial TiO2 nanoparticles, respectively. It has been demonstrated that mesoporous TiO2 microspheres have powerful potential for selective enrichment of phosphorylated peptides. Moreover, the preparation of the mesoporous TiO2 microspheres is easy, simple and low-cost. This mesoporous TiO2 material may be further used as affinity chromatography column packing material for comprehensive phosphorylated proteome research.In Chapter 3, an on-plate selective enrichment method is developed for fast and efficient glycopeptide investigation. Gold nanoparticles were first spotted and sintered on a MALDI-QIT-TOF-MS stainless steel plate, then modified with 4-mercaptophenylboronic acid. These spots were used to selectively capture glycopeptides from peptide mixtures, and the captured target peptides could be analyzed by MALDI MS simply by depositon of DHB matrix. Horseradish peroxidase was employed as a standard glycoprotein to investigate the enrichment efficiency.9 glycopeptides or glycopeptides fragments were identified with HRP concentration as low as 2.5×10-9 M. This on-plate glycopeptide enrichment strategy was further evaluated with other glycoproteins like fetuin, asialofetuin. It also succeeded at binding and identifying glycopeptides from a relatively complicated peptide mixture. The relatively small sample amount needed, low detection limit, and rapid selective enrichment have made this on-plate strategy promising for on-line enrichment of glycopeptides, which could be applied in high-throughput proteome research.In Chapter 4, Con A immobilized magnetic nanoparticles were synthesized for selective separation of glycoproteins. At first, a facile immobilization of Con A on aminophenylboronic acid-functionalized magnetic nanoparticles was performed by forming boronic acid-sugar-Con A bond in sandwich structure using methyl a-D-mannopyranoside as an intermedium. The selective capture ability of Con A modified magnetic nanoparticles for glycoproteins was tested using standard glycoproteins and cell lysate of human hepatocelluar carcinoma cell line 7703. In total 184 glycosylated sites were detected within 172 different glycopeptides corresponding to 101 glycoproteins. Also the regeneration of the protein-immobilized nanoparticles can easily be performed taking advantage of the reversible binding mechanism between boronic acid and sugar chain. The experiment results demonstrated that Con A modified magnetic nanoparticles by the facile and low-cost synthesis provided a convenient and efficient enrichment approach for glycoproteins, and are promising candidates for large scale glycoproteomic research in complicated biological samples.In summary, the main contributes of this dissertation is that we aimed at better solutions of post-translational modified proteome research, and developed several practical techniques for phosphorylated/glycosylated protein or peptide enrichment based on functionalized materials. According to the experiment results, these new techniques offer effective enrichment and identification of phosphorylated and glycosylated protein/peptide even in complicated biological samples, which demonstrated their powerful ability in post-translational proteome research.更多还原