【作者】 曹晶；

【导师】 杨芃原；

【作者基本信息】 复旦大学 ， 化学生物学， 2009， 博士

【摘要】 本博士论文工作的主要贡献为:针对分泌蛋白质组研究的难点问题,开展了一系列的研究。发展了一种快速、简单、高效和普适性的富集方法来浓缩纯化大体积高盐体系中的分泌蛋白质,并且在肝癌细胞的分泌蛋白质组研究中得到了很好的应用;通过特别设计的实验来富集大体积溶液中的低分子量蛋白质,富集到的蛋白质分子量最小只有4 KDa;建立了具有高转移潜能的人肝癌细胞分泌蛋白质组表达谱数据库,这一数据库对肝癌转移相关研究具有很高的价值。应用糖蛋白质组学的方法,对分泌蛋白质的糖基化位点进行了高通量研究,鉴定到了172个新位点,包括CD44和laminin等与肝癌转移密切相关的蛋白质的新位点信息。利用这种方法,获得了高纯度的分泌蛋白（高达96.4%）,解决了分泌蛋白质研究中的污染难题。应用生物信息学分析方法对肝癌细胞分泌蛋白质组数据进行了分子功能、信号通路和蛋白质相互作用等深入的数据分析和挖掘;针对糖基化位点鉴定的可靠性问题,对基于¹⁶O/¹⁸O标记的糖基化位点进行分析比较,发现经两种标记鉴定到的糖基化位点一致性很好,首次用实验数据回答了糖基化位点研究中常见的一个问题。分泌蛋白质被认为包含了疾病早期检测和诊断的标志物,正吸引着越来越多的科研工作者的目光,近年来,分泌蛋白质组研究也取得了长足的进步。除了比较正常的和疾病的,或者不同生理状态的分泌蛋白质的表达差异的研究外,分泌蛋白质的数据库的建立也备受关注。然而,分泌蛋白质组研究存在下面四个亟待解决的问题:1)分泌蛋白容易被胞内蛋白污染,难鉴定到纯的分泌蛋白;2)鉴定低丰度和低分子量的分泌蛋白质还具有很大的挑战;3)有关分泌蛋白质的翻译后修饰研究开展得很少;4)对鉴定到的重要分泌蛋白质的功能研究开展得很少。本论文针对上述难题,开展了一系列的研究,获得了满意的结果。由于蛋白质糖基化在许多重要的生理和病理过程中起着重要的作用,近年来,蛋白质糖基化修饰研究也备受关注。由于糖蛋白/糖肽富集技术和生物质谱技术等的发展,大规模的糖蛋白质组学研究得以实现,尤其对N-型糖蛋白的糖基化位点的鉴定已经积累了不少研究成果。N-型糖蛋白具有和经典的分泌蛋白一样的基于内质网-高尔基体的生物合成转运路径,经典的分泌蛋白几乎都是N-型糖蛋白。基于此,本论文对肝癌细胞分泌蛋白质的糖基化位点进行了大规模的鉴定,对其糖型进行了初步分析。肿瘤相关的研究尽管备受重视,也取得了相当多的成绩,但是肿瘤如何蔓延并破坏它们的宿主器官仍然是一个谜。因实体瘤（包括肝癌）而死亡的人数的90%是由于肿瘤转移造成的,因此,肿瘤转移的问题越来越受到重视。肝癌是一种常见的恶性肿瘤,在我国,肝癌是癌症中的第二大杀手。为了更好的研究肝癌转移的机制,复旦大学中山医院肝癌研究所建立了人肝细胞癌转移裸鼠模型和具有不同转移潜能的细胞模型。本论文的研究以其中具有高转移潜能的HCCLM3和转移潜能相对低的MHCC97L为材料。研究主要是将蛋白质组学和糖蛋白质组学方法应用于肝癌细胞分泌蛋白的研究,在肝癌及其转移的基础研究中获得了非常丰富的蛋白质组信息。本论文由七部分组成。第一章绪论首先概述了分泌蛋白质组研究的七个方面,及其研究进展和研究瓶颈;综述了蛋白质糖基化修饰研究现状,尤其对糖蛋白/糖肽的分离富集研究进展做了很好的综述;概述了肝癌及其转移的蛋白质组学研究进展,说明了本论文选题目的和意义。第二章所述工作对肝癌细胞分泌蛋白进行了初步研究。在第一节,对常用的三种浓缩纯化分泌蛋白质的方法（超滤法,透析法,沉淀法）进行了比较研究,实现了研究的基本目的:探索肝癌细胞系LM3在条件培养基中的最佳培养方法;比较不同的浓缩纯化分泌蛋白质的方法;初步了解转移潜能高的肝癌细胞分泌蛋白质组;找到目前分泌蛋白质组研究的瓶颈;为后续分泌蛋白质组研究打下基础。第三章所述工作发展了一种快速、简单、高效的和普适性的富集方法来浓缩纯化分泌蛋白质。首次采用纳米LTL沸石材料富集大体积培养基中的分泌蛋白,使用SDS-PAGE和Tricine-SDS-PAGE（小分子电泳胶）进行一维电泳分离,切取蛋白条带进行胶内酶解,再进行在线的HPLC-ESI-MS液质联用分析。共鉴定到1474个非冗余蛋白,其中有97个蛋白质的分子量小于15 KDa,这些分子在分泌蛋白质组研究的相关文献中鲜有报道。鉴定到的蛋白质很多是与肝癌的发生发展和侵袭转移密切相关的,如甲胎蛋白（alpha-fetoprotein,AFP）是被广泛应用的肝癌的生物标志物,骨桥蛋白（Osteopontin,OPN）能够促进肝癌细胞的侵袭,基质金属蛋白酶（matrix metalloproteinase,MMPs）是肝癌细胞破坏基底膜,进行侵袭转移的重要分子。结果表明,分泌蛋白质组研究是寻找疾病生物标志物的有效方法,纳米沸石LTL富集分泌蛋白,接1D SDS-PAGE-LC-ESI-MS/MS分离鉴定,是研究分泌蛋白质组的有效的方法。尤其较传统的浓缩纯化方法,纳米沸石富集法是更灵活的方法,它可以特别地、有效地富集低分子量蛋白质,富集鉴定到的蛋白质中,分子量最小的只有4 KDa。第四章所述工作对肝癌细胞分泌蛋白质的糖基化修饰进行了研究。在第一节,研究了肝癌细胞分泌蛋白质的糖基化位点。首先采用亲水法和肼化学法这两种互补的方法富集糖肽,结合nanoLC-ESI-MS/MS分离分析,对分泌蛋白质的糖基化位点进行了高通量研究,鉴定到了194个糖蛋白的300个糖基化位点,其中172个是新位点,包括CD44和laminin等肝癌转移相关重要蛋白的新位点信息。利用这种方法,获得了高纯度的分泌蛋白（高达96.4%）,解答了分泌蛋白研究中的污染问题。首次通过实验直接比较这两种方法。就对糖肽的选择性而言,肼化学法优于亲水法,选择率分别是92.9%和51.3%,然而,就富集鉴定到的糖基化位点而言,亲水法优于肼化学法,分别鉴定到265个和159个糖基化位点。这些发现为今后糖蛋白质组学研究的方法选择提供了很好的参考。在第二节,使用多种凝集素印迹,对肝癌细胞分泌蛋白质的糖型进行了初步的分析,初步比较了具有不同转移潜能的人肝癌细胞（HCCLM3和MHCC97L）分泌蛋白的糖基化差异,为后续研究打下了基础。第五章工作主要是整合了第二、第三和第四章肝癌细胞分泌蛋白质组数据,利用Ingenuity Pathway Analysis（IPA）等软件对整合数据进行了深入的数据挖掘。了解了其可能的亚网络构成和相互作用,其介导肝癌细胞间,胞外与胞内相互作用,及肝癌细胞分泌蛋白参与的重要的信号通路。了解了其重要的节点分子及其与相互作用分子的联络,以便指导进一步研究的开展。第六章工作主要是针对糖基化位点鉴定的可靠性问题,对基于¹⁶O/¹⁸O标记的糖基化位点进行分析比较。发现经两种标记鉴定到的糖基化位点一致性很好,证明在富集糖肽后进行位点标记,基于¹⁶O标记的结果是可信的。基于¹⁶O标记的糖基化位点鉴定结果是否可靠和基于¹⁸O标记是否具有很大的优势是常见的问题,无疑,本章工作首次基于实验数据很好地回答了这一问题。第七章是全文总结和展望。总结了主要的研究结论,提出了进一步的研究设想。更多还原

【Abstract】 The main contributions of this dissertation were: to solve the difficulties on secretome research, a series of studies have been conducted. For the enrichment of secretome in large volume of growth media rich in salt, a simple, fast, effective and universal approach was developed and applied well on secretome research of HCC cells. And highly-efficient enrichment for the low molecular weight proteins （LMWPs） in secretome has been achieved via a specially designed experiment. Notably, the smallest MW touched 4 KDa. A secretomic profile of HCC cells was built up, and this database would exhibit valuable information in the research of HCC metastasis. A large-scale detection of glycosylation sites of secreted proteins was successfully performed. 172 new N-glycosites were determined experimentally, including the new N-glycosites of CD44 and laminin etc, which have been reported to be implicated invasion and metastasis of HCC. Importantly, the utility of N-glycoproteomic strategy has been proved to be a great way to profile the genuinely secreted proteins. The fact that 96.4% of the identified glycoproteins could be secretory proteins positively confirmed this good point. To get the information including molecular function, signal pathways and protein-protein interactions, bioinformatic analyses on the secretome identified were conducted deeply. To exam the reliability of glycosites identified according to the mass increased of 1 Da (¹⁶O labeled) or 3 Da (¹⁸O labeled), the excellent consistence of glycosites from two labeled was founded. Thus, a frequently asked question was answered by experimental data for the first time.Given the importance of secreted proteins as a source for early detection and diagnosis of disease, secreted proteins have been arousing considerable attentions, and the field of secreted proteome （secretome） has achieved substantial advances during recent years. In addition to the comparative proteomic analyses focusing on the characterization of differentially expressed proteins in two different, well controlled states, e.g. normal and disease, the global profiling of secretome received considerable attraction. However, the analysis of secreted proteins represents some challenges in four aspects as following. 1) The contamination of the authentic secreted proteins is unavoidable, resulting in severe interferences for the identification of secreted proteins; 2) those proteins with low molecular weight or with low abundance are difficult to be discovered; 3) little attention has been paid to the post-translational modifications （PTMs） of the secretory proteins; 4) the functional investigation of those detected secreted proteins has been also paid little attention. To find solutions for the questions mentioned above, a series of researches have been performed and some satisfactory results have been achieved.Due to vital roles of glycosylation of proteins in lots of important physiological and pathological processes, interest in investigating glycosylation status of glycoprotein has greatly increased in recent years. Because the great progresses of techniques including enrichment methods for glycoproteins/glycopeptides and mass techniques for biomolecules have been made, the study of glycoproteome is progressing at tremendous speeds and has achieved substantial advances during recent years, special in the large-scale detection of glycosylation sites. N-glycoproteins and classical secreted proteins have the same dependence of the endoplasmic reticulum （ER） - Golgi network, and the classical secreted proteins are almost N-glycoproteins. Thus, a large-scale glycoproteomic identification and N-glycosylation site elucidation has been performed for secreted proteins of HCC cells. Also, the glycoform has been analyzed elementally.How tumors spread and kill their host organism remains an enigma, but not for lack of attention. A renewed focus on the problem of metastasis is now apparent, and for good reason—metastasis remains the cause of 90% of deaths from solid tumors. Hepatocellular Carcinoma （HCC） is a common malignancy worldwide and is a second leading cause of death in our country. To understand better the mechanism of HCC metastasis, the human HCC metastasis nude mice model and HCC cell lines with different metastasis potential were built up by the Liver Cancer Institute of Zhongshan Hospital, Fudan University. This work is made on the two HCC cell lines （HCCLM3 and MHCC97L） with different metastasis potential. The investigation has focused on the application of proteomic and glycoproteomic methods in the secretomic research, and a lot of valuable proteome information has been obtained.This dissertation consists of 7 parts and the contents are summarized as follows:In the first chapter, the seven aspects of secretomic research were summarized, also, the research progresses and the bottle neck. In addition, the advances in the research of glycosylation were summarized, specially, in the separation and enrichment of Glycoproteins/Glycopeptides. Furthermore, a review of liver proteomic study was presented. Importantly, the aims and significance of this dissertation were presented.In the second chapter, primary research of secretome of HCC cells has been made. In the first section, three most often used concentration methods （ultrafiltration, dialysis and precipitation） were used together and compared. The merits and shortcomings of each method were discussed based on the obtained results. Four aims of this section have been achieved: the optimized method for cell culture in conditioned medium was developed; three concentration methods （ultrafiltration, dialysis and precipitation） were compared; the secretome of HCC cells with metastasis potential was studied primarily; the bottle neck of secretomic research was founded our. And all these works were basic for further research.In the third chapter, a simple, fast, efficient and universal enrichment process for secretome was developed. Nanozeolite LTL was used to capture secreted proteins for the first time. Highly-efficient enrichment for the low molecular weight proteins （LMWPs） in secretome has been achieved, and attracted most of attentions in a specially designed experiment. Followed by 1D SDS-PAGE for protein fractionation and then by LC-ESI-MS/MS for protein identification. Totally 1474 unique proteins were confidently identified and 97 proteins of 1474 proteins were those notably with MW less than 15 KDa, which were seldom captured previously by traditional methods. Notably, the smallest MW touches 4 KDa. Many proteins identified above were found to be involved in the processes associated with the cancer development and metastasis. For instance, alpha-fetoprotein （AFP）, a widely known biomarker of HCC, was detected. Osteopontin, a secreted phosphoprotein, is a significant factor in HCC metastasis, and its over-expression correlates with metastatic potential of primary hepatocellular carcinoma, and with invasiveness of liver tumor-derived cell lines in vitro. Elevated levels of matrix metalloproteinase （MMPs） have been shown in many tumors with strong association with the invasive and metastatic potentials. These observations demonstrate the contention that secretomic approach is a great way to the discovery of potential biomarkers. And the strategy that started with a capture of secreted proteins by nanozeolite LTL, followed by a separation of 1D SDS-PAGE and by an identification of LC/ESI-MS/MS has been proven to be perfect. Notably, for special enrichment of LMWPs, compared to conventional methods, this strategy was special and efficient.In the fourth chapter, glycosylation status of HCC cells was investigated. In the first section, a large-scale detection of glycosylation sites of secreted proteins of HCC cells was successfully performed. For the enrichment of glycopeptides, capture methods with hydrophihc affinity （HA） and hydrazide chemistry （HC） were used complementarity. Using both methods in combination with nano-LC-ESI-MS/MS analysis, 300 different glycosylation sites within 194 unique glycoproteins were identified, and 172 glycosites have not been determined experimentally previously, including the new N-glycosites of CD44 and laminin etc, which have been reported to be implicated invasion and metastasis of HCC. Importantly, the utility of N-glycoproteomic strategy is a great way to profile the genuinely secreted proteins. The fact that 96.4% of the identified glycoproteins could be secretory proteins positively confirmed this good point. A direct comparison between HA and HC methods was also investigated for the first time. In brief, in terms of selectivity for glycopeptides, HC is superior to HA （92.9% VS 51.3%）, however, based on the number of glycosites identified, HA outweighs HC （265 VS 159）. This result based on the compared experiment could be useful for the selection of methods. In the second section, glycoforms of secretome of HCC cells were profiled primarily by multi-lectin blot. The difference of glycosylation status of two HCC cell lines （HCCLM3 and MHCC97L） with different metastasis potential were compared primarily, and based on these results more researches would be done in the near future.In the fifth chapter, to get the information including molecular function, signal pathways and protein-protein interactions, bioinformatic analyses on the secretome identified were conducted deeply. Ingenuity Pathway Analysis system was used to excavate the secretome data from chapter two, chapter three and chapter four. The sub-network, protein-protein interactions, cell-cell interactions and crucial pathways were investigated. Also, some key node molecules were discussed. All the explorations are basic and helpful for HCC research in future.In the sixth chapter, to exam the reliability of glycosites identified according to the mass increased of 1 Da (¹⁶O labeled) or 3 Da (¹⁸O labeled), deglycosylation experiments were carried out parallelly H₂¹⁶O in or H₂¹⁸O. As a result, the excellent consistence of glycosites from two labeled was founded. Thus, the results from ¹⁶O labeled were proven to be credible, and a frequently asked question was answered by experimental data for the first time.In the last chapter, a summary was made and a prospect was presented.更多还原