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Discrimination of Aspergillus isolates at the species and strain level by MALDI-TOF mass spectrometry fingerprinting.

Buskirk-AD; Hettick-JM; Green-BJ; Kashon-ML; Slaven-JE; Janotka-E; Schmechel-D; Beezhold-DH
J Am Soc Mass Spectrom 2008 May; 19(5S):S67
Novel Aspect: MALDI-TOF MS in conjunction with discriminant analysis can be used to identify Aspergilli at both the species and strain level. Introduction: The fungal kingdom consists of approximately 1.5 million diverse species. The identification of these fungi is subjective and depends on the expertise of mycologists identifying characteristic morphology of known species as well as phylogenetic analysis. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) has proven useful for discrimination of bacterial species, however, relatively few studies have been applied to fungi. Fungi of the genus Aspergillus have been shown to cause allergy and exacerbate asthma, as well as cause severe disease, especially in children and immunocompromised hosts. In this study, we investigate sixteen isolates from twelve different species of medically important Aspergilli. Methods: Sixteen isolates from twelve different Aspergillus species were subcultured from stock sources and grown 14 days on malt extract agar. Conidia and hyphae of individual fungi were isolated from one plate and transferred to a 2.0 mL microcentrifuge tube containing 100 microL of 0.1 mm zirconium beads. 1.0 mL of 50/50 acetonitrile/4% trifluoroacetic acid was added to each tube and the samples were subjected to three one minute bead disrupting cycles. The resultant solution was centrifuged at 14,500 rpm for 10 minutes and the supernatant was analyzed via MALDI-TOF MS. Samples were mixed 1:1 with 10 mg/mL alpha-cyano-4- hydroxycinnamic acid, and a 1 microL aliquot was spotted onto a target plate and allowed to air dry. Preliminary Results: MALDI-TOF MS was used to generate highly reproducible mass spectral "fingerprints" for sixteen fungal isolates from twelve species of Aspergillus. The resulting spectra were rich in information content, and contain significant peaks between 5-20 kDa. The utility of the method is highlighted by the analysis of A. flavus and A. parasiticus, two fungi with many morphological similarities. The mass spectra from the two species share several m/z, but differ in relative abundance. In spite of the significant similarity between species (and the resultant MALDI-TOF MS fingerprint spectra), statistical analysis was able to correctly identify the fungi. To ensure reproducibility, each fungal isolate was independently cultured eight times, and the MALDI-TOF mass spectrum from each independent culture was added to the database. The fungal isolates can be correctly identified at the species level with 100% accuracy using canonical discriminant analysis with stepwise variable selection. A second database was constructed using five strains of A. flavus. Discriminant analysis on this dataset utilizing all peaks in the spectra yields 0% resubstitution error and 5% cross-validation error. However, strain-level identification utilizing only the most significant peaks results in 0% and 100% error for resubstitution and cross-validation, respectively. This 100 % cross-validation error is presumably due to the fact that the subset of significant peaks chosen for analysis was shared by all strains of A. flavus. Although fungal species identification is generally improved by choosing a subset of m/z values for analysis, this technique fails for datasets with a high degree of redundancy, such as the A. flavus strain dataset. This study demonstrates the utility of mass spectrometry to aid in fungal identification at both the species and strain level.
Fungi; Spectrographic-analysis; Spectroscopes; Biomarkers; Microorganisms; Allergens; Analytical-processes; Mass-spectrometry; Mycology; Morphology
Amada D. Buskirk, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Health Effects Laboratory Division, 1095 Willowdale Road, Morgantown, WV 26505-2888
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Healthcare and Social Assistance; Services
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Journal of the American Society for Mass Spectrometry. 56th ASMS Conference Program June 1 - 5, 2008 Denver, Colorado