基于功能化纳米材料和生物质谱的糖肽富集与鉴定新技术新方法

【作者】 王雅莉；

【导师】 陆豪杰；

【作者基本信息】 复旦大学 ， 分析化学， 2014， 硕士

【摘要】 本论文的研究内容涉及分析化学、化学生物学、材料化学等二级学科,并且还应用到了生物化学的相关技术,属于交叉学科的研究领域。主要是在生物质谱技术的基础上,设计合成了一系列磁性纳米复合材料和聚合物纳米材料,发展了针对蛋白质N-糖基化修饰的高效分离富集与高灵敏度鉴定的新技术与新方法,并将这些新技术新方法成功用于人血清实际样品的研究中。作为一种十分重要的翻译后修饰,糖基化广泛存在于50%以上的蛋白质中,且在分化、发育、肿瘤的发生与转移、免疫、传染及再生中发挥着重要的作用。而且在某些疾病的发生与发展中,例如肿瘤的早期诊断、进程监测、预后评估等阶段都与糖基化息息相关。因此,对于糖基化的研究颇具生物学意义和临床价值,也一直是蛋白质组学领域中的热点。然而,由于糖基化修饰蛋白的天然丰度很低,若想要取得足够量的样本进行分析,则要对样本进行浓缩富集。对于复杂的生物样本,其中所含蛋白质种类极多,动态范围极广,且高丰度的非糖蛋白占绝大多数,严重地抑制了低丰度的糖蛋白的鉴定。其次,糖基化修饰具有微观不均一性的特点,一个蛋白拥有多个糖基化位点和多种糖链结构,使得糖基化的研究十分复杂。在糖基化修饰的质谱分析中,我们通常采用"bottom up"的方法,即在肽段水平上进行研究。但是当糖蛋白经过酶解变成肽段之后,据统计,糖基化肽段仅占总酶解肽段的2-5%。而这些极少数的糖肽在质谱中的信号又会受到大量共存的非糖肽的抑制,导致鉴定困难。因此,研究蛋白质糖基化修饰的首要解决的问题就是要从复杂样品中高效、高灵敏度地分离富集出糖蛋白/肽。目前已有的各类糖蛋白/肽富集方法仍有许多问题有待改善,如适用范围广操作简便的硼酸富集法存在富集选择性不高的问题,而选择性最高的肼化学法存在富集材料价格昂贵、灵敏度低、反应时间长等问题。此外,一般在质谱分析前处理过程中,糖基化修饰的样品要经过各种浓缩富集方法处理,不免引入一些小分子如无机盐等,对质谱信号有抑制作用,干扰糖肽出峰；从而又需要除盐处理,更会导致样品损失,使得上样量达不到仪器鉴定要求。因此,发展针对糖肽的高效富集方法,且无需除盐是一个难点。鉴于此,本论文就围绕这些难题开展研究工作,分为三个章节进行阐述。主要内容如下：第一章是绪论部分。主要阐述了糖基化修饰的特点及重要性,糖蛋白质组学的发展现状,糖蛋白质组学分离富集技术的发展现状和目前存在的问题与挑战,以及纳米材料在糖蛋白质组学中的应用进展。从而引出本论文的研究出发点和研究方向,为发展新的糖蛋白质组学分离富集新方法新技术提供理论依据和实际意义。第二章中介绍了一种基于硼酸功能化的磁性纳米材料Fe3O4@SiO2-APB和聚合物纳米微球PMMA协同作用,富集糖肽和质谱鉴定的新方法。同时采用两种不同纳米材料,利用它们与糖肽和非糖肽之间作用机理的不同,对糖肽进行富集这一想法,在糖蛋白质组学领域中尚属首次。而且,在协同富集糖肽这一新方法中,由于硼酸功能化材料针对糖肽进行富集,另一种聚合物纳米材料是专门针对非糖肽的,根据协同作用的原理,只要找到二者的最佳比例,就可轻松分离糖肽与非糖肽,无需传统方法的清洗步骤,简化实验操作,减少样品损失,达到高效分离富集,一举三得。由于材料都是纳米材料,在溶液中具有很好的分散性；比表面积大,可富集/吸附更多的目标肽段；磁性内核可通过外加磁场实现轻松分离；由于聚合物材料对非糖肽的吸附,富集之后无需清洗,避免样品损失,简化实验操作。我们将材料成功用于仅1μL的人血清样本中,成功鉴定到了66个糖蛋白,实现了快速高效富集。第三章中,我们合成了一种苯胺基团修饰的磁性纳米材料Fe3O4@SiO2@Aniline,用于富集鉴定N-糖基化蛋白/肽段。如前所述,肼化学法是一种特异性很高的糖肽富集方法,原理是将糖链上的羟基氧化成醛基后利用肼基团与之反应,将糖肽富集出来,但是其缺点是材料价格昂贵、富集灵敏度低、反应时间很长。我们受此启发,找到了一种能与糖链氧化后的醛基发生反应的官能团一苯胺基团。在之前的糖链质谱鉴定研究中,常利用还原胺化反应,即利用氨基与糖链氧化后的醛基反应,提高糖链在质谱中的检测灵敏度。但是这一反应需要使用还原剂如氰基硼氢化钠等,操作繁琐且不符合绿色化学的要求。因此,我们发展了一种基于非还原胺化反应原理的富集新方法,使苯胺功能化的磁珠与经高碘酸钠氧化后的糖肽混合孵育,清洗后,用PNGase F酶切糖苷键,释放糖肽进行分析鉴定。此方法无需对氧化后的糖肽进行除盐,亦无需使用还原剂对产物进行还原,反应时间大大缩短至2小时,灵敏度比肼化学法更高,且材料制备简单、廉价易得。最后我们将最优化的富集条件用于鉴定5μL人血清中的N-糖基化蛋白/肽段,成功鉴定到80个糖蛋白,为糖基化修饰大规模的鉴定提供了一种有效的新方法。更多还原

【Abstract】 This thesis presents an interdisciplinary research involved in analytical chemistry, chemical biology, materials chemistry as well as relevant biochemical techniques. Based on the biological mass spectrometry (MS) and a series of newly-designed magnetic nano-composites and polymer nano-materials, the high efficient and high sensitivity enrichment and identification methods have been developed and successfully used in analysis of complex samples such as human serum.As one of the most important and universal protein post-translational and modifications (PTMs), glycosylation is widely existed in more than 50%of all proteins. It plays a significant role in differentiation, development, the metastasis of tumor, immunity, infection and regeneration. It is also closely related to the development of some diseases, such as the early diagnosis of tumor, process monitoring and prognosis evaluation. Therefore, the research on glycosylation has special biological significance and clinical value and has always been the hotspot in the field of proteomics. However, due to the low abundance of glycoproteins, it’s difficult to obtain enough quantity of the samples to further analysis unless the application of pre-enrichment. What’s more, there are enormous number of proteins and extremely wide dynamic range in the very complex biological samples, it’s really a challenge to separate glycoproteins from others. Therefore, the high abundant nonglycosylated proteins would suppress the identification of low abundance glycoproteins seriously. Moreover, glycosylation has the unique characteristic of microheterogeneity which leads to the vast diversity of glycan structures and the difficult determination of glycosylation sites.In MS analysis of glycosylation, we usually adopt the "bottom up" method which means studying it on peptide level. However, the abundance of glycopeptides accountes for only 2-5% in total when glycoprotein is digested into peptides. And the MS signals of nonglycosylated peptides always heavily interfere with those of glycopeptides. As a consequence, the pre-enrichment of glycoproteins/glycopeptides from complex biological samples with high selectivity and high sensitivity is of primary importance.Nowadays, continuous efforts have been made to develop strategies for separation of glycoproteins/glycopeptides from complex biological samples, but any of them are far from perfect. For example, boronic-acid based enrichment method is unbiased to both N-and O-glycoproteins/glycopeptides and facile to handle but has poor specificity. While hydrazide chemistry method owns the best performance of specificity but the poor performance in sensitivity and reaction time, the hydrazide materials are also expensive. Furthermore, the contaminants which were brought during the pretreatment would interference with MS analysis so that desalting or washing is needed. This process would cause loss of samples inevitably. In this thesis, we aimed to develop novel methods to address the above mentioned challsenges.In the first chapter, we give a brief introduction of glycosylation, the proceedings and challenges of glycolproteomics and the application of nano-materials in glycoproteins/glycopeptides enrichment. All of the background information provides theoretical and practical support on our research.In the second chapter, we introduce a new method for highly efficient and specific enrichment of glycopeptides using two different nanomaterials synergistically. It’s the first time to use two different nanomaterials for enriching glycopeptides in glycoproteomics field. One is boronic-acid functionalized nanoparticles Fe3O4@SiO2-APB to enrich glycopeptides, the other is conventional poly(methyl methacrylate)(PMMA) nanobeads which have strong adsorption towards non-glycopeptides. By optimizing the proportion of these two materials, extremely high sensitivity and selectivity are achieved. Since the washing step is not necessary for these conditions, the enrichment process is simplified and the recovery efficiency of target glycopeptides reaches 90%. The superiority of this technique also comes from the good dispersibility and high specific surface area of nanoparticles and easy separation by external magnetic field. Of course, we applied it successfully into characterization of glycoproteome of human serum samples. Finally,147 different N-glycosylation peptides within 66 unique glycoproteins are identified in the sample volume as little as 1μL.In the third chapter, aniline functionalized magnetic nanoparticles Fe3O4@SiO2@Aniline has been successfully synthesized for the isolation of glycopeptides. Due to the unsurpassable specificity of solid phase extraction by hydrazide chemistry, we have been inspired by its mechanism and develop a novel technique based on the conjugation of aldehydes from oxidized glycopeptides and aniline group functionalized on the magnetic nanoparticles via non-reductive amination reaction. Compared with the hydrazide chemistry method, desalting is no longer needed which facilitates the realization of high sensitivity. Through the formation of stable Schiff base or glycosylamine, the reducing regent is also eliminated in this method. The whole experiment can be completed in just 2 hours. In addition, aniline functionalized nanomaterial is easily prepared and cheap. At last, we use this technique to map as much as 80 N-linked glycoproteins in only 5μL human serum sample.In summary, these two brand-new protocols have shed new light upon the study of glycoproteomics.更多还原