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  • 學位論文

同位素標記電灑法質譜於不同狀態下醣蛋白變化之研究

Comparative Studies of Glycoprotein Using Isotope Labeling and Electrospray Ion Trap Mass Spectrometry

指導教授 : 何國榮

摘要


醣蛋白之表現會隨著生理狀態和疾病型式而改變,並與疾病發展相關,因此研究不同狀態下醣蛋白之變化,對於疾病之診斷可提供有用的資訊。醣蛋白濃度、醣鏈分佈及醣化程度皆可能影響醣蛋白之表現,因此本研究主要目的在於提出一分析的策略可用於評估不同狀態下,醣蛋白濃度、醣鏈分佈和醣化程度的相對變化量。藉由比較醣胜肽和胜肽在不同狀態時的質譜訊號相對強度,以獲得醣蛋白濃度、醣化程度、醣鏈分佈之相對變化。利用d0-甲醛與d2-甲醛同位素標記兩不同狀態的醣胜肽,以纖維素微晶體純化醣胜肽,並以電灑法質譜儀偵測以逹到相對定量之分析。為了證實策略之可行性,首先以核糖核酸酶B作為探討對象,探討新策略在以下不同狀態之可行性:(1).醣蛋白濃度改變、醣鏈分佈和醣化程度皆不變;(2).醣鏈分佈改變、醣蛋白濃度與醣化程度皆不變;(3).醣化程度改變、醣蛋白濃度與醣鏈分佈皆不變;(4).醣蛋白濃度、醣鏈分佈、醣化程度均改變。證實所提出之相對定量策略方法可有效地區辨不同狀態下醣蛋白表現之變化,來自於醣蛋白濃度改變、醣化程度改變或醣鏈分佈改變之貢獻分別為何。此外,利用電灑法離子阱質譜分析醣胜肽,藉由離子阱質譜儀具有多次質譜/質譜之能力,於正離子模式下,以質譜/質譜(MS2)獲得醣鏈組成資訊,並以質譜/質譜/質譜(MS3)獲得醣化位置之資訊;於負離子模式下,以多次質譜/質譜(MSn)方式可獲得醣鏈鍵結資訊。 進一步測試策略於複雜樣品具有一個以上醣化位置且醣鏈含有唾液酸之醣蛋白之適用性,選用乳鐵蛋白作為分析目標,以逆相液相毛細管液相層析取代纖維素微晶體純化步驟。實驗結果證實策略於複雜樣品可獲得醣蛋白濃度、醣鏈分佈和醣化程度的相對變化量。 將分析策略應用於真實樣品,選擇母乳中的乳鐵蛋白為真實樣品之例子。利用乳鐵蛋白標準品及市面上購得之鮮乳評估純化方法的可行性,且將各個純化步驟做最佳化的探討,再將收集得到之不同哺乳階段母乳樣品進行純化。最後將分析策略應用於不同哺乳期間母乳中的乳鐵蛋白,成功區別醣蛋白表現之變化來自醣蛋白濃度變化、醣化程度變化、或醣鏈分佈變化之貢獻為何。

並列摘要


Glycoproteins are vared with regulatory states, type of disease, and even with disease progression. Therefore, comparative studies of the change of glycoproteins may provide useful information for the diagnosis of disease. Several causes including the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile may account for the difference if the signal of a glycopeptide was found to be changed. A new strategy was proposed for comparative analyses of glycoprotein in which the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile could be differentiated. Comparative analysis was performed by stable isotope labeling using formaldehyde-d0 and formaldehyde-d2 along with cellulose microcrystalline enrichment and mass spectrometry analysis. The utility of the proposed strategy was demonstrated with ribonuclease B. The feasibility of the new strategy under different situations was studied as following: (1)glycoprotein concentration changed, glycan profile and glycosylation site occupancy fixed; (2)glycan profile changed, glycoprotein concentration and glycosylation site occupancy fixed; (3)glycosylation site occupancy changed, glycoprotein concentration and glycan profile fixed; (4) glycoprotein concentration, glycan profile and glycosylation site occupancy all changed. As a consequence, the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile could be obtained simultaneously. ESI-MS analysis of glycopeptides was performed on a linear ion trap mass spectrometer. With the capability of MS2 and MS3 of the instrument, the site of glycosylation and the glycan sequence may also be obtained in positive-ion mode. The negative-ion mode of MSn was useful for glycan structural analysis. To test the feasibility of the proposed strategy in complex glcoprotein with more than one glycosylation site and sialylated N-glycans, lactoferrin was chosen as the model compound. Cellulose microcrystalline enrichment was replaced by reversed-phase liquid chromatography. The results showed that the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile could be obtained in complex glycoprotein sample. To apply the strategy on lactoferrin from human milk of different stages of lactation period, first optimize purification method by lactoferrin standard and market milk. Lactoferrin was successfully purified form human milk. The glycoprotein change of lactoferrin from different lactation stages due to glycoprotein concentration, glycosylation site occupancy and glycan profile could be differentiated by the strategy.

參考文獻


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