Development of a quantitative proteomics method to study the adaption of fungi to extreme environments

Abstract

Originally black fungi - also called dematiaceous fungi - were characterized as inhabitants of living and dead plant material. In the last 30 years they have been isolated from hypersaline waters, acidic environments, radioactive areas, as human pathogens or opportunists and as a dominant part of the epi- and endolithic microbial communities. Recent studies have shown that their stress resistance against solar radiation, radioactivity, desiccation and oligotrophic conditions even allows them to survive in space and under Martian conditions. Due to all these facts this organism is of great interest for many studies to acquire knowledge about their biological behavior in extreme environments. Findings of the regulation of cell products will help to understand the enormous stress tolerance. For this a method for relative protein quantitation can be helpful to get deeper insight into biological functions. To develop an appropriate method Bovine Serum Albumin (BSA) has been used to establish a quantitation method based on iTRAQ (isobaric Tags for Relative and Absolute Protein Quantitation) labeling. With molecular weight matched iTRAQ labels peptides in different samples were labeled and up to 4 samples were measured and relatively quantified in parallel by matrix-assisted laser desorption/ionization reflectron time-of-flight mass spectrometry (MALDI-RTOF-MS). Analytical parameters like reproducibility of peptide labeling, sample clean-up and relative quantities were gathered and the dynamic range for concentration differences for different samples was evaluated. To adapt the established method to black fungi samples of the strain Exophiala dermatitidis, different clean-up steps necessary to remove interfering substances have been tested. The implementation of a suitable method proved to be difficult because of the high amount of disturbing buffer substances necessary for efficient protein extraction of Exophiala dermatitidis samples as well as the thick melanized cell walls. Nevertheless, the established iTRAQ based quantitation method gave insights into biological up- and down regulation of some peptides of Exophiala dermatitidis samples. Although the developed method still requires improvement, it has high potential for the final analysis. To get deeper insights into biological functions of black fungi also protein identification of separated proteins is essential. Therefore protein identification after two dimensional (2D) gel electrophoresis was carried out. 25 spots of a 2D gel of an extract of microcolonial black fungi were cut out, trypsinized and measured by MALDI mass spectrometry. An online available gel of Saccharomyces cerevisiae was used as a reference gel to get a first idea of protein identity. Using this approach some proteins of the strain Exophiala dermatitidis could be identified with the demanded significance.