Proteome analysis of Aspergillus ochraceus

Abstract

Genome sequencing for many important fungi has begun during recent years; however, there is still some 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. The cell walls of fungi are exceptionally resistant to destruction, therefore two lysis protocols were tested: (1) lysis via manual grinding using liquid nitrogen, and (2) mechanical lysis via rapid agitation with glass beads using MagNalyser. Mechanical grinding with mortar and pestle using liquid nitrogen was found to be a more efficient extraction method for our purpose, resulting in extracts with higher protein content and a clear band pattern in SDS-PAGE. 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. We could identify 31 spots representing 26 proteins, most of them involved in metabolic processes and response to stress. Seventeen spots were identified by de novo sequencing due to a lack of DNA and protein database sequences of A. ochraceus. The proteins identified in our study have been reported for the first time in A. ochraceus and this represents the first proteomic approach with identification of major proteins, when the fungus was grown under submerged culture.

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant

“…While grinding in liquid nitrogen ensured a better cooling of the sample, cell disruption via beads has a very low personal deviation. Extending previous studies [ 23 , 42 , 43 ], we were able to show that the subsequent extraction protocol has a higher impact on the total protein yield than the initial cell disruption. We demonstrated that a higher number of proteins was obtained when solubilisation of the hardly soluble protein fraction was achieved by additional steps.…”

“…Nearly all new studies needed an adaptation of the protein extraction methodology. In general, fungi are known to have rigid and complex cell walls, which is why a special focus on cell wall disruption is required during sample preparation [ 17 , 22 , 23 ].…”

Comprehensive proteomic analysis of Penicillium verrucosum

“…A 5600+ TripleTOF (Sciex, USA) was used for identification (Information dependent acquisition, IDA) and quantification (sequential window acquisition of all theoretical fragment‐ion spectra, SWATH) runs. For the IDA experiments, a 140 ms survey scan (TOF‐MS) was performed, from which the top 20 ions were selected for MS/MS analysis, each a 100 ms scan .…”

A Review beyond the borders: Proteomics of microclonial black fungi and black yeasts

“…Several approaches are described and optimized by different researchers; however there are still no general protocols available to extract proteins from any given sample [27]. The combination of TCA and acetone is reported as effective and easy for precipitation of fungal proteins [28,29]. Other strategies like 100 mM ammonium acetate in methanol were described also for fungal tissues [21].…”