An Efficient Method of Filamentous Fungal Vesicle Extraction and Protein Preparation for High-resolution Mass Spectrometry
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.
Cryphonectria parasiticaVesicleScanning electron microscopyMass spectrometry identification
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