發明
中華民國
105121435
I 568746
用以評估自由基產生能力的寡肽及方法
高雄醫學大學
2017/02/01
本研究設計了一段可用來篩選活性氧化物質的胜肽引子,代號是ACAR,其含有特殊官能基可與活性氧化物質反應形成胜肽-OH, 胜肽-O2H及胜肽-O3H。當樣品(化合物)與ACAR混合後,在有UV或無UV下進行反應10分鐘,反應後可以基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF MS)進行分析。在質譜圖上觀察ACAR是否接上一個氧 (m/z 791)、二個氧(m/z 807) 或接上三個氧 (m/z 823)的反應,待測樣品(化合物)經上述反應,可計算其具誘發活性氧化物質之能力。此檢測方法僅需微量樣本(1 μL),所需反應時間只需10分鐘,反應後立即分析,不僅快速且使用方便,更可節省細胞培養所需耗費的時間。此外,ACAR因設計上具有一個螢光標籤,尚可另外選擇高壓液相層析(high performance liquid chromatography, HPLC)搭配此外光(UV)、螢光或串聯式質譜儀(tandem mass spectrometry, MS/MS)來偵測樣品中的活性氧化物質。 The design of this peptide probe, ACAR, contains a specific group that could react with ROS to form the products of peptide-OH, peptide-O2H and peptide-O3H. After mixing the test samples (compounds) with ACAR, the reaction could be initiated with or without UV light for 10 min. Then sample solutions are detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The signals of ACAR are m/z 791, 807 and 823 for mono- di- and trioxidation after ACAR reacted with ROS. We could use these signals to calculate the ROS induced ability for test samples (compounds) without time-consuming experiment for cell culture. Only a small volume of sample (1 μL) is sufficient for ROS screening. Furthermore, this peptide probe contains a fluorescent tag and the oxidative products could be detected by high performance liquid chromatography (HPLC) coupled with UV, fluorescent and tandem mass spectrometry (MS/MS).
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