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A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in  Aspergillus fumigatus . Here, implementation of a shotgun proteomic strategy led to the identification of non-redundant mycelial proteins ( n  = 414) from  A. fumigatus  including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and p I . Indirect identification of secondary metabolite cluster expression was also achieved, with proteins ( n  = 18) from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical analysis then revealed that gliotoxin significantly attenuates H 2 O 2 -induced oxidative stress in  A. fumigatus  ( p >0.0001), confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly increased abundance ( p <0.05) of proliferating cell nuclear antigen (PCNA), NADH-quinone oxidoreductase and the gliotoxin oxidoreductase GliT, along with significantly attenuated abundance ( p <0.05) of a heat shock protein, an oxidative stress protein and an autolysis-associated chitinase, when gliotoxin and H 2 O 2  were present, compared to H 2 O 2  alone. Moreover, gliotoxin exposure significantly reduced the abundance of selected proteins ( p <0.05) involved in  de novo  purine biosynthesis. Significantly elevated abundance ( p <0.05) of a key enzyme, xanthine-guanine phosphoribosyl transferase Xpt1, utilised in purine salvage, was observed in the presence of H 2 O 2  and gliotoxin. This work provides new insights into the  A. fumigatus  proteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the organism.

A Proteomic Approach to Investigating Gene Cluster Expression and Secondary Metabolite Functionality in Aspergillus fumigatus