【作者】 王秦哲；

【导师】 李铮；

【作者基本信息】 西北大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 研究背景：肝细胞癌是最常见的成人肝脏原发性肿瘤、世界第五大实体瘤和第三大癌症死亡原因,肝癌细胞系HepG2和正常肝细胞系L02是研究人肝细胞极有价值的模型,并得到广泛应用。异常糖基化在癌症发生发展中的重要作用已逐渐得到认识,然而,异常糖蛋白在肝细胞癌中的作用还未阐明。本研究运用糖蛋白质组学研究手段鉴定细胞全N-糖蛋白质及糖基化位点,并运用酰肼化学唾液酸化糖肽选择性提取方法初步研究了细胞样品中唾液酸化N-糖蛋白质及唾液酸化位点。方法：酰肼化学固相萃取方法和亲水相互作用固相萃取方法能够高效富集糖肽,且两种方法具有互补性。富集到的糖肽经PNGase F去糖基化、串联质谱鉴定糖肽和糖基化位点。利用一系列生物信息学工具分析癌症细胞与正常细胞糖蛋白差异。结果：实验结果分为两部分：(1)从HepG2和L02细胞中共鉴定到394个糖肽,代表235个糖蛋白,包含401个糖基化位点。酰肼方法在鉴定糖蛋白数目和富集特异性上高于亲水方法,且两者存在很好的互补性。两种方法每个糖蛋白平均鉴定到1.6个糖肽。按照ratio大于2和小于0.5统计,两种方法从HepG2和L02均鉴定到的170个糖肽中,21个表达上调,17个表达下调。与L02细胞相比,HepG2细胞糖蛋白糖结合活性、受体活性较高,控制生物学质量和细胞加工过程、参与细胞分化过程、信号转导、信号传递和信号处理的糖蛋白有所增加。两种细胞鉴定的糖蛋白在KEGG通路数据库中对应139条通路,其中细胞外基质受体通路、癌症通路、黏着斑、吞噬体通路、细胞粘附因子、血细胞生成通路、内质网蛋白加工通路、肌动蛋白细胞骨架调节通路、蛋白消化和吸收通路匹配较高。鉴定到木糖基转移酶、UDP-葡萄糖醛酸转移酶A1、糖基转移酶25和前胶原赖氨酸2-氧化戊二酸5-双加氧酶3这4个O-糖链合成通路酶。(2)从HepG2和L02细胞中共鉴定到60个唾液酸化糖肽,64个糖基化位点,对应41个唾液酸化糖蛋白。与全糖蛋白类似,L02细胞中鉴定到的唾液酸化糖蛋白略多于HepG2细胞。但共同鉴定的唾液酸化糖蛋白只有3个。可能反映两种细胞唾液酸化模式存在较大差异。本实验中鉴定的唾液酸化糖蛋白在Uniprot数据库中标注为已知、潜在和未知的比例为58%、13%和29%。膜蛋白分别占HepG2和L02鉴定到的唾液酸化N-糖蛋白的48%和64%。两种细胞中鉴定到的唾液酸化糖蛋白均分布在细胞、细胞器、大分子复合体等位置。HepG2和L02细胞鉴定到的糖蛋白的分子功能方而主要集中在结合活性、催化活性和分子转导活性。更多还原

【Abstract】 Background:Hepatocellular carcinoma is one of the most commonly human cancers and the third cause of death in the world. The cancer cell line HepG2and normal hepatocellular cell line L02are valuable cell models and are already widely used in the world. The important role of abnormal glycosylation in the development and progressing of cancer is more and more recognized in different cancers, while it is not fully revealed in hepatocellular carcinoma. This research focuses on identifying N-linked glycoproteins in HepG2and L02cells by mass spectrometry based glycoproteomics.Method:Hydrazide chemistry and Hydrophilic Interaction methods are two complemental methods which can isolate and enrich glycopeptide in the high throughput manner. After releasing of glycan by PNGase F, the enriched formerly glycopeptides are identified by LC-MS/MS as well as their glycosylation sites. Later on, we use a series of bioinformatic tools to uncover the difference of glycoproteins between cancer cell line and normal cell line.Result:We obtained the following results:Part1:394N-linked glycopeptides, containing401glycosylation sites and representing235glycoproteins, were identified from HepG2and L02cells. More glycopeptides were identified by Hydrazide chemistry method than by Hydrophilic Interaction method. More glycopeptides were identifed when combining the two methods.21of170glycopeptides identified by both Hydrazide chemistry and Hydrophilic Interaction methods in both HepG2and L02cells were up-regulated between HepG2and L02cells according to spectral counts.17glycopeptides were down-regulated. Gene ontology analysis showed that carbohydrate binding ability and recepter ability were higher in glycoproteins of HepG2cells, as well as elevated glycoproteins which may participate in biological quality, cellular process, developmental process, signal transduction, and cellular homeostasis.139pathways were mapped in KEGG database. ECM-receptor interaction, pathways in cancer, focal adhesion, phagosome, cell adhesion molecules, protein processing in endoplasmic reticulum, regulation of actin cytoskeleton and protein and absorption are the highest mapped pathways. Four enzymes, glucoside xylosyltransferase1, UDP glucuronosyltransferase1family Al, glycotransferase25domain containing1and procollagen-lysine-2-oxoglutarate5-dioxygenase3, in O-glycan biosynthesis were also identified by this research.Part2:60glycopeptides bearing sialic acids, with64glycosylation sites, were identified by a modified Hydrazide chemistry method. Like the whole glycopeptide extraction method, sialic acid focused method identified more glycopeptides in L02cells than in HepG2cells. But only3glycoproteins with sialic acids were identified in both the two cell lines, which may reflect a major change of sialic acid in glycoprotein or due to the relatively small list of identification.58%,13%and29%identified glycoproteins with sialic acid were recorded as Known, potential and no record in Uniprot database, respectively. Most of glycoproteins identified in both cells have binding ability, catalytic ability and molecular transduction ability.更多还原