Purpose: Early diagnosis of life-threatening invasive aspergillosis (IA) 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. Methods: We have constructed multiple reporter peptides (RPs) that are cleaved by endoproteases, sectreted from Aspergillus species with high specificity. These RPs were added to serum specimens from patients with invasive aspergillosis, healthy control individuals and patients with systemic inflammation. Spiked serum specimens were incubated under standardized conditions and cleaved fragments RPs were extracted with affinity chromatography prior to MALDI-TOF mass spectrometry. Results: Patients with IA could be classified with high accuracy based on the MS-profiles of fragments generated from the cleavage of RPs. The diagnostic sensitivity of RP-spiking was above 90%, however specificity ranged from 59% to 92% depending on the collectives that were compared. However, accuracy of RP-spiking was superior to the galactomannan assay that was performed in parallel. Conclusions: RP-spiking has been proposed to improve MS-based clinical proteomic profiling (1). The cleavage of specific RPs is indicating Aspergillus-specific proteolytic activity in serum specimens and serves as surrogate marker for invasive Aspergillosis. However, up to now the knowledge of substrate specificity of secreted proteases from Aspergillus species is rather poor (2, 3) and systematic investigations will be a prerequisite for new activity-based diagnostic approaches with the potential to improve the diagnostic approaches for IA. References: 1. Findeisen P, Post S, Wenz F, Neumaier M. Addition of exogenous reporter peptides to serum samples before mass spectrometry-based protease profiling provides advantages over profiling of endogenous peptides. Clinical chemistry 2007;53:1864-6. 2. Neustadt M, Costina V, Kupfahl C, Buchheidt D, Eckerskorn C, Neumaier M, Findeisen P. Characterization and identification of proteases secreted by Aspergillus fumigatus using free flow electrophoresis and MS. Electrophoresis 2009;30:2142-50. 3. Schaal R, Kupfahl C, Buchheidt D, Neumaier M, Findeisen P. Systematic identification of substrates for profiling of secreted proteases from Aspergillus species. Journal of microbiological methods 2007;71:93-100.
Full conference title:
4th Advances Against Aspergillosis
- AAA 4th (2010)