MALDI-TOF Analysis of Fungal Protein Extracts

Following the protocol of Djalali Farahani-Kofoet et al. [31 (link)] for the MALDI-TOF analysis, 1 µL of the protein extract was spotted on a polished steel target (Bruker Daltonik, Bremen, Germany) and allowed to dry. Then, 1 µL of the saturated HCCA (α-cyano-4-hydroxycinnamic acid solution) was added as matrix and allowed to dry. The MALDI method was calibrated using a bacterial test standard (Bruker Daltonik). The MALDI target plate containing the protein extracts was read with an ultrafleXtreme MALDI-TOF mass spectrometer (Bruker Daltonik) working in linear positive mode and acquiring mass spectra in the range of m/z 200–20,000. Measurements were performed by flexControl v3.4 software (Bruker Daltonik). The protein spectra served as the fingerprint of the test fungi and were compared with the reference database (1832 entries) of the Bruker Filamentous Fungi Library, including 13 entries for Trichoderma sp. The MALDI Biotyper v3.1 software (Bruker Daltonik) was used to process the raw spectra, generate peak lists and perform a database search for isolate identification. A dendrogram based on PCA clustering of m/z values was constructed using the hierarchical method, correlation distance measure, and average linkage algorithm.