Background: Filamentous fungi have a robust cell wall that requires particular sample preparation to ensure good-quality mass spectra for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). In this study, we compared five unique sample preparation methods to obtain extracted protein from filamentous fungi.
Materials/methods: Seven strains of Aspergillus fumigatus including ATCC 204305 and three of Schizophyllum commune were isolated in clinical samples from our hospital. These strains were grown at 30°C on Sabouraud dextrose broth. Then, the following five protein extraction methods were performed: ethanol/formic acid standard extraction, freezing in liquid nitrogen, cell disruption with silica beads, combination of liquid nitrogen and silica beads, and homogenization by non-freezing. We compared the number of common specific peaks used for identification in A. fumigatus/S. commune databases (Microflex version 3.1, Bruker Daltonics Inc.) with the number of peaks obtained by the five methods.
Results: Thirteen specific peaks were found in the mass spectra of A. fumigatus and 9 specific peaks were found in those of S. commune. In the ethanol/formic acid standard extraction method, the average appearance rates in A. fumigatus and S. commune were 66% and 4%, respectively. The methods with the best rates were the combination of liquid nitrogen and silica beads method and the freezing in liquid nitrogen method. In the combination of liquid nitrogen and silica beads method, the rates were 74% and 59% for A. fumigatus and S. commune, respectively, whereas the rates with the freezing in liquid nitrogen method were 74% and 54%, respectively.
Conclusions:The two methods using liquid nitrogen obtained a larger number of mass spectra than the other methods including the ethanol/formic acid standard extraction method. These methods have high protein extraction efficiency and increase the identification accuracy of A. fumigatus and S. commune.
Presenter email address: [email protected]
Full conference title:
- ECCMID 30th (2020)