Analysis of secreted proteins from Aspergillus flavus

Abstract

MS/MS techniques in proteomics make possible the identification of proteins from organisms with little or no genome sequence information available. Peptide sequences are obtained from tandem mass spectra by matching peptide mass and fragmentation information to protein sequence information from related organisms, including unannotated genome sequence data. This peptide identification data can then be grouped and reconstructed into protein data. In this study, we have used this approach to study protein secretion by Aspergillus flavus, a filamentous fungus for which very little genome sequence information is available. A. flavus is capable of degrading the flavonoid rutin (quercetin 3-O-glycoside), as the only source of carbon via an extracellular enzyme system. In this continuing study, a proteomic analysis was used to identify secreted proteins from A. flavus when grown on rutin. The growth media glucose and potato dextrose were used to identify differentially expressed secreted proteins. The secreted proteins were analyzed by 1- and 2-DE and MS/MS. A total of 51 unique A. flavus secreted proteins were identified from the three growth conditions. Ten proteins were unique to rutin-, five to glucose- and one to potato dextrose-grown A. flavus. Sixteen secreted proteins were common to all three media. Fourteen identifications were of hypothetical proteins or proteins of unknown functions. To our knowledge, this is the first extensive proteomic study conducted to identify the secreted proteins from a filamentous fungus.

Development of monoclonal antibodies to recombinant terrelysin and characterization of expression in Aspergillus terreus

“…Recently, Asphaemolysin was identified as one of the most abundant proteins in the A. fumigatus secretome even though this protein does not possess a signal peptide (Wartenberg et al, 2011). Other studies have highlighted secretion of proteins with no predicted signal sequence and have proposed nonconventional secretory mechanisms (Medina et al, 2005;Nickel, 2010;Rubartelli & Sitia, 1997). At day 6 in room temperature cultures and at day 4 at 37 u C, small increases in levels of terrelysin were observed in CSN.…”

The Use of Mass Spectrometry for Characterization of Fungal Secretomes

“…Limiting factors in the identification were the lack of the genome sequence of this fungus, the presence of glycosylation (a common occurrence in extracellular fungal enzymes; (Peberdy 1994))which affects detection by MALDI-TOF, or the lack of similar sequences from other organisms in the databases. In a later publication (Medina et al 2005), by incorporating the nanoLC-MS/MS technique, which does not have the limitations found in MALDI-TOF, they were able to identify 51 proteins. The extracellular proteome of Aspergillus oryzae was analyzed by comparing fungal cultures in liquid and solid media utilizing wheat bran as carbon source.…”

Hunting down fungal secretomes using liquid‐phase IEF prior to high resolution 2‐DE

“…Yet, several studies on fungal secretomics used TCA/acetone method to precipitate proteins from liquid media [14,18,19,23], with one study reporting the use of TCA in water followed by multiple ethanol washes [15]. Here, protein content could not be assayed; however, 1-and 2-D profiles were improved and showed intense bands corresponding to 266 spots (Fig.…”

“…The separation of basic secreted proteins along 7-10 and 6.3-8.3 pH gradients resulted in low-quality 2-D profiles. The vast majority of fungal secretome results based on 2-DE published to date only reported acidic proteins [12,15,[18][19][20][21][22][44][45][46]. Oda et al [16] mentioned that pIs of proteins in the Aspergillus oryzae secretome fell exclusively within 4-7 range.…”

“…To date, lyophilization has only been reported as a processing step to reduce and concentrate dilute liquid protein samples prior to classic protein extraction methods [15,19,21], with the exception of Cao et al [12] who used it to recover secreted proteins from Pyrenophora tritici-repentis without any further extraction and resolved 67 spots along 4-7 IPG strips. In our study, secretome samples were lyophilized and resolubilized in solutions compatible with downstream separation techniques (1-or 2-DE).…”