响应面法优化-高分辨质谱鉴定玛卡中蛋白

Response Surface Methodology Optimization-High Resolution Mass Spectrometry for the Identification of Proteins in Maca

本文建立了液相色谱-高分辨质谱鉴定玛卡中蛋白的方法。从缓冲液浓度、提取料液比、提取温度、提取pH以及提取时间5个因素优化了提取条件,通过响应面试验预测其蛋白提取最优方案并验证。最终确定在1 g样品中,加入45 mL pH=10的Tris-HCl缓冲液,提取温度70℃,时间40 min,酸沉pH=2.5,提取率为45.69%,与预测值46.69%基本一致。以UPLC-Q-Exactive四极杆-静电场轨道阱高分辨质谱联用仪分析,所得肽段在Uniprot上进行Blast序列对比,其中明确生物来源与分子功能的有6条。本文建立的方法提取效率高,可为玛卡多肽的结构与活性功能之间的研究提供技术手段。

In this paper,a method for the identification of proteins in Maca by liquid chromatography-high resolution mass spectrometry was established.The extraction conditions were optimized from five factors including buffer concentration,extraction ratio,extraction temperature,extraction pH and extraction time,and response surface analysis was used to predict and verify the optimal protein extraction scheme.The results show that when the ratio of material to liquid was 1∶45,the extraction temperature was 70℃,the extraction time was 40 min and the pH of acid precipitation was 2.5,the maximum extraction efficiency of Maca protein was 46.69%,which was basically consistent with the predicted value of 46.69%.The ultra-high performance liquid chromatography(UPLC)-Q-Exactive quadrupole-electrostatic field orbitrap high-resolution mass spectrometer was used to analyze the peptide fragments.Blast sequences were compared on Uniprot,and six of them were identified as biological sources and molecular functions.The method established in this paper has high extraction efficiency and can provide technical means for the study of the relationship between the structure and the active function of Maka polypeptides.