Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi

National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV 26505, USA.
Analytical Biochemistry (Impact Factor: 2.22). 04/2011; 411(1):122-8. DOI: 10.1016/j.ab.2010.11.025
Source: PubMed


Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to discriminate moniliaceous fungal species; however, darkly pigmented fungi yield poor fingerprint mass spectra that contain few peaks of low relative abundance. In this study, the effect of dark fungal pigments on the observed MALDI mass spectra was investigated. Peptide and protein samples containing varying concentrations of synthetic melanin or fungal pigments extracted from Aspergillus niger were analyzed by MALDI-TOF and MALDI-qTOF (quadrupole TOF) MS. Signal suppression was observed in samples containing greater than 250ng/μl pigment. Microscopic examination of the MALDI sample deposit was usually heterogeneous, with regions of high pigment concentration appearing as black. Acquisition of MALDI mass spectra from these darkly pigmented regions of the sample deposit yielded poor or no [M+H](+) ion signal. In contrast, nonpigmented regions within the sample deposit and hyphal negative control extracts of A. niger were not inhibited. This study demonstrated that dark fungal pigments inhibited the desorption/ionization process during MALDI-MS; however, these fungi may be successfully analyzed by MALDI-TOF MS when culture methods that suppress pigment expression are used. The addition of tricyclazole to the fungal growth media blocks fungal melanin synthesis and results in less melanized fungi that may be analyzed by MALDI-TOF MS.

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Available from: Don H Beezhold, Jun 13, 2014
    • "During the analysis of darkly pigmented spores or cell material the melanin diminishes the ionization capability of the MALDI matrix by interfering with the energy transfer from the matrix to the sample molecules. The melanin compounds act as an energy sink (Buskirk et al., 2011). Thus, sample molecules are ionized poorly due to an insufficient energy transfer. "
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    ABSTRACT: Stachybotrys chartarum and S. chlorohalonata are two closely related species. Unambiguous identification of these two species is a challenging task if relying solely on morphological criteria and therefore smarter and less labour-intensive approaches are needed. Here we show that even such closely related species of fungi as S. chartarum and S. chlorohalonata are unequivocally discriminated by their highly reproducible MALDI-TOF-MS fingerprints (matrix assisted laser desorption/ionization time-of-flight mass spectrometry fingerprints). We examined 19 Stachybotrys and one Aspergillus isolate by MALDI-TOF-MS. All but one isolate produced melanin containing conidia on malt extract agar. Mass spectra were obtained in good quality from the analysis of hyaline and darkly pigmented conidia by circumventing the property of melanin which causes signal suppression. MALDI-TOF fingerprint analysis clearly discriminated not only the two morphologically similar species S. chartarum and S. chlorohalonata from each other but separated them precisely from S. bisbyi and A. versicolor isolates. Furthermore, even S. chartarum chemotypes A and S could be differentiated into two distinct groups by their MALDI-TOF fingerprints. The chemotypes of S. chartarum isolates were identified by Trichodiene synthase 5 (tri5) sequences prior to mass spectra analysis. Additionally, species identities of all isolates were verified by their 18S rRNA and tri5 gene sequences. Copyright © 2015. Published by Elsevier B.V.
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    • "Identification of a few dematiaceous species belonging to Haplographium, Exophiala and Madurella was also problematic. Pigmentation is indeed known to interfere with the quality of the spectra by decreasing or suppressing the ion signal, resulting in the inhibition of the desorption/ionization process during MALDI-TOF MS (Buskirk et al. 2011). Strains incorrectly identified by MALDI-TOF MS resulted essentially from cross-identifications within the genera Trichophyton and Aspergillus. "
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    • "Knowing that the black or dark brown pigmented fungi are almost always associated with melanin which is known to absorb portion of visible and UV light, the lack of data may be attributed to the characteristics of the pigment. Attempts to analyze them by massspectroscopy by other researchers yield poor fingerprint spectra (Buskirk et al., 2011; Bochenek and Gudowska- Nowak, 2003). Advancements of phytophotobiology will benefit other fields concerned with dark pigmented fungi. "
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