開發凝集素-苯亞硼酸修飾奈米探針輔助親和性質譜法應用於快速醣蛋白分析

陳政揚

指導教授 ： 洪偉修 ； 陳玉如

國立臺灣師範大學/理學院/化學系/碩士(2010年)

摘要

醣基化是一個重要而且普遍的蛋白質轉譯後修飾。醣蛋白參與許多重要的生物功能，例如蛋白質分選(protein sorting)、免疫辨識(immune recognition)、受體結合(receptor binding)、發炎反應(inflammation)，以及致病性(pathogenicity)等。更重要的是，在體液中存在有許多醣蛋白，可以用來作為診斷和治療的依據。但由於醣蛋白中醣鏈結構的高度複雜性，導致每一種醣蛋白異構物在自然界中皆呈現相對低濃度。因此，為深入分析完整的醣蛋白質體，有效的萃取步驟是不可或缺的。如何能快速、有效率且正確的檢測醣蛋白，迄今仍面臨著極大的挑戰。為了解決這個問題，我們開發奈米探針輔助親和性質譜法(nanoprobe-based affinity mass spectrometry ; NBAMS)，集結了新設計的雙功能磁性奈米粒子以及利用飛行時間質譜法(MALDI-TOF MS)，提供快速且高效能的蛋白質萃取及鑑定。相較於傳統上個別利用凝集素或苯亞硼酸萃取醣蛋白的方式，我們設計了凝集素(lectin)-亞硼酸修飾的雙功能磁性奈米粒子(BA-lectin@MNP)。其優點在於，凝集素可提供專一性親和力，而醣鏈上的1,2-順二醇(1,2-cis diol)和苯亞硼酸(phenylboronic aicd)之間形成共價鍵結；前者能萃取特定的醣蛋白，而後者則能進一步的透過共價鍵結穩固奈米粒子與醣蛋白之間的作用力，並顯著的增進醣蛋白萃取的效果。這些功能性修飾的磁性奈米粒子在蛋白質混合物中展現專一選擇性，捕捉特定的醣蛋白。而不同的濃度之下，使用BA-lectin@MNP對於特定醣蛋白的萃取效能超越lectin@MNP 2-7倍，並為BA@MNP的6-28倍。我們發現，在凝集素上修飾苯亞硼酸可以在低濃度下增強醣蛋白萃取的效果。藉由MALDI-TOF MS的偵測，BA-lectin@MNP可萃取最低濃度到0.005 μg/μL的醣蛋白。至此，我們期望以亞硼酸修飾為主的磁性奈米粒子可以廣泛的應用於醣蛋白萃取研究，並發現臨床診斷上具有代表意義的生物標記醣蛋白。

關鍵字

磁性奈米粒子 ； 苯亞硼酸 ； 醣蛋白萃取

並列摘要

Glycosylation is an important and perhaps most abundant form of protein post-translational modification. Glycoproteins participate in diverse biological functions such as protein sorting, immune recognition, receptor binding, inflammation, and pathogenicity. More importantly, many glycoproteins present in body fluids are used for diagnostic and therapeutic purposes. However, it is still a great challenge to analyze the glycoproteins due to the facts that glycoproteins usually bear enormous structural complexity of glycans and present at a relatively low concentration. An efficient enrichment step is essential for complete characterization of a glycoproteome. To address this issue, we developed a nanoprobe-based affinity mass spectrometric method which integrates the newly designed bi-functional MNPs and direct protein identification by MALDI-TOF MS for rapid and efficient enrichment of glycoproteins. Compared with conventional tag by lectin or phenylboronic acid (BA), we design hybrid conjugation of BAdecorated lectin on magnetic nanoparticle. The bi-functional MNPs, named lectin-phenylboronic acid functionalized magnetic nanoparticle (BA-lectin@MNP), took advantages of the specific affinity interaction of lectins with their ligands and the covalent binding between 1,2-cis diol from glycans and the phenylboronic acid; the former provides enrichment specificity while the latter further stabilize the binding affinity. These functionalized MNPs demonstrated selective capture of glycoproteins from a mixture of proteins and glycoproteins. The enrichment efficiency of BA-lectin@MNP showed 2- to 7-fold higher intensities than those isolated by lectin@MNP and 6- to 28-fold by BA@MNP under different concentrations. Decoration of BA on lectin improves the enrichment performance under low concentration and the detection limit of glycoprotein enrichment using BA-lectin@MNP and MALDI-TOF MS was as low as 0.005 μg/μL. We expect that the use of BA-lectin@MNP-based mass spectrometric method provides a powerful tool for glycoprotein enrichment, facilitating the subsequent analysis of glycoproteins and discovery of new potential diagnostic and therapeutic markers.

參考文獻

[45] Lin, C. C., Yeh, Y. C., Yang, C. Y., Chen, G. F., et al., Quantitative analysis of multivalent interactions of carbohydrate-encapsulated gold nanoparticles with concanavalin A. Chem Commun (Camb) 2003, 2920-2921.

[1] Hsu, D. K., Chen, H. Y., Liu, F. T., Galectin-3 regulates T-cell functions. Immunol Rev 2009, 230, 114-127.

[2] Gupta, G., Sinha, S., Mitra, N., Surolia, A., Probing into the role of conserved N-glycosylation sites in the Tyrosinase glycoprotein family. Glycoconj J 2009, 26, 691-695.

[3] Wulff, B. B., Chakrabarti, A., Jones, D. A., Recognitional specificity and evolution in the tomato-Cladosporium fulvum pathosystem. Mol Plant Microbe Interact 2009, 22, 1191-1202.

[5] Shimizu, K., Taniichi, T., Satomura, S., Matsuura, S., et al., Establishment of assay kits for the determination of microheterogeneities of alpha-fetoprotein using lectin-affinity electrophoresis. Clin Chim Acta 1993, 214, 3-12.