title: Characterization and identification of proteases secreted by Aspergillus fumigatus using free flow electrophoresis and MS.
abstract: Early diagnosis of life-threatening invasive aspergillosis in neutropenic patients remains challenging because current laboratory methods have limited diagnostic sensitivity and/or specificity. Aspergillus species are known to secrete various pathogenetically relevant proteases and the monitoring of their protease activity in serum specimens might serve as a new diagnostic approach.For the characterization and identification of secreted proteases, the culture supernatant of Aspergillus fumigatus was fractionated using free flow electrophoresis (Becton Dickinson). Protease activity of separated fractions was measured using fluorescently labeled reporter peptides. Fractions were also co-incubated in parallel with various protease inhibitors that specifically inhibit a distinct class of proteases e.g. metallo- or cysteine-proteases. Those fractions with high protease activity were further subjected to LC-MS/MS analysis for protease identification. The highest protease activity was measured in fractions with an acidic pH range. The results of the 'inhibitor-panel' gave a clear indication that it is mainly metallo- and serine-proteases that are involved in the degradation of reporter peptides. Furthermore, several proteases were identified that facilitate the optimization of reporter peptides for functional protease profiling as a diagnostic tool for invasive aspergillosis.
authors: Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany.
authors: Madlen
authors: M
authors: Neustadt
authors: Victor
authors: V
authors: Costina
authors: Claudio
authors: C
authors: Kupfahl
authors: Dieter
authors: D
authors: Buchheidt
authors: Christoph
authors: C
authors: Eckerskorn
authors: Michael
authors: M
authors: Neumaier
authors: Peter
authors: P
authors: Findeisen
mesh_terms: D001232:Aspergillus fumigatus / Q000737:chemistry / Q000201:enzymology*; D002853:Chromatography, Liquid; D003470:Culture Media; D004586:Electrophoresis / Q000379:methods*; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D053719:Tandem Mass Spectrometry
chemical_list: D003470:Culture Media; D005656:Fungal Proteins; D010455:Peptides; D010447:Peptide Hydrolases