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IDENTIFICATION OF ALLERGENS FROM METARHIZIUM ANISOPLIAE USING MASS SPECTROMETRY
Citation:
Shoemaker, J A. AND M J. Donohue. IDENTIFICATION OF ALLERGENS FROM METARHIZIUM ANISOPLIAE USING MASS SPECTROMETRY. Presented at 5th International Symposium on the Interface between Analytical Chemistry and Microbiology, Richland, WA, April 19-21, 2004.
Impact/Purpose:
To understand children's risks from exposure to molds in their environment and to explore risk management options for mitigating those risks.
Description:
Background
The U.S. EPA, under the "Children at Risk" Program, is currently addressing the problem of indoor fungal bioaerosol contamination. The fungus Metarhizium Anisopliae has been used as a bio-pesticide for insect control since the 1800's. Recent studies have shown that exposure to this micoroorganism can cause an immediate hyper-immunosensitivity or Type I allergenic response. Currently, the proteins which cause this allergenic response to M. Anisopliae are unknown. The goal of this project is the identification and characterization of allergens from M. Anisopliae using mass spectrometry (MS). Matrix assisted laser desorption ionization MS (MALDI-MS) and electrospray tandem MS (ESI-MS/MS) were used to analyze the fungal peptides and proteins isolated by 2D gel electrophoresis and immuno-blot analysis.
Methods
Total protein extracts of M. Anisopliae were separated using 2-D gel electrophoresis. Allergenic proteins were identified by immuno-blot analysis, using mouse hyperimmune serum against M. anisopliae (primary antibody) and anti-mouse immunoglobulin E (secondary antibody) as the detection system. Gel spots that reacted with the IgE were excised and in-gel digested. The peptides were extracted and analyzed by MALDI-MS and ESI-MS/MS. The MS data on molecular weight, peptide profiles and amino acid sequences were used to mine databases for potential sequence or domain homology to known allergens. Peptide sequences were analyzed using the MASCOT� data mining algorithm.
Results
Eight proteins were identified as allergenic proteins based on IgE reactivity in the immuno-blot assay. MALDI-MS peptide fingerprints have been obtained on all eight gel spots. Using these mass fingerprints to mine non-redundant databases yielded no significant matches. Five of the allergenic gel spots were partially sequenced by ESI-MS/MS and tentatively identified as catalases. The identification was confirmed on four of the five spots using a polyclonal catalase antibody. Catalase serves to protect the cell from the toxic effects of hydrogen peroxide by catalyzing its decomposition into molecular oxygen and water.
Conclusions
Eight proteins were identified in M. Anisopliae as allergenic by gel electrophoresis and immuno-blot analysis. Five of the allergic proteins in M. Anisopliae have been partially sequenced by MS and identified as catalases. The next step will be to study other fungi or molds to determine potential similarities in the allergenic proteins identified. The identification of fungal proteins that induce IgE responses can eventually aid in the prevention or treatment of allergies.