【作者】 沈雯雯；

【导师】 陆豪杰；

【作者基本信息】 复旦大学 ， 化学生物学， 2010， 博士

【摘要】 本博士学位论文的主要贡献在于：针对蛋白质组学分离富集技术中的三大难点——低丰度蛋白、糖基化后修饰蛋白和高分子量蛋白的分离分析,开创性地设计合成了一系列无机/聚合物纳米复合材料[SnO2@PMMA, TiO2@PMMA, ZnO@PMMA, SnO2@Poly(HEMA-co-St-co-VPBA)],发展了一种新型凝胶电泳技术(HEAG/PAM),实现了蛋白质高效率分离富集和高灵敏质谱鉴定,并将这些新技术新方法成功应用于人类大肠癌的生物靶标研究。过去的二十年,也是蛋白质组学发展的二十年。这项依附于迅速革新的生物质谱技术及生物信息学的学科越来越被发展和认可,并被广泛地应用于临床疾病生物标志物的筛选和研究,在临床早期诊断中发挥着重要的作用。然而,蛋白质组学中所运用的分离富集技术相对于快速发展的分析仪器技术来说,显得过于缓慢。传统的蛋白质分离富集方法已经无法满足日益增长的技术需求,尤其体现在低丰度蛋白、糖基化后修饰蛋白和高分子量蛋白的分离分析方面。因此,发展蛋白质分离富集和质谱鉴定新方法新技术,是目前蛋白质组学研究中的一个重要方向。首先,低丰度蛋白的分析与鉴定是蛋白质组学研究的重点和难点之一。与疾病相关的蛋白质往往分布在低丰度蛋白质的范围内,这些低丰度蛋白质是执行重要生物功能的蛋白质,如调控蛋白、信号传导蛋白和受体蛋白等。主要是由两方面因素导致低丰度蛋白鉴定困难：一方面,在质谱分析前处理过程中,由于缺乏高效的浓缩技术而导致样品损失,从而使得上样量达不到鉴定要求；另一方面,在样品前处理过程中,一些对质谱信号有抑制作用的小分子如无机盐类、离液剂、表面活性剂(去垢剂)等会吸附在样品上,与多肽/蛋白样品同时进入质谱检测,严重干扰多肽/蛋白样品的出峰。因此,发展低丰度蛋白的富集和除盐技术,是蛋白质组学的一项重要研究课题。其次,在已知的400多种翻译后修饰中,糖基化是目前研究的重点之一。蛋白的糖基化调控着细胞分子机制,包括细胞黏附、受体激活、信号传导、分子运输和清除、胞吞作用。很多疾病与蛋白的糖基化或者糖苷酶的缺失有关,例如自身免疫失调或者婴儿初期癫痫症。美国食品与药物管理局(FDA)目前确认的癌症靶标有半数是糖蛋白。越来越多基于质谱的糖蛋白分析策略被不断发展和提出,用以确定糖基化位点信息和糖链信息。但是,糖蛋白的位点相对不多,带有糖链的多肽(称为糖肽)在整个蛋白酶解液中所占很少,并且这些糖肽的质谱信号也会受到同时存在的大量的非糖肽信号的抑制,导致鉴定困难。糖链结构的确定也是另一难点,这是因为单糖的类型多样且连接方式多样,因此很难通过一级质谱上简单的分子量差值比对来确定复杂的天线结构。因此,如何选择性地富集糖肽且实现多级质谱确定糖链结构是目前糖蛋白分析的研究方向。最后,高分子量蛋白的分离分析受到现有技术的制约。高分子量蛋白质(分子量大于100 kDa)在生物体中执行非常重要的生物功能,如参与细胞骨架组成、免疫应激功能、参与转录及在高等真核生物体内发生翻译后修饰。并且,高分子量蛋白质和很多重大疾病相关,如迪谢纳-贝克肌肉萎缩症、原发性心肌炎等。目前高分子蛋白质组学的研究一个关键的难点在于缺乏从复杂样品中分离高分子量蛋白的有效方法。适用于分离较大蛋白的琼脂糖凝胶在大规模高分辨分离方面表现很差,因此需要发展新的有效的分离技术。高分子量蛋白在MALDI质谱分析中的鉴定率低是另一个不利于高分子量蛋白组学研究的主要因素。一方面,高分子量蛋白质具有很长的多肽链,增加了质谱污染物如无机盐、离液剂和表面活性剂在前处理过程中被蛋白表面吸附的可能性。而且,高分子量蛋白往往会有更多的疏水性肽段,在使用商品化的MALDI基质如CHCA时,这些疏水性肽段的离子化效率较差。因此,在鉴定高分子量蛋白之前,较之其他蛋白需要增加除盐和质谱信号增强的相应技术。针对以上蛋白质组学分离分析三大问题,本博士学位论文分为五章节开展研究工作。各章节主要内容如下：第一章是绪论部分。本章主要论述蛋白质组学的发展现状,蛋白质组学分离富集技术的发展现状和遇到的挑战,以及纳米材料在蛋白质组学方面的应用进展。从而引出本论文工作的研究方向,为设计发展蛋白质分离富集的新方法新思路提出理论依据和实际意义。第二章主要介绍低丰度蛋白质的纳米核壳材料SnO2@PMMA和TiO2@PMMA富集和质谱鉴定的新方法研究。我们设计合成了含无机纳米粒子核的高分子微球SnO2@PMMA和TiO2@PMMA。这种方法结合了PMMA与蛋白质、多肽间的亲和力、高分子微球在水中的悬浮能力以及无机纳米粒子与高分子壳的较强作用力三大特点,并且在合成过程中避免了表面活性剂的使用。PMMA微球可以稳定悬浮于水中并有较大的表面积从而大量捕捉溶液中的目标分子,吸附后的材料可从样品溶液中分离并且能在水中再分散从而能够与MALDI靶板上的基质行成共结晶薄膜,同时微球内部纳米颗粒能够紧紧的抓住包裹的聚合材料,防止其在质谱分析中解离。我们以标准蛋白质和多肽溶液为测试样品,优化不同材料核壳的条件、富集溶剂、富集时间、离心速度、离心时间、干扰背景条件和离子化条件等,建立了复合材料进行痕量多肽或蛋白质的富集新方法。该方法采用吸附蛋白/多肽后剔除上清,实现一步富集除盐。材料富集只需10分钟,实现了高效快速富集。第三章主要介绍低丰度蛋白质的纳米核壳材料ZnO@PMMA富集和质谱鉴定的新方法研究。我们在第二章所介绍的两种复合纳米材料的基础上,又设计合成了一种含量子点无机纳米粒子核的高分子微球ZnO@PMMA。这种新型材料的优势在于,一方面利用高聚物的强吸附能力来吸附肽段,从而实现肽段的富集,另一方面利用纳米粒子在所用MALDI激光吸收波长条件下有较强的吸收强度及有效的传递给分析样品能量的能力这一特殊的光电性能来增强质谱信号。这种材料不仅可以富集低丰度蛋白肽段,还可以对质谱信号起到增强的作用,从而全面提高低丰度蛋白的检测限。这种材料吸附能力很好,操作简单易行,操作过程基本上实现低丰度蛋白的零损失。我们对该材料的富集除盐步骤进行了优化,目前对于极低浓度的体系(1amol/μL),可达到的富集效率在2个数量级；低浓度的体系(1fmol/μL)的富集效率则在3-4个数量级。在除盐的功能上,该复合纳米材料更显示了强大的脱盐功能,不仅在高浓度的NH4HCO3、NaCl、Urea和KCl中实现高效富集和脱盐,而且能够避免一定量的SDS等表面活性剂的干扰,可实现忍耐饱和NaCl体系(6.2 M)的盐的干扰。我们将此材料成功地应用于人类大肠癌的低丰度蛋白鉴定,鉴定到超过70个偏低丰度蛋白,其中有8个与癌症相关的蛋白第一次在大肠癌中被报道。第四章主要介绍基于新型凝胶电泳HEAG/PAM的高分子量蛋白质组分析新方法研究。我们发明了一种新型凝胶作为电泳支持介质,并设计了针对高分子量蛋白质组学的分析策略,即基于该新型凝胶分离、结合纳米复合材料ZnO@PMMA质谱鉴定的分析路线。该分离凝胶是一种乙基羟基化的琼脂糖和聚丙烯酰胺的混合凝胶,其特点在于既具有琼脂糖凝胶平均孔径大、分辨率高、机械强度好的特点,又具有聚丙烯酰胺凝胶质谱兼容型的特点,最重要的是采用了衍生化的琼脂糖代替普通琼脂糖,熔点和凝固点都大大低于普通琼脂糖,便于操作和充分混匀,适用于普通薄板垂直电泳,可形成均一的、重现性好的混合凝胶。与此同时,我们使用纳米复合材料ZnO@PMMA对蛋白进行富集除盐和MALDI质谱信号增强,解决了高分子量蛋白由于量少、易带质谱污染物及疏水性肽段较多引起的MALDI质谱鉴定困难的问题。第五章主要介绍糖蛋白的纳米核壳材料SnO2@Poly(HEMA-co-St-co-VPBA)富集和质谱鉴定的新方法研究。我们利用硼酸根在一定的PH条件下与顺势二羟基发生可逆反应的原理,设计合成了富含硼酸根官能团的无机/聚合物纳米复合材料,在实现对糖肽的高效选择性富集的同时,利用多级质谱实现对糖链的解析。该功能化纳米复合材料的合成过程较之以往的化学合成方法更简单,主要包括三个步骤：水热法合成纳米SnO2核,SnO2表面和HEMA发生羟基交换反应,以及VPBA、PS和修饰了HEMA的SnO2发生自由基共聚反应。整个合成过程不到3个小时,比起以往的需要16个小时以上的化学合成引入硼酸根的方法更加高效。实验表明,这种方法合成的含硼酸根功能团的材料较之之前的材料来说,表面硼酸根含量更高,用于糖肽富集时检测限减低了5倍。并且,我们将此含硼酸根功能团的纳米复合材料成功应用于人类血清样本中的糖蛋白研究,成功富集并解析了血清样本中的结合珠蛋白和酸性糖蛋白的糖肽。更多还原

【Abstract】 There are three main academic values of this doctoral thesis. First, we have developed a new method for enriching and desalting low-abundant proteins based on three kinds of newly designed core-shell nanobeads subsequently. Second, we have designed core-shell boronic-acid functionalized nanoparticles for selectively enriching glycopeptides, followed by multistage MS to characterize glycosylation sites and glycan structures simultaneously. Third, we have established a new mass spectrometry based analysis strategy for high-molecular-weight (HMW) proteome research, which contains a new gel enhanced electrophoresis separation and core-shell nanobeads assisted identification.Nowadays, three challenges in proteomic have been faced.First of all is low-abundant protein research. In the clinical proteome research, disease associated proteins, which would be biomarkers, expressed commonly in low abundance. Identification of low-abundant proteins by mass spectrometry (MS) is still a challenge due to the sample loss and contaminants interference with MS during the sample pretreatment. Several types of nanomaterials, which have been developed to enrich and desalt peptides/proteins from mixtures, still have some drawbacks in practical applications.The second challenge is analyzing glycoprotein. Protein glycosylation, as one of the most important posttranslational modifications (PTMs), regulates cellular mechanisms, including cell adhesion, receptor activation, signal transduction, molecular trafficking and clearance, and endocytosis. Many diseases are caused by glycosylation or glycosidase deficiencies, such as autoimmune disorders, infantile-onset symptomatic epilepsy. In fact, the US Food and Drug Administration has agreed that over half of the recent cancer biomarkers are glycoproteins. To identify glycosylation sites, as well as the primary structures of the glycans, mass spectrometric strategies have been developed. However, as glycopeptides are always low in abundance when the glycoprotein is digested into peptides, the MS signals of nonglycosylated peptides always heavily interfere with those of glycopeptides. Moreover, determination of glycan structures is usually difficult because of their vast diversity. Therefore, selective enriching methods followed by multistage MS technique are necessary for glycopeptide analysis. Last but not the least, high-molecular-weight (HMW) proteome research is still a challenge. Proteins with the molecular weights above 100 kDa, which are commonly defined as HMW proteins, are known to be involved in a number of human diseases and some of them have been approved as cancer biomarkers, such as CA125 for monitoring ovarian cancer in serum, HMW CEA and mucin for monitoring bladder cancer in urine. One bottleneck is lack of highly efficient separation of HMW proteins from complex real samples. Low success rate of identification is the following problem for the HMW proteome analysis. The MS signals could be suppressed by the contaminants due to the long polypeptide chains of HMW proteins, which would be easily attacked by inorganic salts, chaotropes and detergents during separation procedure. Besides, a large number of hydrophobic peptides of the HMW proteins make their ionization in matrix-assisted laser desorption/ionization (MALDI)-systems difficult by using the commercial organic matrices, such as a-cyano-4-hydroxycinnamic acid (CHCA). Therefore, extra two steps for HMW protein identification are needed, including desalting and signal enhancement.In this thesis, we introduce our researches in five chapters.In the first chapter, we give a brief introduction on the proceedings and challenges of proteomics and its related technolegies. Furthermore, we review the nanomaterials and their applications in proteomics. All of the background information provides theotical and applicable support on doing researches.In the second and third chapters, we introduce a developed method based on three kinds of core-shell nanobeads, which were designed for enriching and desalting low-abundant proteins with low limits of detection, rapid enrichment, good reproducibility, high recovery, and powerful desalting ability. These core-shell nanobeads were synthesized without using surfactants. For the SnO2@poly (methyl methacrylate) (PMMA) and TiO2@PMMA nanobeads synthesis, SnO2 and TiO2 nanoparticles modified with poly (ethylene glycol) methyl ether (PEGME) were synthesized by solvothermal methods to prevent them from aggregation firstly, hydroxy-exchange reactions then took place on the PEGME-SnO2 and PEGME-TiO2 surfaces, and MMA monomers were simultaneously polymerized in boiling water to form PMMA beads. After the two nanobeads enrichment procedure, the MS signal intensities and the corresponding signal-to-noise (S/N) ratios of the intact horse heart myoglobin (MYO,400 fmol/μL) were increased by one order of magnitude, while those of the digested MYO (1 fmol/μL) were enhanced by three orders of magnitude. For the ZnO@PMMA nanobeads, the difference from the uppers in synthesis was that ZnO-cores had been prepared through polymerization of methyl methacrylate initiated by the inherent free radicals on the ZnO surface. These optimized ZnO@PMMA nanobeads displayed more powerful enriching and desalting abilities:-80% bovine serum albumin digests were enriched by ZnO@PMMA from 100 amol/μL solution within 10-min incubation; high-quality mass spectra were obtained, even with the presence of saturated NaCl (6.2 M), saturated NH4HCO3 (2.6 M), or 1 M urea. This method was successfully applied to human colorectal cancer proteome research, and eight new proteins have been found.In the fourth chapter, we introduce a new mass spectrometry based analysis strategy has been established here for high-molecular-weight (HMW) proteome research. First, a 2-hydroxyethyl agarose/polyacrylamide (HEAG/PAM) electrophoresis gel was designed for the first time to realize an easy-handling separation method with high spatial resolution for HMW proteins, good reproducibility and mass spectrometry-compatible sliver staining. Second, ZnO@PMMA nanobeads were applied here for enriching and desalting the peptides from the HMW proteins. Third, the peptides were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) with the presence of the ZnO@PMMA nanobeads, and their MS signals were enhanced markedly. The success rate of identification for HMW proteins was significantly increased due to high enriching efficiency and salt tolerance capability as well as signal enhancing capability of the ZnO@PMMA nanobeads. We believe that this analysis strategy will inspire and accelerate the HMW proteome studies.In the fifth chapter, we introduce core-shell boronic-acid functionalized nanoparticles SnO2@Poly(HEMA-co-St-co-VPBA) designed for selectively enriching glycopeptides, followed by multistage MS analysis. Such 60-nm sized core-shell nanoparticles are prepared bv means of copolymerization between 2-hydroxyethyl methacrylate (HEMA) grafted on SnO2 nanoparticles, styrene and 4-vinylphenylboronic acid (VPBA). All of the synthesis procedures are completed within 3 h. Cyclic boronate esters form between boronic-acid groups on the polymer chains and cis-diol groups on glycopeptides, and thus almost all intact glycopeptides from low-abundant horseradish peroxidase (HRP) and bovine asialofetuin (ASF) are enriched with high selectivity and efficiency. After enrichment, both intact N-and O-glycopeptides are characterized by multistage MS. Furthermore, we successfully apply this method to the human serum sample for characterizing the target glycoproteins haptoglobin and alpha-1-acid-glycoprotein. The present selective enriching method followed by multistage-MS analysis is proved to be a good choice for routine glycopeptide characterization.更多还原