以質譜技術評估在不同酸鹼值環境水解Avastin之雙硫鍵錯接變化

Abstract

正確的雙硫鍵摺疊影響蛋白質的活性及結構穩定性，因此對蛋白質類藥物而言，鑑定雙硫鍵的連接情況至關重要。隨著質譜技術的成熟，現今多以生化質譜方法鑑定蛋白質雙硫鍵，在此方法中需使用酵素水解蛋白質。酵素活性範圍多為弱鹼性環境，然而在弱鹼性環境下可能使蛋白質雙硫鍵還原再重新摺疊形成不同構型的雙硫鍵，造成在結構鑑定上無法判斷是樣品本身的雙硫鍵摺疊錯誤還是因實驗過程而產生的雙硫鍵重組。在本研究中，以溶菌酶（Lysozyme）及Bevacizumab（Avastin）作為樣品，控制酵素在中性以及弱酸性的溶液下進行水解，再使用雙甲基化標記結合質譜方法搭配RADAR軟體鑑定雙硫鍵，進而觀察雙硫鍵連接情形的變化，以提供最佳化鑑定雙硫鍵的實驗條件，降低雙硫鍵重組應造成的干擾。以trypsin及Lys-C在pH 6環境水解樣品，可完整鑑定到預期的含雙硫鍵胜肽，且皆未鑑定到錯接雙硫鍵。而以trypsin + Glu-C搭配變性劑rapigest在pH 6環境下水解Avastin，也可完整鑑定到預期的含雙硫鍵胜肽，且未觀察到錯接雙硫鍵。以thermolysin進行水解，不論在pH 5, 6, 7環境下都有觀察到錯接雙硫鍵的存在。本研究結果顯示，選用適當的酵素、在弱酸性的環境下進行水解反應，可有效的減少雙硫鍵重組反應的發生，雖然使用的酵素活性受到抑制，但依舊可以成功鑑定到溶菌酶及Bevacizumab中所有的雙硫鍵。Disulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds to ensure quality and functions of protein drugs. Protein digestion procedures cannot be avoided for disulfide linkage analysis in conventional manner. In order to preserve enzyme activity during protein digestion, it is commonly carried out at basic environment which increases the possibilities of disulfide bond scrambling. However; when disulfide bond rearrangement occurs, it is not quite easy to differentiate whether by sample itself or digestion process cause the scrambling disulfide linkages. In this study, optimization on digestion pH was realized for the reduction of disulfide bond rearrangement. Three sets of proteases, including trypsin plus Glu-C, thermolysin and Lys-C were used, followed by dimethyl labeling and mass spectrometry for bevacizumab (Avastin) disulfide linkage analysis. There was no scrambled disulfide bond identified at pH 6 when using Lys-C or trypsin plus Glu-C as enzymes. When thermolysin was applied, there were still scrambled disulfide bonds identified either at pH 5, pH 6 or pH 7. Nevertheless, there was fewer scrambled disulfide bonds observed at low pH. All disulfidebonds on bevacizumab can be solved with this approach. The results demonstrated that by choosing the proper enzymes, using lower digestion pH environment could reduce the degree of scrambled disulfide linkages.