一种适用于高分辨质谱分析的高效丝状真菌囊泡提取及蛋白制样方法

An Efficient Method of Filamentous Fungal Vesicle Extraction and Protein Preparation for High-resolution Mass Spectrometry

使用液氮冷冻研磨破碎真菌菌丝细胞,结合醋酸钠沉淀和超速离心纯化,获得的囊泡样品进一步使用超滤法浓缩和过滤,扫描电镜观察和蛋白电泳分析,实验结果显示所制备的真菌囊泡完整性好,蛋白纯度高,未见明显杂质污染,符合后续实验要求。进一步使用串联质谱标签技术联用高分辨生物质谱仪对真菌囊泡蛋白组进行分析,在提取的低毒病毒侵染后的真菌囊泡中成功鉴定到病毒蛋白的存在。实验方法的建立和优化,为丝状真菌囊泡提取、囊泡蛋白组学和病毒宿主相互作用研究提供了方法学保障。

The fungal mycelium was ground by liquid nitrogen,and then purified by sodium acetate precipitation and ultracentrifugation.The selected vesicle samples were further concentrated and filtered through ultrafiltration.Scanning electron microscope observation and protein electrophoresis analysis showed that the prepared fungal vesicles had good integrity,high protein purity,and no obvious impurity contamination,which met the requirements of subsequent experiments.Furthermore,tandem mass tags(TMT)combined with high-resolution biological mass spectrometry was used to analyze the fungal vesicle proteome.The viral proteins was successfully identified in the fungal vesicles upon hypovirus infection.The establishment and optimization of the experimental method in this paper provides methodological support for vesicle extraction,proteomic analysis and virus-host interaction research in filamentous fungi.