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標題: | 建立二氧化鈦磁性奈米粒子純化方法以研究淋巴癌細胞之唾液酸醣蛋白體 Magnetic Fe3O4@TiO2 Core/shell Nanospheres for the Sialoproteomics Analysis in B Lymphoma Cells |
作者: | Yi-Ju Chen 陳怡儒 |
指導教授: | 陳玉如(Yu-Ju Chen) |
關鍵字: | 唾液酸醣蛋白體,二氧化鈦,磁性奈米粒子,B淋巴癌細胞, Sialoproteome,Titanium dioxide,Magnetic nanoparticles,B lymphoma cells, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 結構異常的醣基化修飾已被報導與癌症的發生及轉移極為相關,尤其是位於醣鏈最末端帶負電的單醣唾液酸,大量存在於癌症細胞中。此唾液酸(尤以聚唾液酸)在細胞中具有保護和參與細胞及細胞間結合、癌症轉移等功能。為了系統性地剖析唾液酸醣蛋白體與疾病的關係,從生物樣品中有效地純化唾液酸醣胜肽是質譜分析醣蛋白體之一重要關鍵。由近來研究指出,利用帶負電的唾液酸與二氧化鈦之間交互作用機制,已被發展為純化唾液酸醣胜肽的一種方法。
藉由結合了二氧化鈦與磁性奈米粒子易分離的優點,在此研究中,我們將使用具有二氧化鈦修飾的氧化鐵磁性奈米粒子去純化唾液酸醣胜肽。以富含唾液酸的醣蛋白胎球蛋白作為標準品,進行實驗條件如調整緩衝液的極性、酸度與粒子堆疊環境等優化後,比較一般市售的二氧化鈦粒子,我們可成功鑑定到胎球蛋白中18條含唾液酸醣胜肽與11條聚唾液酸胜肽,此外,於不同蛋白混合液中,我們也可鑑定到胎球蛋白中18條醣胜肽,其中含有14條唾液酸醣胜肽與8條聚唾液酸醣胜肽,此結果表示此奈米探針對唾液酸醣胜肽具有好的專一性與純化效果。 我們進而將此策略應用在分析淋巴癌細胞(HBL-1與DHL-6)之唾液酸醣蛋白體。先使用100微克DHL-6胜肽去優化實驗條件,經串聯式液相層析質譜儀分析後,可以鑑定到66個含N醣基化位置,專一性為9.36%。經由改善專一性實驗,約有59條(專一性25.43%)與和32條(專一性16.84%)N醣胜肽分別在HBL-1與DHL-6細胞中鑑定而得。為了獲得N醣胜肽上完整醣結構,我們進一步鑑定二次質譜圖譜中醣型斷片水合氫離子,在HBL-1細胞中,約有1124醣胜肽與含有591張唾液酸醣胜肽圖譜;而在DHL-6細胞中,約有572張醣胜肽圖譜與237張含有唾液酸醣胜肽圖譜,其中許多已知的醣蛋白,包括CD98hc和CD79b可在HBL-1細胞中鑑定到。重要的是,我們利用Orbitrap Fusion質譜儀成功鑑定到Transmembrane 9 superfamily member 3 protein (跨膜超家族)蛋白(在細胞中扮演訊號來源),帶有雙分支且含兩個唾液酸醣型。總括而言,未來我們將進一步鑑定完整的醣型結構及唾液酸修飾,去探討其與淋巴癌功能與變異的作用關係。 Abnormal glycosylation has been reported to associate with malignant transformation of tumor cells. Among different carbohydrates, the terminal position of glycans such as sialic acid, a negatively charged carbohydrate residue, usually exhibits a high level in tumor cells. The sialic acids, particularly polysialyl glycans, play a protective role in living cells and organisms and may participate in cell-cell interaction and migration in cancer metastasis. For systematic profiling of the sialoproteome associated with disease, effective enrichment of sialylated peptides from biological samples still presents an analytical challenge for MS-based glycoproteomic analysis. Among recent developments, the interaction between the highly negatively charged sialic acid and titanium dioxide (TiO2) has been explored for enrichment of sialylated glycopeptides. By combining the advantages of hydrophilic surface of TiO2 and magnetic separation of Fe3O4 magnetic nanoparticles, in this study, TiO2 coated Fe3O4 magnetic nanoparticles (Fe3O4@TiO2) with well-defined core/shell structure were fabricated to enrich the N-linked sialylated glycopeptides. Using tryptic digests of sialo-glycoprotein fetuin as a model study, optimum enrichment performance was evaluated with several experimental conditions including loading buffer polarity, acidity and binding environments. With MALDI-MS analysis, all of 18 sialylated glycopeptides containing 11 polysialylation of fetuin can be captured by Fe3O4@TiO2., When fetuin was spiked into protein mixture (BSA, HRP, fetuin), 14 sialylated glycopeptides (with 8 polysialylation) of 18 intact glycopeptides from fetuin were enriched by Fe3O4@TiO2 core-shell nanospheres, demonstrating that the Fe3O4@TiO2 nanoprobe had good specificity and efficiency for enrichment of sialylated glycopeptides. To study the role of sialylation to B cell lymphoma, we further attempted to apply this strategy for analyzing sialoproteome in B lymphoma cells, HBL-1 and DHL-6. Using 100 μg tryptic digest of DHL-6 cells, 66 N-glycosylation sites with 9.36% specificity were identified by Q-TOF LC-MS/MS analysis. Furthermore, 59 and 32 N-glycosylation sites were identified and the specificity was 25.43% and 16.84% for HBL-1 and DHL-6 cells, respectively by applying published protocol. To obtain the information of intact glycosylation, we further analyzed and confirmed oxonium ions in MS/MS spectra. Here, a total of 1124 and 572 MS/MS scans of glycopeptides as well as 597 and 237 scans containing sialylation were identified in HBL-1 and DHL-6 cells, respectively. The results identified some well-known glycoproteins, including CD98hc and CD79b, in HBL-1 cell. Most importantly, the intact sialylated glycopeptide of transmembrane 9 superfamily member 3 protein (Q9HD45, sequence: IVDVNLTSEGK), which associated with cell surface signaling, had biantennary glycan structure with 2 sialic acids, was also explored by Orbitrap Fusion MS analysis. In conclusion, the detailed analyses to delineate the glycan structure of intact glycopeptides are still needed for exploration the molecular pathways involved in sialylation modulation and formation of B-cell lymphoma. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52395 |
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