利用聚精胺酸修飾鑽石奈米粒子為親和性探針於磺酸化胜肽的質譜分析

MALDI-TOF Mass Spectrometric Analysis of Sulfonated Peptides Using Poly-arginine-coated Diamond Nanoparticles as Affinity Probe

黃建寧(Chien-Ning Huang)

指導教授 ： 吳志哲

國立暨南國際大學/科技學院/應用化學系/碩士(2008年)

摘要

在人體中的磺酸化扮演著重要的角色，磺酸化可以活化人體中的分子進而進行不同的生化功能。目前對於分析磺酸化生物分子的最主要工具是基質輔助脫附游離-飛行時間-質譜儀，由於磺酸化分子是屬於強酸性的化合物，因此此類分子游離的過程並不容易形成帶電荷的分子，因此目前許多研究如何改善或提升磺酸化分子於基質輔助脫附游離法的游離效率。
本篇實驗主要利用聚精胺酸和磺酸化分子擁有非常專一且穩固的鍵結力將磺酸化分子濃縮萃取，進而以利於基質輔助脫附游離法的質譜分析。實驗的首先合成了以鑽石奈米粒子為基材的聚精胺酸親合性探針，之後以胰島素、磺酸化胰島素鏈A、磺酸化胰島素鏈B當標準品來做測試並找出最佳的實驗流程，實驗結果顯示所合成的親合性探針成功的濃縮萃取磺酸化胜肽，並且對於磺酸化胜肽於基質輔助脫附游離法的偵測極限可達10 fentomole。然後利用已確立的實驗流程對過甲酸化的胰島素做磺酸化胜肽的濃縮萃取，實驗結果跟利用標準品混合液一致，表示對於經過過甲酸化的樣品可直接進行濃縮萃取，樣品不需要再經過前處理。最後將牛血清蛋白做過甲酸化後再做酵素水解，最後濃縮萃取磺酸化胜肽並於線上資料庫做比對其磺酸化半胱氨酸，此實驗結果磺酸化半胱氨酸個數的涵蓋率可達62.86%，此涵蓋率和利用陽離子交換液相層析法一致，但是相較起陽離子交換液相層析法此親合性探針是較為簡便且快速的方法。

關鍵字

鑽石奈米粒子 ； 基質輔助雷射脫附游離法 ； 聚精胺酸 ； 磺酸化 ； 半胱胺酸

並列摘要

Some cysteine residues in proteins are sensitive to oxidation by peroxides such as hydrogen peroxide and lipid peroxides that are generated by normal cellular metabolism. However, in the presence of excess peroxide, cysteine will be overoxidized to cysteine sulfonic acid form. Recent proteome analyses have revealed that overoxidation of the cysteine residue could alter the activities or functions of several proteins, such as matrix metalloproteinase-7 and glyceraldehyde-3-phosphatedehydrogenase. Even under physiological conditions, the overoxidation can occur without an exogenous oxidant. For example, ubiquitin carboxyl-terminal hydrolase L1 in the brains of patients with Alzheimer’s and Parkinson’s diseases.
In general, the strong negative charge caused by sulfonic acid in acidic compounds has been reported to influence the signal intensity in MALDI, because the ionization efficiency is low and some suppression effects.
In this study, we present a protocol to extract and enrich the sulfonated peptide from a mixture solution that included insulin, insulin chain A oxidized and insulin chain B oxidized from bovine pancreas. The approach is produced by taking the advantage of that guanidinium group of arginine molecule exhibits exceptionally high affinity toward sulf[on]ate group. Hence, we employed polyarginine coated on the diamond nanoparticles as affinity probes to extract and isolate sulfonated peptides from a dilute solution, and applied in MALDI-TOF Mass analysis.

並列關鍵字

Diamond nanoparticles ； MALDI ； Poly-arginine ； Sulfonation ； Cysteine

參考文獻

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