臺灣博碩士論文加值系統

蛋白質上的醣基化異常與一些疾病息息相關。相較於N醣基化，O-醣基化的位置與醣基結構上的分析方法依舊是很困難的，其原因主要有三個:(1)沒有保留序列可用來預測醣基化位置；(2)目前沒有一個切醣酵素可以直接把醣從O-醣化位置上移除；(3)發生O醣化的區域其位置非常密集靠近，酵素很難進行有效率的消化。在此我們使用化學方法(β-Elimination)消去醣胜肽上的O-醣基並搭配液相層析二次質譜(LC-MS/MS)以鑑定O-醣化位置(由非專一性酵素Proteinase K消化成醣胜肽)。同時也對intact protein(未移除O醣基)進行消化，接著以LC-MS/MS做偵測。基於滯留時間的差別，由β-eliminated peptides中可判斷出O醣化位置，並由相對應的intact glycopeptides比對至碰撞誘導解離(collision-induced dissociation CID)或電子轉移解離(electron capture dissociation ETD)的MS/MS圖，由CID中的特徵離子如291(SA)，365(GalNAc+Gal)，657(GalNAc+Gal+SA)以鑑定出醣基結構。在此我們使用標準醣蛋白(Fetuin)做測試，利用此方法能正確的解出O醣基化位置S271，T281，S282，S296，S341。此外，由初步結果顯示，此β-elimination的方法能成功的移去O醣基且並不會造成太嚴重的蛋白質水解，在未來可望應用在未知醣蛋白上。

Altered protein O-glycosylation is known to associate with many human diseases. Studying protein O-glycosylaion, however, is difficult due to the lack of analytical methods. Compared to N-glycosylation, analyzing O-glycosylation site and structure is more challenging since there is no consensus sequence to predict its binding sites, no universal or specific enzymes to break O-glycans from the protein backbone; and it is hard to digest due to the high O-glycan binding density along a short sequence. In this study, we combine β elimination to cleave O-glycans from the peptide backbone and non-specific proteinase K to digest the cleaved peptides for analyzing O-glycosylation sites through mass mapping via LC-MSMS analysis. The intact protein without O-glycan release is also digested and analyzed by LC-MSMS for comparison. Based on the retention time matchings, potential O-glycosylation sites can be deduced from the β-eliminated peptides and further confirmed by the corresponding intact mass. The glycan structure can be identified by MSMS spectra of the intact peptide via either CID or ETD. Using fetuin, a O-glycosylated protein, as a model, we show all the known glycosylation sites, S271,T281,S282,S296,S341, can be successfully identified and their glycan structures can be unambiguously identified by the characteristic fragments such as 291(SA), 365(GalNAc+Gal), 657(GalNAc+Gal+ SA) detected via CID. Furthermore, the data indicate that β-elimination can lead to O-glycan eliminated peptides without extreme hydrolysis. Our preliminary data show that the developed method can be applied to identify unknown O-glycosylation sites and structure.

中文摘要: I
英文摘要 : II
謝誌 IV
目錄 . V
表目錄... VIII
圖目錄...IX
第一章 序論 1
1.1醣質體學 1
1.2醣基的類型 (Types of glycans) 2
1.3醣化現象的多樣性(heterogeneity) 3
1.4分析醣蛋白的挑戰性 3
1.5分析O-醣胜肽(O-glycopeptide)與常見的方法 4
1.6以串聯式質譜儀分析醣胜肽(glycopeptides) 6
第二章 實驗方法 19
2.1實驗藥品與儀器 19
2.2一級結構序列的鑑定 19
2.2.1酵素消化 (Enzymatic Digestion)鑑定其一級結構酵素消化之實驗流程19
2.2.2胜肽序列之鑑定 (Peptide Assignment) 20
2.3N-醣基化的位置與N-醣基結構的鑑定 20
2.3.1利用切醣酵素去醣基之實驗流程 20
2.3.2 N-醣基化位置之鑑定 20
2.3.3 N-醣基結構之鑑定 21
2.4O-醣基化的位置與O-醣基結構的鑑定 23
2.4.1分析流程與方法 23
2.4.2 專一性(非專一性)酵素消化 23
2.4.3化學消去(ß- Elimination: After Enzymatic Digestion ) 23
2.4.4 O-醣基化位置之鑑定 24
2.4.5 O-醣基結構之鑑定 24
2.5奈流液相層析質譜儀(nano-LC-MS/MS) 25
第三章 結果與討論 29
3.1測試β-Elimination kit在peptide level中的水解程度 29
3.2測試不同溫度下O型醣基化發生b-Elimination的程度 29
3.3利用β-Elimination kit搭配LTQ-orbitrap以鑑定標準醣蛋白的O 型醣基化位置 30
3.4利用β-Elimination kit搭配LTQ-orbitrap質譜技術(CID，ETD mode)以鑑定未知醣蛋白的醣基化位置以及醣基結構 32
3.4.1胜肽圖譜 (Peptide mapping) 鑑定其一級結構序列 32
3.4.2 N-醣化位置之鑑定與其醣基結構的分析 33
3.4.3 N-醣基化位置之鑑定 34
3.4.4 N-醣基結構的鑑定 (N-Glycan structure identification) 34
3.4.5 O型醣化位置之鑑定與其醣基結構的分析 35
3.4.6利用化學消去方法(β-elimination)初步推測O型醣基化位置 35
3.4.7 利用醣胜肽(intact glycopeptides)直接進行CID-MS2或ETD-MS2證明醣化位置 36
3.4.8高度醣基化胜肽分析最大的困難處 40
第四章 結論 41
參考文獻 81

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