| Summary: | 碩士 === 國立臺灣師範大學 === 化學系 === 101 === 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.
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