Aspergillus ochraceus is a filamentous fungus, which produces toxic and carcinogenic secondary metabolite Ochratoxin A (OTA). Genome sequencing for many important fungi has been started during recent years, however there is still deficiency in proteome profiling of Aspergilli. To obtain a comprehensive overview of proteins and their expression, a proteomic approach based on 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry was used to investigate A. ochraceus.
Maximum OTA concentration was determined on the 20th day of cultivation of A. ochraceus NRRL 5175 by HPLC. The cell walls of fungi are exceptionally resistant to destruction, therefore two lysis protocols were tested: (i) lysis via manual grinding using liquid nitrogen and (ii) mechanical lysis via rapid agitation with glass beads using MagNalyser. Mechanical grinding with mortar and pestle using liquid nitrogen was found to be more efficient extraction method for our purpose, resulting in extracts with higher protein content and a clear band pattern in SDS-PAGE. The proteome of A. ochraceus NRRL 5221 and NRRL 5175 cultivated in ME broth were compared using DIGE without protein identification. Furthermore, the proteome of A. ochraceus NRRL 5221 cultivated in different media (YES and ME broth) was studied and showing differentially regulated proteins. Two-dimensional electrophoresis gave a complex spot pattern comprising proteins of a broad range of isoelectric points and molecular masses. The most abundant spots were subjected to mass spectrometric analysis. Forty one spots representing 26 proteins were identified. Most of the identified proteins were involved in metabolic processes and response to stress.
Twenty spots were identified by MS/MS spectra and MASCOT search.
Proteins, which resulted with low confidence score were sequenced either manually or automatically with the help of PEAKS software. The most abundant peptide fragment ions ``y-ions'' and ``b-ions'', less abundant peptide fragments ions ``a-ions'' as well as immonium ions were used to complete the de novo sequences from MS/MS spectra. The generated sequences were subjected to MS homology search for the identification of proteins. Twenty one spots were identified by de novo sequencing due to a lack of DNA and protein database sequences of A. ochraceus. This proteome profiling of A. ochraceus will significantly accelerate understanding of this organism. Most of the identified proteins in our study have been reported for the first time in A. ochraceus, but in the future proteome profiling could be helpful to find out more about triggering factors for mycotoxin production under different environmental conditions.
Proteome / Aspergillus ochraceus / MALDI-TOF TOF mass spectrometer / Two dimensional gel electrophoresis / Proteins / Ochratoxin A (OTA) / HPLC / De novo sequencing / DIGE / Proteomics