| Proteomic studies of filamentous fungi are severely lacking, particularly of secreted proteins expressed via dispensable metabolic pathways. Protein secretion plays an important role in fungi and more studies on the global analysis of secreted proteins need to be conducted. Proteomic analysis has proven to be the most powerful method for identification of proteins in complex mixtures and is suitable for the study of alteration of protein expression in an organism under varying environmental conditions. New techniques in proteomics are making possible the identification of proteins from mass spectrometry data, even from organisms whose genomes have not been sequenced.; The filamentous fungus Aspergillus flavus is capable of degrading the flavonoid rutin (quercetin 3-O-glycoside) as the only source of carbon via an extracellular enzyme system. The enzyme quercetin 2,3-dioxygenase is responsible for the degradation of quercetin, the aglycone of rutin. It is a copper-containing enzyme that oxidatively cleaves the O-heterocyclic ring of quercetin with concomitant release of carbon monoxide. In this study, a proteomic analysis was conducted to identify differentially expressed secreted proteins from A. flavus when grown on rutin, glucose and potato dextrose media. The secreted proteins were analyzed by two-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization time-of-flight and tandem mass spectrometry, and identified using several databases including an online database, a database containing all available fungal protein sequences, and unannotated genome sequences of two Aspergillus species and other organisms. Additionally, quercetin 2,3-dioxyhenase was isolated and purified from A. flavus using a novel three-step purification scheme; which is different from previously published protocols. Tandem mass spectrometry has provided the first information regarding its amino acid sequence by homology BLAST searching of a region of the unannotated genome sequence of A. nidulans .; This study has been conducted to expand the knowledge of proteins and enzymes secreted by A. flavus under varying conditions of growth and to gain a more complete understanding of the metabolism of rutin. It demonstrates the high capability of combined proteomic techniques for the identification of proteins from unsequenced genomes such as A. flavus . It is the first complete proteomic study performed of secreted proteins from a filamentous fungus. |
