研發新型醣探針及其在醣蛋白質體學之應用

Abstract

醣化是一種重要的轉錄後修飾作用，其具有調節細胞內、外不同生物現象的功能。然而，研究醣類在生物角色是相當困難。究其原因主要在於細胞醣蛋白上，相比於蛋白質，醣類的含量通常較為稀少且呈現非均相的分布。除此之外，醣化作用複雜性高且和基因模版相關性低。造成要得到足夠的量及純度進行分析相當不易。為了克服這些問題，我們開發出新型醣探針，主要結合螢光性質與生物素。其主要三個功能分別為；疊氮基進行點擊化學，專一性結合至生物體內特殊醣類。同時，藉螢光的高靈敏特性偵測目標分子。並將其結構設計為螢光產生探針，具有反應後才產生螢光訊號的特性。利用這樣的性質來降低背景雜訊，減少非專一性的訊號產生。最後，藉由生物素(biotin)與蛋白素(streptavidin)之間的專一性親合力來純化並濃縮標定之醣蛋白。但由於生物素與蛋白素之間作用力太強而不易沖提。因此，進一步改進；在螢光基團與生物素間導入雙硫鍵結構，生物素的部分可由化學試劑切除，例如TCEP或DTT，藉此快速並方便的得到探針標定之醣蛋白。 由實驗結果証實，新型醣探針可在細胞中作用。接著，我們更進一步結合其同時具有三種功能之優點，應用到醣蛋白質體學的研究，希望可以得到單一醣蛋白的序列。 未來的實驗將會著重在比較性的醣蛋白質體學研究，尤其是正常細胞與癌症細胞醣蛋白的比較，並對其差異做進一步的研究。最終，我們希望這樣的新型醣探針可被應用到診斷與治療上。

Glycosylation is an important post-translational modification. It regulates the function of cells inside and outside of different biological phenomena. However, research in the biological role of carbohydrates is very difficult. The reason lies mainly in the glycoprotein, compared to protein, carbohydrate content are usually more scarce and display a heterogeneous distribution on the cell surface. In addition, the complexity of glycosylation and no template exist, make difficult to obtain pure and enough amount of carbohydrates to do analytical study. To tackle these problems, We have designed a new probe to combine the fluorescent with biotin to amplify the signal. It has three major functions: To do click chemistry via azido group, Specificity in vivo binding to labeled carbohydrates and monitor the targeted molecule by high sensitive fluorescent. Moreover, this type of probe only show high fluorescence after click reaction. This property can reduce the background and non-specific signal. We also used the high specific binding of biotin and streptavidin to enrich the labeled glycoprotein. However, the tight binding of biotin and streptavidin make the elution very difficult. To improve that, between fluorescence and biotin group, we introduced a disulfide linkage that can be cleavage by DTT or TCEP to help the purification of labeled glycoprotein. By using this method, we have succeeded to label cell by tri-functional probe and this strategy can be used in the glycomics study and hope someday we can identify the sequence of single glycoprotein. Future experiments will focus on comparative glycomics, especially the studies of glycoprotein differences in normal cells and cancer cells. Ultimately, we hope that this new type of sugar probe can be applied to the diagnosis and treatment.