Aim of this study was to analyze the variability of mass-spectra, indicators of the identification and applicability of intraspecific grouping under MALDI-TOF-mass-spectrometry of the protein extracts from A. fumigatus cultures. Materials and methods. We were used 60 clinical isolates of A. fumigatus identified by 18S-rDNA-sequencing. They were deposited in Russian Collection of Pathogenic Fungi (Saint-Petersburg). Culture preparation for extraction was made as previously described (Ryabinin I.A., 6th Advances Against Aspergillosis). For isolation of proteins from cultures standard Bruker protocol was performed. Each extract was examined twice on Autoflex speed TOF/TOF (Bruker Daltonics) with using α-cyano-4-hydroxycynnamic acid solution and spectra base «Fungi Library» for species identification in Biotyper 3.1 software. Procedures of subculturing, extraction and mass-spectrometry were repeated 1 to 5 times to collect several mass-spectra for revealing the accuracy of intraspecific grouping (the number of repetitions for each strain were picked randomly). Mass-spectra identified with score value >1,7 only were used for further processing. Dendrogram constructed by hierarchical principal component analysis (HPCA) was used for grouping. Results: Currently were collected 114 mass-spectra. The typical features of A. fumigatus proteome mass-spectra were described. Also we identified specific deviation of original mass-spectra-profiles from reference MSP. We have obtained the following distribution of selection by range of score value (percentage of mass-spectra is in brackets): 2,300-3,000 (5,3%); 2,000 – 2,299 (44,7%); 1,700 – 1,999 (50%). Categorization of the identification was as follows (in percentage of mass-spectra): A and B – by 48,2%; C – 3,6%.The greatest matches were found with 9 out of 12 mass-spectra of A. fumigatus from Fungi Library. Similarity with A. fumigatus DSM(Z) 15966 and MUZ 136033 occurred more frequently than with others. HPCA-dendrogram allowed to divide mass-spectra selection into 7 groups, but we have marked 19 intergroup relations because same strains were placed in different groups.
Full conference title:
- ISHAM 19th (2015)