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

• Main Author: 陳政揚
• Other Authors: 洪偉修
• Format: Others
• Language: en_US
• Online Access: http://ndltd.ncl.edu.tw/handle/82410853647743234592

碩士 === 國立臺灣師範大學 === 化學系 === 98 === 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.