Proteomic Analysis of the Filamentous Fungus: Aspergillus fumigatus
EMSL Project ID
2407
Abstract
Fungi, with the possible exception of yeast, are less well studied than other microbes. One objective of this proposal is to generate a unique proteome database for a filamentous fungus directly important to humans. Aspergillus fumigatus, a haploid, asexual filamentous mold, has a dual nature, causing a serious human disease and contributing positively to humanity as an important biomass degrading organism. A. fumigatus is the most common species associated with human aspergillosis, while at the same time it is isolated frequently from decomposing biomass such as composts and manure. Several important aspects of the biology of A. fumigatus can be addressed now that the genomic sequence is available.* We propose to examine the differences between the proteomes of two strains, the clinical isolate used in the genome sequence determination coordinated by the Sanger Centre and a strain isolated from a compost niche. We hypothesize that unique proteins are expressed at temperatures above 37C and not at lower temperatures and that the proteomes of the clinical isolate and the compost isolate are different. The unique capability at EMSL will allow us to perform mass spectrometry and data analysis on protein samples prepared from the cell fractions of the two A. fumigatus isolates grown at high and low temperatures. The samples containing equivalent amounts of protein from 14N- and the 15N- labeled samples will be digested with trypsin and analyzed. A database of peptides will be created by analyzing the digested proteins, separated by capillary liquid chromatography (LC) and analyzed with the Finnegan LCQ quadrupole ion trap mass spectrometer. The database will be used to examine protein expression levels. Since the proteins will be combined in equivalent amounts, the relative abundance of each peptide can be determined by comparison of the signals from the 14N-peptide and the 15N-peptide. Using a combination of 14N and 15N for both temperatures (25oC and 37oC) allows for direct comparison and eliminates ambiguities introduced by the use of isotopic labeling. We would like to determine the difference in expression of the proteins at the high and low temperatures. Unique proteins expressed at the high temperature only are candidates for pathogenicity factors. Ultimately, we would extend this work with cells from an active infection to define new and specific fungal targets for drug discovery programs. The database generated in this study will be useful to a diverse group of researchers. It is the intention of the authors to manage the database so that it is easily accessible and generally available. In particular, scientists with the Agricultural Research Services, USDA and DOE working on biomass conversion will find this a useful resource. * A partial sequence is available now at TIGR; the complete genome sequence for Aspergillus fumigatus will be published soon.
Project Details
Project type
Exploratory Research
Start Date
2002-01-01
End Date
2002-11-12
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members