利用雙甲基化搭配液相層析串聯式質譜技術進行蛋白質藥物Trastuzumab之雙硫鍵結鑑定分析
Abstract
雙硫鍵在穩定蛋白質的三級結構和維持其生化活性上扮演重要的角色,同時,雙硫鍵的完整鍵結與否,常常關係到蛋白質藥物的品質.傳統上利用質譜分析蛋白質雙硫鍵的方法,常採用間接鑑定方式,比較經過還原前和還原以後含有cysteine胜肽質譜圖的差異,雙硫鍵的兩個胺基酸cysteine經還原後會接上兩個氫,而使該胜肽鏈的分子量多2 Da,但當同一個蛋白質裡含有複雜且多條的雙硫鍵時,就會產生更多樣的排列組合,所以這樣的方法,並不適用於分析具有多條複雜雙硫鍵的蛋白質.
在本篇研究中,我們提出一套鑑定雙硫鍵的方法,應用於蛋白質藥物trastuzumab上.在非還原的條件下,利用雙甲基化胜肽在Q/TOF質譜中,CID碎片會有a1離子訊號被提升的現象,搭配計算軟體RADAR掃描a1離子及其對應分子量,並與資料庫中含有cysteine的胜肽組合去比對,藉此鑑定出雙硫鍵的位置.此外,將針對不同的蛋白酶搭配,多種蛋白質水解與雙甲基化的最佳條件作測試,以期可以在最短的時間內,鑑定到最多正確的雙硫鍵數目.最後,在最佳化實驗條件下,利用RADAR對a1離子作自動化的掃描,成功的鑑定出trastuzumab上七條雙硫鍵位置.
本篇研究提供一套快速且正確鑑定雙硫鍵的方法,適用於例行的蛋白質藥物結構的鑑定,可以發展成一套完整有效的蛋白藥物品管方式.
Disulfide bond formation plays a critical role in stabilizing protein tertiary structure thus maintaining its biological activity. It also infers the quality of recombinant protein pharmaceuticals. In this study, a simple and rapid method was demonstrated for disulfide bond identification of an IgG drug trastuzumab. Dimethyl labeling was applied to proteolytic peptides in nonreduced condition, which exhibit enhanced a1 ion signals in CID; multiple a1 ions can be observed due to multiple N-termini. Computational software RADAR was also developed to perform the automatic a1 ion screening followed by searching for molecular weight match among the cysteine-containing peptides. Selections of appropriate proteases combination and buffer pH for dimethyl labeling were investigated. The results (peak lists) were dealt with custom-made software RADAR to screen a1 ions, and found the corresponding molecular weight for disulfide bond assignment. Seven unique disulfide linkages of trastuzumab were successfully identified using automatic a1 ion screening and RADAR with optimized experimental conditions. This approach provides accurate and efficient solution, and it has great potential for routine structural characterization of protein pharmaceuticals.
Disulfide bond formation plays a critical role in stabilizing protein tertiary structure thus maintaining its biological activity. It also infers the quality of recombinant protein pharmaceuticals. In this study, a simple and rapid method was demonstrated for disulfide bond identification of an IgG drug trastuzumab. Dimethyl labeling was applied to proteolytic peptides in nonreduced condition, which exhibit enhanced a1 ion signals in CID; multiple a1 ions can be observed due to multiple N-termini. Computational software RADAR was also developed to perform the automatic a1 ion screening followed by searching for molecular weight match among the cysteine-containing peptides. Selections of appropriate proteases combination and buffer pH for dimethyl labeling were investigated. The results (peak lists) were dealt with custom-made software RADAR to screen a1 ions, and found the corresponding molecular weight for disulfide bond assignment. Seven unique disulfide linkages of trastuzumab were successfully identified using automatic a1 ion screening and RADAR with optimized experimental conditions. This approach provides accurate and efficient solution, and it has great potential for routine structural characterization of protein pharmaceuticals.
Description
Keywords
單株抗體, 雙硫鍵, a1離子, 蛋白質藥物, Monoclonal antibody, Disulfide bond, a1 ion, Protein pharmaceutical