|Identification of Aspergillus spores by preparative IEF and MALDI-TOF MS
|Exploiting Separation Science
|Dr Jiri Salplachta
|Dr Marie Horka
Prof Karel Slais
|Czech Academy of Sciences, Institute of Analytical Chemistry
Abstract Information :
Fungi play an important role in many ecosystems including human environment. Aspergillus is one of the most common and widely distributed genera of fungi. Aspergillus species have a great impact on various fields of interest. Some species of Aspergillus are used in food production and in industrial bioprocesses. Others species produce toxic secondary metabolites (aflatoxins) in infected crops. Many species can cause opportunistic fungal disease in hosts with weakened immune system. In this respect, identification of Aspergillus spp. is of significant interest. Fungal identification is currently performed by micro- and macroscopic examination of morphological and culture characteristics. However, these methods are often time consuming and tend to fail. In recent years, matrix-assisted laser desorption/ionization time-of-ﬂight mass spectrometry (MALDI-TOF MS) has become a fast and reliable technique for identification of various microorganisms including several fungal species.
This study presents a reliable method, a combination of recently developed preparative isoelectric focusing (IEF) in a cellulose-based separation medium with MALDI-TOF MS analysis, for identification of Aspergillus spores in different sample matrices. Four Aspergillus species, A. niger, A. flavus, A. fumigatus, and A. parasiticus, were chosen for this purpose. In the proposed method, fungal samples were first treated by preparative IEF and the separated spores were then identified by MALDI-TOF MS. Isoelectric points of the spores were determined by capillary IEF. Preparative IEF was used as an efficient technique to separate fungal spores from each other as well as from other sample components that could affect identification of the examined spores. The separation medium was designed so that it allows separation of large molecules and microorganisms and it is inert to the separated analytes. Final positions of the focused spores were indicated by the colored pI markers. Fractions containing the separated spores were easily collected with a spatula, the spores were then extracted from the separation medium and further processed in a simple way for subsequent mass spectrometric analysis. Since the final samples contained individual spores and virtually no interfering compounds, the spores were identified unambiguously by MALDI-TOF MS.