利用雙甲基化標記搭配質譜儀技術自動化鑑定蛋白質雙硫鍵鍵結

Abstract

驗證生物藥物的正確摺疊結構，以及正確雙硫鍵鍵結，越來越受到重視。正確蛋白質構型可維持蛋白質活性，而蛋白質後轉譯修飾所產生的雙硫鍵鍵結，主要扮演著維持蛋白質構型的角色。本研究中，利用雙甲基化標記以及質譜鑑定方法，搭配自製軟體RADAR自動化計算篩選a1離子的策略，將RADAR軟體應用於鑑定蛋白質藥物Bevacizumab與Trastuzumab的雙硫鍵鍵結，以及台南與新竹兩地區的複雜眼鏡蛇蛇毒蛋白的雙硫鍵鍵結。由於經雙甲基化標記胜肽後於MSMS質譜分析圖譜中，將產生a1離子訊號增強現象，藉此現象可鑑定其雙硫鍵鍵結胜肽的N端胺基酸殘基。RADAR軟體可自動化與數據庫中含半胱胺酸之胜肽進行比對，首先比對其a1離子，若符合N端胺基酸再比對其分子量，接著進一步比對其離子碎片，藉此策略快速鑑定雙硫鍵鍵結。應用RADAR軟體自動比對其a1離子策略，已成功鑑定出蛋白質藥物Bevacizumab與Trastuzumab所有的雙硫鍵鍵結。另外，於台南眼鏡蛇蛇毒蛋白上鑑定得到18個蛋白質的雙硫鍵鍵結；新竹眼鏡蛇蛇毒蛋白上鑑定得到17個蛋白質的雙硫鍵鍵結。針對雙甲基化反應使用之還原劑氰基硼氫化鈉，探討還原劑濃度及反應時間是否會造成非預期雙硫鍵鍵結產生實驗中，證明還原劑氰基硼氫化鈉不影響非預期雙硫鍵鍵結產生。本研究主要提供一個簡單以及快速鑑定雙硫鍵鍵結的方法，不但可作為檢查蛋白質藥物結構品管工具，同時也適用於鑑定複雜蛋白質混合物的雙硫鍵鍵結。

Increasing interest in production of bio-pharmaceuticals is accompanied by an increasing need for verification of protein folding and correct disulfide bonding. Correct protein conformation is often essential to the protein’s activity, and post-translational modifications such as disulfide bonds have substantial roles in maintaining the native fold. In this study, we demonstrate a method that using dimethyl labeling, mass spectrometry and computational screening of a1 ions with customized software, RADAR, which was applied for assigning the disulfide-bonding network of protein pharmaceuticals Bevacizumab, Trastuzumab and complex protein mixture of Tainan and Hsinchu cobra snake venoms. Labeled peptides which exhibit enhanced a1 ion signals during MS/MS fragmentation, the N-terminal amino acids from disulfide-linked peptides can be determined. Customized software, RADAR, can perform the automatic a1 ion screening followed by searching for molecular weight match further comparing the fragment ions against the cysteine-containing peptides. All disulfide peptides in Bevacizumab and Trastuzumab were completely assigned, by the automatic a1 ion screening and RADAR. Additionally, eighteen unique protein disulfide bonds of Tainan cobra snake venom and seventeen unique protein disulfide bonds of Hsinchu cobra snake venom were successfully identified. To evaluate whether the disulfide bond scrambling caused by sodium cyanoborohydride, we compared different concentrations and reaction time for sodium cyanoborohydride used in dimethyl labeling reaction. The result demonstrated that it would not affect the disulfide scrambling. This presented approach is simple and rapid, offering a quality examination tool for protein pharmaceuticals. It can be also a powerful tool to determine disulfide linkage of complex protein mixture.