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将纯化的天然虎纹捕鸟蛛毒素 Ⅴ经盐酸胍变性 30min后 ,在pH 3 0、反应温度为 37℃的条件下与三羧甲基磷酸 (TCEP)反应 12min ,用反相高效液相色谱分离得到其全部去折叠中间体 ,通过基体辅助激光解吸电离飞行时间质谱 (MALDI TOFMS)进行鉴定 ,并利用烷基化反应对这些去折叠中间体予以进一步确证。根据其保留时间 ,分析虎纹毒素 Ⅴ各去折叠中间体的色谱行为 ,初步探讨了多肽或蛋白质构象异构体反相色谱行为的多样性。
The purified natural tiger woodwort toxin Ⅴ denatured with guanidine hydrochloride 30min, at pH 30, the reaction temperature was 37 ℃ conditions with tricarboxymethyl acid (TCEP) reaction 12min, using reverse phase high performance liquid chromatography All the unfolded intermediates were isolated and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOFMS). These unfolded intermediates were further confirmed by alkylation. According to their retention time, the chromatographic behavior of each deconstructed intermediate of tiger toxin V was analyzed, and the diversity of reverse phase chromatographic behavior of peptides or protein conformers was discussed.